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Sample records for albumin human serum

  1. Large fragments of human serum albumin.

    PubMed

    Geisow, M J; Beaven, G H

    1977-03-01

    Large fragments of human serum albumin were produced by treatment of the native protein with pepsin at pH3.5. Published sequences of human albumin [Behrens, Spiekerman & Brown (1975) Fed. Proc. Fed. Am. Soc. Exp. Biol. 34, 591; Meloun, Moravek & Kostka (1975) FEBSLett.58, 134-137]were used to locate the fragments in the primary structure. The fragments support both the sequence and proposed disulphide-linkage pattern (Behrens et al., 1975). As the pH of a solution of albumin is lowered from pH4 to pH3.5, the protein undergoes a reversible conformational change known as the N-F transition. The distribution of large fragments of human albumin digested with pepsin in the above pH region was critically dependent on pH. It appeared that this distribution was dependent on the conformation of the protein at low pH, rather than the activity of pepsin. The yields of the large fragments produced by peptic digestion at different values of pH suggested that the C-terminal region of albumin unfolds or separates from the rest of the molecule during the N-F transition. The similarity of peptic fragments of human and bovine albumin produced under identical conditions supports the proposed similar tertiary structure of these molecules.

  2. Polymerized soluble venom--human serum albumin

    SciTech Connect

    Patterson, R.; Suszko, I.M.; Grammer, L.C.

    1985-03-01

    Extensive previous studies have demonstrated that attempts to produce polymers of Hymenoptera venoms for human immunotherapy resulted in insoluble precipitates that could be injected with safety but with very limited immunogenicity in allergic patients. We now report soluble polymers prepared by conjugating bee venom with human serum albumin with glutaraldehyde. The bee venom-albumin polymer (BVAP) preparation was fractionated on Sephacryl S-300 to have a molecular weight range higher than catalase. /sup 125/I-labeled bee venom phospholipase A was almost completely incorporated into BVAP. Rabbit antibody responses to bee venom and bee venom phospholipase A were induced by BVAP. Human antisera against bee venom were absorbed by BVAP. No new antigenic determinants on BVAP were present as evidenced by absorption of antisera against BVAP by bee venom and albumin. BVAP has potential immunotherapeutic value in patients with anaphylactic sensitivity to bee venom.

  3. Interaction of Citrinin with Human Serum Albumin.

    PubMed

    Poór, Miklós; Lemli, Beáta; Bálint, Mónika; Hetényi, Csaba; Sali, Nikolett; Kőszegi, Tamás; Kunsági-Máté, Sándor

    2015-12-01

    Citrinin (CIT) is a mycotoxin produced by several Aspergillus, Penicillium, and Monascus species. CIT occurs worldwide in different foods and drinks and causes health problems for humans and animals. Human serum albumin (HSA) is the most abundant plasma protein in human circulation. Albumin forms stable complexes with many drugs and xenobiotics; therefore, HSA commonly plays important role in the pharmacokinetics or toxicokinetics of numerous compounds. However, the interaction of CIT with HSA is poorly characterized yet. In this study, the complex formation of CIT with HSA was investigated using fluorescence spectroscopy and ultrafiltration techniques. For the deeper understanding of the interaction, thermodynamic, and molecular modeling studies were performed as well. Our results suggest that CIT forms stable complex with HSA (logK ~ 5.3) and its primary binding site is located in subdomain IIA (Sudlow's Site I). In vitro cell experiments also recommend that CIT-HSA interaction may have biological relevance. Finally, the complex formations of CIT with bovine, porcine, and rat serum albumin were investigated, in order to test the potential species differences of CIT-albumin interactions.

  4. Interaction of Citrinin with Human Serum Albumin

    PubMed Central

    Poór, Miklós; Lemli, Beáta; Bálint, Mónika; Hetényi, Csaba; Sali, Nikolett; Kőszegi, Tamás; Kunsági-Máté, Sándor

    2015-01-01

    Citrinin (CIT) is a mycotoxin produced by several Aspergillus, Penicillium, and Monascus species. CIT occurs worldwide in different foods and drinks and causes health problems for humans and animals. Human serum albumin (HSA) is the most abundant plasma protein in human circulation. Albumin forms stable complexes with many drugs and xenobiotics; therefore, HSA commonly plays important role in the pharmacokinetics or toxicokinetics of numerous compounds. However, the interaction of CIT with HSA is poorly characterized yet. In this study, the complex formation of CIT with HSA was investigated using fluorescence spectroscopy and ultrafiltration techniques. For the deeper understanding of the interaction, thermodynamic, and molecular modeling studies were performed as well. Our results suggest that CIT forms stable complex with HSA (logK ~ 5.3) and its primary binding site is located in subdomain IIA (Sudlow’s Site I). In vitro cell experiments also recommend that CIT-HSA interaction may have biological relevance. Finally, the complex formations of CIT with bovine, porcine, and rat serum albumin were investigated, in order to test the potential species differences of CIT-albumin interactions. PMID:26633504

  5. (PCG) Protein Crystal Growth Human Serum Albumin

    NASA Technical Reports Server (NTRS)

    1989-01-01

    (PCG) Protein Crystal Growth Human Serum Albumin. Contributes to many transport and regulatory processes and has multifunctional binding properties which range from various metals, to fatty acids, hormones, and a wide spectrum of therapeutic drugs. The most abundant protein of the circulatory system. It binds and transports an incredible variety of biological and pharmaceutical ligands throughout the blood stream. Principal Investigator on STS-26 was Larry DeLucas.

  6. Atomic structure and chemistry of human serum albumin

    NASA Technical Reports Server (NTRS)

    He, Xiao M.; Carter, Daniel C.

    1992-01-01

    The three-dimensional structure of human serum albumin has been determined crystallographically to a resolution of 2.8 A. It comprises three homologous domains that assemble to form a heart-shaped molecule. Each domain is a product of two subdomains that possess common structural motifs. The principal regions of ligand binding to human serum albumin are located in hydrophobic cavities in subdomains IIA and ILIA, which exhibit similar chemistry. The structure explains numerous physical phenomena and should provide insight into future pharmacokinetic and genetically engineered therapeutic applications of serum albumin.

  7. 99M-technetium labeled macroaggregated human serum albumin pharmaceutical

    DOEpatents

    Winchell, Harry S.; Barak, Morton; Van Fleet, III, Parmer

    1977-05-17

    A reagent comprising macroaggregated human serum albumin having dispersed therein particles of stannous tin and a method for instantly making a labeled pharmaceutical therefrom, are disclosed. The labeled pharmaceutical is utilized in organ imaging.

  8. [Detection of autoantibodies to polymerized human serum albumin].

    PubMed

    Manns, M; Müller, M; Meyer zum Büschenfelde, K H

    1984-11-01

    Binding activity of antibodies against polymerized human serum albumin (pHSA) was measured in the serum of 348 patients with various hepatic and non-hepatic diseases and in the serum of 108 control persons. The methods used were passive hemagglutination (PH) with antigen loaded human erythrocytes and radial immunodiffusion (ID). In the PH-method only HBsAg-positive sera reacted. Blocking experiments with pHSA, polymerized bovine serum albumin (pBSA) and monomeric human serum albumin (mHSA) showed, that the PH-method measures HBsAG associated receptors for pHSA. In HBeAG-positive cases titers were significantly higher than in anti-HBe-positive sera. Using the ID-method it could be shown, that 40% of sera of patients with liver diseases (n = 272), 37% of patients with LED (n = 27), 72% of patients with rheumatoid arthritis (n = 32), 6% of patients with glomerulonephritis (n = 17) and 2% of normal persons (n = 108) reacted. These sera reacted in the immunodiffusion assay with pHSA and pBSA but not with mHSA. Autoantibodies against non species specific determinants of pHSA which are not specific for liver diseases and possibly due to disturbed immunoregulation can be demonstrated by immunodiffusion. They may possibly be modulators of the pHSA mediated binding of hepatitis B-virus to hepatocytes.

  9. Sequences Of Amino Acids For Human Serum Albumin

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C.

    1992-01-01

    Sequences of amino acids defined for use in making polypeptides one-third to one-sixth as large as parent human serum albumin molecule. Smaller, chemically stable peptides have diverse applications including service as artificial human serum and as active components of biosensors and chromatographic matrices. In applications involving production of artificial sera from new sequences, little or no concern about viral contaminants. Smaller genetically engineered polypeptides more easily expressed and produced in large quantities, making commercial isolation and production more feasible and profitable.

  10. Three-dimensional structure of human serum albumin

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C.; He, Xiao-Min; Munson, Sibyl H.; Twigg, Pamela D.; Gernert, Kim M.; Broom, M. Beth; Miller, Teresa Y.

    1989-01-01

    The three-dimensional structure of human serum albumin has been solved at 6.0 A resolution by the method of multiple isomorphous replacement. Crystals were grown from solutions of polyethylene glycol in the infrequently observed space group P42(1)2 and diffracted X-rays to lattice d-spacings of less than 2.9 A. The electron density maps are of high quality and revealed the structure as a predominantly alpha-helical globin protein in which the course of the polypeptide can be traced. The binding loci of several organic compounds have been determined.

  11. Analysis of Aged Human Serum Albumin Affinity for Doxazosin.

    PubMed

    Chudzik, Mariola; Równicka-Zubik, Joanna; Pożycka, Jadwiga; Pawelczak, Bartosz; Sulkowska, Anna

    2016-01-01

    Structural changes of human serum albumin (HSA) caused by old age and coexisting diseases result in differences in the binding of doxazosin (DOX). DOX is a postsynaptic α1- adrenoreceptor antagonist used for treatment of hypertension and benign prostatic hyperplasia. In elderly people suffering from various renal or hepatic diseases the significant portion of N-form of human serum albumin (normal) is converted to A-form (aged). The differences in binding of doxazosin to N- and Aform of albumin are an important factor, which may determines therapeutic dosage and toxicity of the test drug. To indicate these differences, the technique of fluorescence spectroscopy was used. The association constant (Ka) obtained from fluorescence quenching demonstrated that doxazosin has higher affinity for AHSA than for HSA. In order to describe the cooperativity in binding process, the values of the Hill's coefficient has been analysed. For DOX-HSA system (λex 295 nm) Hill's coefficient is close to 1 and it indicates that there is a single class of binding sites. For DOX-HSA (λex 275 nm) and DOX-AHSA (λex 275 nm and λex 295 nm) systems we observed positive cooperativity (nH>1). A greater red shift of fluorescence emission maximum of AHSA than HSA in the presence of DOX was observed. This suggests that the binding of DOX to AHSA was accompanied by a stronger increase in polarity around the fluorophores in comparison to HSA. The binding interaction between DOX and HSA has been also studied by molecular docking simulation.

  12. Interaction of drugs with bovine and human serum albumin

    NASA Astrophysics Data System (ADS)

    Sułkowska, Anna

    2002-09-01

    The study on the interaction of antithyroid drugs: 2-mercapto-1-methylimidazole (Methimazole, MMI) and 6 n-propyl-2-thiouracil (PTU) with two kinds of serum albumin: bovine (BSA) and human (HSA) has been undertaken. Fluorescence emission spectra of serum albumin in the presence of MMI or PTU, recorded at the excitation wavelengths 280 and 295 nm, clearly show that the studied drugs act as quenchers. A decrease in fluorescence intensity at 340 or 350 nm, when excited at 280 or 295 nm, respectively, is attributed to changes in the environment of the protein fluorophores caused to the presence of the ligand. The 295 nm lights excites tryptophan residues, while the 280 nm lights excites both tryptophan and tyrosine residues. A comparison of quenching effects, when protein is excited at 295 and 280 nm, reveals that the tryptophanyl group interacts with the ligand. The differences in interactions of pyrimidine derivatives with HSA and BSA were observed using spectrofluorimetry technique. As the HSA structure contains only one tryptophanyl residue (Trp 214), while BSA has two ones (Trp 135 and Trp 214), the similar decrease of fluorescence points at the subdomain IIA, where Trp 214 was located, as a binding site of the studied drugs.

  13. Human serum albumin crystals and method of preparation

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C. (Inventor)

    1989-01-01

    Human serum albumin (HSA) crystals are provided in the form of tetragonal plates having the space groups P42(sub 1)2, the crystals being grown to sizes in excess of 0.5 mm in two dimensions and a thickness of 0.1 mm. Growth of the crystals is carried out by a hanging drop method wherein a precipitant solution containing polyethylene glycol (PEG) and a phosphate buffer is mixed with an HSA solution, and a droplet of mixed solution is suspended over a well of precipitant solution. Crystals grow to the desired size in 3 to 7 days. Concentration of reagents, pH and other parameters are controlled within prescribed limits. The resulting crystals exhibit a size and quality such as to allow performance of x ray diffraction studies and enable the conduct of drug binding studies as well as genetic engineering studies.

  14. Heme-based catalytic properties of human serum albumin

    PubMed Central

    Ascenzi, P; di Masi, A; Fanali, G; Fasano, M

    2015-01-01

    Human serum albumin (HSA): (i) controls the plasma oncotic pressure, (ii) modulates fluid distribution between the body compartments, (iii) represents the depot and carrier of endogenous and exogenous compounds, (iv) increases the apparent solubility and lifetime of hydrophobic compounds, (v) affects pharmacokinetics of many drugs, (vi) inactivates toxic compounds, (vii) induces chemical modifications of some ligands, (viii) displays antioxidant properties, and (ix) shows enzymatic properties. Under physiological and pathological conditions, HSA has a pivotal role in heme scavenging transferring the metal-macrocycle from high- and low-density lipoproteins to hemopexin, thus acquiring globin-like reactivity. Here, the heme-based catalytic properties of HSA are reviewed and the structural bases of drug-dependent allosteric regulation are highlighted. PMID:27551458

  15. Three-dimensional structure of human serum albumin

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C.; He, Xiao-Min; Twigg, Pamela D.; Casale, Elena

    1991-01-01

    The binding locations to human serum albumin (HSA) of several drug molecules were determined at low resolution using crystallographic methods. The principal binding sites are located within subdomains IIA and IIIA. Preliminary studies suggest that an approach to increasing the in vivo efficacy of drugs which are rendered less effective or ineffective by virtue of their interaction with HSA, would be the use of competitive displacement in drug therapies and/or the development of a general inhibitor to the site within subdomain IIIA. These findings also suggest that the facilitated transfer of various ligands across organ/circulatory interfaces such as liver, kidney, and brain may be associated with binding to the IIIA subdomain.

  16. [Study on the interaction of doxycycline with human serum albumin].

    PubMed

    Hu, Tao-Ying; Chen, Lin; Liu, Ying

    2014-05-01

    The present study was designed to investigate the interaction of doxycycline (DC) with human serum albumin (HSA) by the inner filter effects, displacement experiments and molecular docking methods, based on classic multi-spectroscopy. With fluorescence quenching method at 298 and 310 K, the binding constants Ka, were determined to be 2. 73 X 10(5) and 0. 74X 10(5) L mol-1, respectively, and there was one binding site between DC and HSA, indicating that the binding of DC to HSA was strong, and the quenching mechanism was a static quenching. The thermodynamic parameters (enthalpy change, AH and enthropy change, delta S) were calculated to be -83. 55 kJ mol-1 and -176. 31 J mol-1 K-1 via the Vant' Hoff equation, which indicated that the interaction of DC with HSA was driven mainly by hydrogen bonding and van der Waals forces. Based on the Föster's theory of non-radiation energy transfer, the specific binding distance between Trp-214 (acceptor) and DC (donor) was 4. 98 nm, which was similar to the result confirmed by molecular docking. Through displacement experiments, sub-domain IIA of HSA was assigned to possess the high-affinity binding site of DC. Three-dimensional fluorescence spectra indicated that the binding of DC to HSA induced the conformation change of HSA and increased the disclosure of some part of hydrophobic regions that had been buried before. The results of FTIR spectroscopy showed that DC bound to HSA led to the slight unfolding of the polypeptide chain of HSA. Furthermore, the binding details between DC and HSA were further confirmed by molecular docking methods, which revealed that DC was bound at sub-domain IIA through multiple interactions, such as hydrophobic effect, polar forces and pi-pi interactions. The experimental results provide theoretical basis and reliable data for the study of the interaction between small drug molecule and human serum albumin PMID:25095435

  17. Binding and hydrolysis of soman by human serum albumin.

    PubMed

    Li, Bin; Nachon, Florian; Froment, Marie-Thérèse; Verdier, Laurent; Debouzy, Jean-Claude; Brasme, Bernardo; Gillon, Emilie; Schopfer, Lawrence M; Lockridge, Oksana; Masson, Patrick

    2008-02-01

    Human plasma and fatty acid free human albumin were incubated with soman at pH 8.0 and 25 degrees C. Four methods were used to monitor the reaction of albumin with soman: progressive inhibition of the aryl acylamidase activity of albumin, the release of fluoride ion from soman, 31P NMR, and mass spectrometry. Inhibition (phosphonylation) was slow with a bimolecular rate constant of 15 +/- 3 M(-1) min (-1). MALDI-TOF and tandem mass spectrometry of the soman-albumin adduct showed that albumin was phosphonylated on tyrosine 411. No secondary dealkylation of the adduct (aging) occurred. Covalent docking simulations and 31P NMR experiments showed that albumin has no enantiomeric preference for the four stereoisomers of soman. Spontaneous reactivation at pH 8.0 and 25 degrees C, measured as regaining of aryl acylamidase activity and decrease of covalent adduct (pinacolyl methylphosphonylated albumin) by NMR, occurred at a rate of 0.0044 h (-1), indicating that the adduct is quite stable ( t1/2 = 6.5 days). At pH 7.4 and 22 degrees C, the covalent soman-albumin adduct, measured by MALDI-TOF mass spectrometry, was more stable ( t1/2 = 20 days). Though the concentration of albumin in plasma is very high (about 0.6 mM), its reactivity with soman (phosphonylation and phosphotriesterase activity) is too slow to play a major role in detoxification of the highly toxic organophosphorus compound soman. Increasing the bimolecular rate constant of albumin for organophosphates is a protein engineering challenge that could lead to a new class of bioscavengers to be used against poisoning by nerve agents. Soman-albumin adducts detected by mass spectrometry could be useful for the diagnosis of soman exposure. PMID:18163544

  18. Human Serum Albumin Complexed with Myristate and AZT

    SciTech Connect

    Zhu, Lili; Yang, Feng; Chen, Liqing; Meehan, Edward J.; Huang, Mingdong

    2008-06-16

    3'-Azido-3'-deoxythymidine (AZT) is the first clinically effective drug for the treatment of human immunodeficiency virus infection. The drug interaction with human serum albumin (HSA) has been an important component in understanding its mechanism of action, especially in drug distribution and in drug-drug interaction on HSA in the case of multi-drug therapy. We present here crystal structures of a ternary HSA-Myr-AZT complex and a quaternary HSA-Myr-AZT-SAL complex (Myr, myristate; SAL, salicylic acid). From this study, a new drug binding subsite on HSA Sudlow site 1 was identified. The presence of fatty acid is needed for the creation of this subsite due to fatty acid induced conformational changes of HSA. Thus, the Sudlow site 1 of HSA can be divided into three non-overlapped subsites: a SAL subsite, an indomethacin subsite and an AZT subsite. Binding of a drug to HSA often influences simultaneous binding of other drugs. From the HSA-Myr-AZT-SAL complex structure, we observed the coexistence of two drugs (AZT and SAL) in Sudlow site 1 and the competition between these two drugs in subdomain IB. These results provide new structural information on HSA-drug interaction and drug-drug interaction on HSA.

  19. Human plasma lipocalins and serum albumin: Plasma alternative carriers?

    PubMed

    di Masi, Alessandra; Trezza, Viviana; Leboffe, Loris; Ascenzi, Paolo

    2016-04-28

    Lipocalins are an evolutionarily conserved family of proteins that bind and transport a variety of exogenous and endogenous ligands. Lipocalins share a conserved eight anti-parallel β-sheet structure. Among the different lipocalins identified in humans, α-1-acid glycoprotein (AGP), apolipoprotein D (apoD), apolipoprotein M (apoM), α1-microglobulin (α1-m) and retinol-binding protein (RBP) are plasma proteins. In particular, AGP is the most important transporter for basic and neutral drugs, apoD, apoM, and RBP mainly bind endogenous molecules such as progesterone, pregnenolone, bilirubin, sphingosine-1-phosphate, and retinol, while α1-m binds the heme. Human serum albumin (HSA) is a monomeric all-α protein that binds endogenous and exogenous molecules like fatty acids, heme, and acidic drugs. Changes in the plasmatic levels of lipocalins and HSA are responsible for the onset of pathological conditions associated with an altered drug transport and delivery. This, however, does not necessary result in potential adverse effects in patients because many drugs can bind both HSA and lipocalins, and therefore mutual compensatory binding mechanisms can be hypothesized. Here, molecular and clinical aspects of ligand transport by plasma lipocalins and HSA are reviewed, with special attention to their role as alterative carriers in health and disease. PMID:26951925

  20. [Preparation of Human Serum Albumin Micro/Nanotubes].

    PubMed

    Jiao, Pei-pei; Guo, Yan-li; Niu, Ai-hua; Kang, Xiao-feng

    2016-01-01

    In this research, protein micro/nanotubes were fabricated by alternate layer-by-layer (LbL) assembly of human serum albumin (HSA) and polyethyleneimine (PEI) into polycarbonate (PC) membranes. The experimental conditions of pH values, ionic strength, the depositions cycles and the diameter of porous membrane were discussed. The morphology and composition of tubes were characterized by scanning electron microscope (SEM), transmission electron microscope (TEM), fourier transform infrared spectroscopy (FTIR) and energy dispersive spectroscopy (EDS). The results show that pH and ionic strength of the solution are the key factors that influence the effect of assembly. Micro/nanotubes with good opening hollow tubular structure were obtained when pH 7.4 HSA solution and pH 10.3 PEI solution without NaCl were used in synthesis procedure. The outer diameter of tube was dependent on the PC template, thus the micro/nanotubes size was controlled by the wall thickness, which can be adjusted by the number of layers of the HSA and PEI deposited along the pore walls. To avoid the thin wall being damaged in dissolving the template and vacuum drying, the PEI/HSA bilayer number should not be less than 3. The polar solvent N,N-dimethylformamide (DMF) can dissolve PC template to release the micro/nanotubes. PMID:27228766

  1. Hydrophobically derivatized hyperbranched polyglycerol as a human serum albumin substitute.

    PubMed

    Kainthan, Rajesh K; Janzen, Johan; Kizhakkedathu, Jayachandran N; Devine, Dana V; Brooks, Donald E

    2008-04-01

    There is a huge clinical demand for Human Serum Albumin (HSA), with a world market of approximately $1.5B/year. Concern over prion and viral transmission in the blood supply has led to a need for safer substitutes and offers the opportunity for development of materials with enhanced properties over the presently available plasma expanders. We report here the synthesis and testing of a new synthetic plasma expander that can replace not only the osmotic and volume expansion properties of HSA but, uniquely, its binding and transport properties. We have synthesized several hyperbranched polyglycerols derivatized with hydrophobic groups and short poly(ethylene glycol) (PEG) chains. The hydrophobic groups provide regions for binding fatty acids and other hydrophobic materials while PEG imparts the necessary protection from host defense systems and enhances circulation longevity. These polymers, being hyperbranched, have only a small effect on plasma viscosity. We have shown in vitro that our materials bind 2-3 moles palmitic acid per mole, do not activate the platelet, coagulation or complement systems and do not cause red cell aggregation. In mice these materials are non-toxic with circulation half-lives as high as 34h, controllable by manipulating the molecular weight and the degree of PEG derivatization. PMID:18194812

  2. Cooperative binding of drugs on human serum albumin

    NASA Astrophysics Data System (ADS)

    Varela, L. M.; Pérez-Rodríguez, M.; García, M.

    In order to explain the adsorption isotherms of the amphiphilic penicillins nafcillin and cloxacillin onto human serum albumin (HSA), a cooperative multilayer adsorption model is introduced, combining the Brunauer-Emmet-Teller (BET) adsorption isotherm with an amphiphilic ionic adsorbate, whose chemical potential is derived from Guggenheim's theory. The non-cooperative model has been previously proved to qualitatively predict the measured adsorption maxima of these drugs [Varela, L. M., García, M., Pérez-Rodríguez, M., Taboada, P., Ruso, J. M., and Mosquera, V., 2001, J. chem. Phys., 114, 7682]. The surface interactions among adsorbed drug molecules are modelled in a mean-field fashion, so the chemical potential of the adsorbate is assumed to include a term proportional to the surface coverage, the constant of proportionality being the lateral interaction energy between bound molecules. The interaction energies obtained from the empirical binding isotherms are of the order of tenths of the thermal energy, therefore suggesting the principal role of van der Waals forces in the binding process.

  3. Thermodynamic analysis of hydration in human serum heme-albumin

    SciTech Connect

    Baroni, Simona; Pariani, Giorgio; Fanali, Gabriella; Longo, Dario; Ascenzi, Paolo; Aime, Silvio; Fasano, Mauro

    2009-07-31

    Ferric human serum heme-albumin (heme-HSA) shows a peculiar nuclear magnetic relaxation dispersion (NMRD) behavior that allows to investigate structural and functional properties. Here, we report a thermodynamic analysis of NMRD profiles of heme-HSA between 20 and 60 {sup o}C to characterize its hydration. NMRD profiles, all showing two Lorentzian dispersions at 0.3 and 60 MHz, were analyzed in terms of modulation of the zero field splitting tensor for the S = {sup 5}/{sub 2} manifold. Values of correlation times for tensor fluctuation ({tau}{sub v}) and chemical exchange of water molecules ({tau}{sub M}) show the expected temperature dependence, with activation enthalpies of -1.94 and -2.46 {+-} 0.2 kJ mol{sup -1}, respectively. The cluster of water molecules located in the close proximity of the heme is progressively reduced in size by increasing the temperature, with {Delta}H = 68 {+-} 28 kJ mol{sup -1} and {Delta}S = 200 {+-} 80 J mol{sup -1} K{sup -1}. These results highlight the role of the water solvent in heme-HSA structure-function relationships.

  4. [Preparation of Human Serum Albumin Micro/Nanotubes].

    PubMed

    Jiao, Pei-pei; Guo, Yan-li; Niu, Ai-hua; Kang, Xiao-feng

    2016-01-01

    In this research, protein micro/nanotubes were fabricated by alternate layer-by-layer (LbL) assembly of human serum albumin (HSA) and polyethyleneimine (PEI) into polycarbonate (PC) membranes. The experimental conditions of pH values, ionic strength, the depositions cycles and the diameter of porous membrane were discussed. The morphology and composition of tubes were characterized by scanning electron microscope (SEM), transmission electron microscope (TEM), fourier transform infrared spectroscopy (FTIR) and energy dispersive spectroscopy (EDS). The results show that pH and ionic strength of the solution are the key factors that influence the effect of assembly. Micro/nanotubes with good opening hollow tubular structure were obtained when pH 7.4 HSA solution and pH 10.3 PEI solution without NaCl were used in synthesis procedure. The outer diameter of tube was dependent on the PC template, thus the micro/nanotubes size was controlled by the wall thickness, which can be adjusted by the number of layers of the HSA and PEI deposited along the pore walls. To avoid the thin wall being damaged in dissolving the template and vacuum drying, the PEI/HSA bilayer number should not be less than 3. The polar solvent N,N-dimethylformamide (DMF) can dissolve PC template to release the micro/nanotubes.

  5. Interaction of Human Serum Albumin with Metal Protoporphyrins

    NASA Astrophysics Data System (ADS)

    Hu, Jie; Brancaleon, Lorenzo

    2015-03-01

    Fluorescence spectroscopy is widely used in biotechnology, nanotechnology, and molecular biophysics, since it can provide information on a wide range of molecular processes, e.g. the interactions of solvent molecules with fluorophores, conformational changes, and binding interactions etc. In this study, we present the photophysical properties of the interaction of human serum albumin (HSA) with a series of metal compound of Protoporphyrin IX (PPIX), including ZnPPIX, FePPIX, MgPPIX, MnPPIX and SnPPIX respectively, as well as the free base PPIX. Binding constants were retrieved independently using the Benesi-Hildebrand analysis of the porphyrin emission or absorption spectra and the fluorescence quenching (i.e. Stern-Volmer analysis) and reveal that the two methods yield a difference of approximately one order or magnitude between the two. Fluorescence lifetimes was used to probe whether binding of the porphyrin changes the conformation of the protein or if the interaction places the porphyrin at a location that can prompt resonance energy transfer with the lone Tryptophan residue. In recent years it has been discovered that HSA provides a specific binding site for metal-chelated protoporphyrins in subdomain IA. This has opened a novel field of study over the importance of this site for biomedical applications but it has also created the potential for a series of biotechnological applications of the HSA/protoporphyrin complexes. Our study provides a preliminary investigation of the interaction with metal-chelated protoporphyrins that had not been previously investigated.

  6. Serum Albumin Domain Structures in Human Blood Serum by Mass Spectrometry and Computational Biology*

    PubMed Central

    Belsom, Adam; Schneider, Michael; Fischer, Lutz; Brock, Oliver; Rappsilber, Juri

    2016-01-01

    Chemical cross-linking combined with mass spectrometry has proven useful for studying protein-protein interactions and protein structure, however the low density of cross-link data has so far precluded its use in determining structures de novo. Cross-linking density has been typically limited by the chemical selectivity of the standard cross-linking reagents that are commonly used for protein cross-linking. We have implemented the use of a heterobifunctional cross-linking reagent, sulfosuccinimidyl 4,4′-azipentanoate (sulfo-SDA), combining a traditional sulfo-N-hydroxysuccinimide (sulfo-NHS) ester and a UV photoactivatable diazirine group. This diazirine yields a highly reactive and promiscuous carbene species, the net result being a greatly increased number of cross-links compared with homobifunctional, NHS-based cross-linkers. We present a novel methodology that combines the use of this high density photo-cross-linking data with conformational space search to investigate the structure of human serum albumin domains, from purified samples, and in its native environment, human blood serum. Our approach is able to determine human serum albumin domain structures with good accuracy: root-mean-square deviation to crystal structure are 2.8/5.6/2.9 Å (purified samples) and 4.5/5.9/4.8Å (serum samples) for domains A/B/C for the first selected structure; 2.5/4.9/2.9 Å (purified samples) and 3.5/5.2/3.8 Å (serum samples) for the best out of top five selected structures. Our proof-of-concept study on human serum albumin demonstrates initial potential of our approach for determining the structures of more proteins in the complex biological contexts in which they function and which they may require for correct folding. Data are available via ProteomeXchange with identifier PXD001692. PMID:26385339

  7. Serum Albumin Domain Structures in Human Blood Serum by Mass Spectrometry and Computational Biology.

    PubMed

    Belsom, Adam; Schneider, Michael; Fischer, Lutz; Brock, Oliver; Rappsilber, Juri

    2016-03-01

    Chemical cross-linking combined with mass spectrometry has proven useful for studying protein-protein interactions and protein structure, however the low density of cross-link data has so far precluded its use in determining structures de novo. Cross-linking density has been typically limited by the chemical selectivity of the standard cross-linking reagents that are commonly used for protein cross-linking. We have implemented the use of a heterobifunctional cross-linking reagent, sulfosuccinimidyl 4,4'-azipentanoate (sulfo-SDA), combining a traditional sulfo-N-hydroxysuccinimide (sulfo-NHS) ester and a UV photoactivatable diazirine group. This diazirine yields a highly reactive and promiscuous carbene species, the net result being a greatly increased number of cross-links compared with homobifunctional, NHS-based cross-linkers. We present a novel methodology that combines the use of this high density photo-cross-linking data with conformational space search to investigate the structure of human serum albumin domains, from purified samples, and in its native environment, human blood serum. Our approach is able to determine human serum albumin domain structures with good accuracy: root-mean-square deviation to crystal structure are 2.8/5.6/2.9 Å (purified samples) and 4.5/5.9/4.8Å (serum samples) for domains A/B/C for the first selected structure; 2.5/4.9/2.9 Å (purified samples) and 3.5/5.2/3.8 Å (serum samples) for the best out of top five selected structures. Our proof-of-concept study on human serum albumin demonstrates initial potential of our approach for determining the structures of more proteins in the complex biological contexts in which they function and which they may require for correct folding. Data are available via ProteomeXchange with identifier PXD001692.

  8. Iron absorption in humans: bovine serum albumin compared with beef muscle and egg white

    SciTech Connect

    Hurrell, R.F.; Lynch, S.R.; Trinidad, T.P.; Dassenko, S.A.; Cook, J.D.

    1988-01-01

    We studied the influence of bovine serum albumin and beef meat on nonheme iron absorption in humans and on dialyzable iron in vitro. The addition of serum albumin to a maize gruel had no significant effect on nonheme Fe absorption whereas the addition of beef meat caused a threefold increase. When added to a bread meal, serum albumin caused a modest 60% increase in nonheme Fe absorption and beef meat had no effect. When added to a protein-free meal, serum albumin reduced Fe absorption by 47% compared with a 72% reduction on addition of egg white. The bioavailability of nonheme Fe from meals containing serum albumin was consistently overestimated by the in vitro technique. We conclude that the facilitation of nonheme Fe absorption by meat is not a general property of all animal protein but is better explained by the action of one or more specific animal tissues.

  9. Structural basis of transport of lysophospholipids by human serum albumin

    SciTech Connect

    Guo, Shihui; Shi, Xiaoli; Yang, Feng; Chen, Liqing; Meehan, Edward J.; Bian, Chuanbing; Huang, Mingdong

    2010-10-08

    Lysophospholipids play important roles in cellular signal transduction and are implicated in many biological processes, including tumorigenesis, angiogenesis, immunity, atherosclerosis, arteriosclerosis, cancer and neuronal survival. The intracellular transport of lysophospholipids is through FA (fatty acid)-binding protein. Lysophospholipids are also found in the extracellular space. However, the transport mechanism of lysophospholipids in the extracellular space is unknown. HSA (human serum albumin) is the most abundant carrier protein in blood plasma and plays an important role in determining the absorption, distribution, metabolism and excretion of drugs. In the present study, LPE (lysophosphatidylethanolamine) was used as the ligand to analyse the interaction of lysophospholipids with HSA by fluorescence quenching and crystallography. Fluorescence measurement showed that LPE binds to HSA with a K{sub d} (dissociation constant) of 5.6 {micro}M. The presence of FA (myristate) decreases this binding affinity (K{sub d} of 12.9 {micro}M). Moreover, we determined the crystal structure of HSA in complex with both myristate and LPE and showed that LPE binds at Sudlow site I located in subdomain IIA. LPE occupies two of the three subsites in Sudlow site I, with the LPE acyl chain occupying the hydrophobic bottom of Sudlow site I and the polar head group located at Sudlow site I entrance region pointing to the solvent. This orientation of LPE in HSA suggests that HSA is capable of accommodating other lysophospholipids and phospholipids. The study provides structural information on HSA-lysophospholipid interaction and may facilitate our understanding of the transport and distribution of lysophospholipids.

  10. The influence of fatty acids on theophylline binding to human serum albumin. Comparative fluorescence study

    NASA Astrophysics Data System (ADS)

    Maciążek-Jurczyk, M.; Sułkowska, A.; Bojko, B.; Równicka-Zubik, J.; Szkudlarek-Haśnik, A.; Zubik-Skupień, I.; Góra, A.; Dubas, M.; Korzonek-Szlacheta, I.; Wielkoszyński, T.; Żurawiński, W.; Sosada, K.

    2012-04-01

    Theophylline, popular diuretic, is used to treat asthma and bronchospasm. In blood it forms complexes with albumin, which is also the main transporter of fatty acids. The aim of the present study was to describe the influence of fatty acids (FA) on binding of theophylline (Th) to human serum albumin (HSA) in the high affinity binding sites. Binding parameters have been obtained on the basis of the fluorescence analysis. The data obtained for the complex of Th and natural human serum albumin (nHSA) obtained from blood of obese patients qualified for surgical removal of stomach was compared with our previous studies on the influence of FA on the complex of Th and commercially available defatted human serum albumin (dHSA).

  11. Influence of Millimeter Electromagnetic Waves on Fluorescence of Water-Saline Solutions of Human Serum Albumin

    NASA Astrophysics Data System (ADS)

    Vardevanyan, P. O.; Antonyan, A. P.; Shahinyan, M. A.; Mikaelyan, M. S.

    2016-07-01

    The effect of electromagnetic waves of the millimeter region on the conformation and fluorescence characteristics of human serum albumin was studied. It is shown that the irradiation of the albumin solution leads to an increase of the fluorescence intensity depending on the duration of irradiation. At an irradiation frequency of 48 GHz the fluorescence intensity of albumin hardly changes at all, while at 41.8 and 51.8 GHz it increases. It is also shown that when the irradiation frequency is 51.8 GHz, the intensity of the albumin solution fluorescence increases with increase of the irradiation time.

  12. Isolation of human anti-serum albumin Fab antibodies with an extended serum-half life.

    PubMed

    Kang, Hyeon-Ju; Kim, Hye-Jin; Cha, Sang-Hoon

    2016-01-01

    The serum albumin (SA) has been exploited to generate long-acting biotherapeutics by taking advantage of the FcRn-mediated recycling mechanism in a direct or an indirect way. Since Fab fragments have been proven to be clinically safe for human usage, we assumed that human anti-SA Fab antibodies could have a great potential as a carrier molecule to extend the serum half-life of therapeutic proteins. We, herein, had attempted to isolate anti-SA Fab antibodies from HuDVFab-8L antibody library via a phage display technology, and identified eight discrete human Fab antibodies. One of the Fab antibodies, SL335, showed the strongest binding reactivity to human SA with nM range of affinity at both pH 6 and pH 7.4, and cross-reacted to SAs from various species including rat, mouse, canine and monkey. The in vivo pharmacokinetic assay using a rat model indicated that SL335 has approximately 10 fold longer serum half-life and 26 to 44-fold increase in AUC0 → ∞ compared to the negative control Fab molecule in both intravenous and subcutaneous administrations. Knowing that Fabs have proven to be safe in clinics for a long time, SL335 seems to have a great potential in generating long-acting protein drugs by tagging effector molecules with either chemical conjugation or genetic fusion.

  13. A retrospective analysis of 25% human serum albumin supplementation in hypoalbuminemic dogs with septic peritonitis

    PubMed Central

    Horowitz, Farrah B.; Read, Robyn L.; Powell, Lisa L.

    2015-01-01

    This study describes the influence of 25% human serum albumin (HSA) supplementation on serum albumin level, total protein (TP), colloid osmotic pressure (COP), hospital stay, and survival in dogs with septic peritonitis. Records of 39 dogs with septic peritonitis were evaluated. In the HSA group, initial and post-transfusion TP, albumin, COP, and HSA dose were recorded. In the non-supplemented group, repeated values of TP, albumin, and COP were recorded over their hospitalization. Eighteen dogs survived (53.8% mortality). Repeat albumin values were higher in survivors (mean 23.9 g/L) and elevated repeat albumin values were associated with HSA supplementation. Repeat albumin and TP were higher in the HSA supplemented group (mean 24 g/L and 51.9 g/L, respectively) and their COP increased by 5.8 mmHg. Length of hospitalization was not affected. Twenty-five percent HSA increases albumin, TP, and COP in canine patients with septic peritonitis. Higher postoperative albumin levels are associated with survival. PMID:26028681

  14. Experimental and theoretical investigation on the interaction between cyclovirobuxine D and human serum albumin

    NASA Astrophysics Data System (ADS)

    Yue, Yuanyuan; Liu, Ren; Liu, Jianming; Dong, Qiao; Fan, Jing

    2014-07-01

    Cyclovirobuxine D is an active compound extracted from the plant Buxux microphylla, and widely available as medications; however, its abuse may casts potential detrimental effects on human health. By using multispectroscopic techniques and molecular modeling, the interaction of cyclovirobuxine D with human serum albumin was investigated. The fluorescence results manifested that static type was the operative mechanism for the interaction with human serum albumin. The structural investigation of the complexed HSA through CD, three-dimensional, FT-IR and synchronous fluorescence shown the polypeptide chain of HSA partially destabilizing. Docking studies revealed the molecule to be bound in the subdomain IIA. Finally, we investigated the distance between the bound ligand and Trp-214 of human serum albumin.

  15. Human serum albumin homeostasis: a new look at the roles of synthesis, catabolism, renal and gastrointestinal excretion, and the clinical value of serum albumin measurements.

    PubMed

    Levitt, David G; Levitt, Michael D

    2016-01-01

    Serum albumin concentration (CP) is a remarkably strong prognostic indicator of morbidity and mortality in both sick and seemingly healthy subjects. Surprisingly, the specifics of the pathophysiology underlying the relationship between CP and ill-health are poorly understood. This review provides a summary that is not previously available in the literature, concerning how synthesis, catabolism, and renal and gastrointestinal clearance of albumin interact to bring about albumin homeostasis, with a focus on the clinical factors that influence this homeostasis. In normal humans, the albumin turnover time of about 25 days reflects a liver albumin synthesis rate of about 10.5 g/day balanced by renal (≈6%), gastrointestinal (≈10%), and catabolic (≈84%) clearances. The acute development of hypoalbuminemia with sepsis or trauma results from increased albumin capillary permeability leading to redistribution of albumin from the vascular to interstitial space. The best understood mechanism of chronic hypoalbuminemia is the decreased albumin synthesis observed in liver disease. Decreased albumin production also accounts for hypoalbuminemia observed with a low-protein and normal caloric diet. However, a calorie- and protein-deficient diet does not reduce albumin synthesis and is not associated with hypoalbuminemia, and CP is not a useful marker of malnutrition. In most disease states other than liver disease, albumin synthesis is normal or increased, and hypoalbuminemia reflects an enhanced rate of albumin turnover resulting either from an increased rate of catabolism (a poorly understood phenomenon) or enhanced loss of albumin into the urine (nephrosis) or intestine (protein-losing enteropathy). The latter may occur with subtle intestinal pathology and hence may be more prevalent than commonly appreciated. Clinically, reduced CP appears to be a result rather than a cause of ill-health, and therapy designed to increase CP has limited benefit. The ubiquitous occurrence of

  16. Human serum albumin homeostasis: a new look at the roles of synthesis, catabolism, renal and gastrointestinal excretion, and the clinical value of serum albumin measurements

    PubMed Central

    Levitt, David G; Levitt, Michael D

    2016-01-01

    Serum albumin concentration (CP) is a remarkably strong prognostic indicator of morbidity and mortality in both sick and seemingly healthy subjects. Surprisingly, the specifics of the pathophysiology underlying the relationship between CP and ill-health are poorly understood. This review provides a summary that is not previously available in the literature, concerning how synthesis, catabolism, and renal and gastrointestinal clearance of albumin interact to bring about albumin homeostasis, with a focus on the clinical factors that influence this homeostasis. In normal humans, the albumin turnover time of about 25 days reflects a liver albumin synthesis rate of about 10.5 g/day balanced by renal (≈6%), gastrointestinal (≈10%), and catabolic (≈84%) clearances. The acute development of hypoalbuminemia with sepsis or trauma results from increased albumin capillary permeability leading to redistribution of albumin from the vascular to interstitial space. The best understood mechanism of chronic hypoalbuminemia is the decreased albumin synthesis observed in liver disease. Decreased albumin production also accounts for hypoalbuminemia observed with a low-protein and normal caloric diet. However, a calorie- and protein-deficient diet does not reduce albumin synthesis and is not associated with hypoalbuminemia, and CP is not a useful marker of malnutrition. In most disease states other than liver disease, albumin synthesis is normal or increased, and hypoalbuminemia reflects an enhanced rate of albumin turnover resulting either from an increased rate of catabolism (a poorly understood phenomenon) or enhanced loss of albumin into the urine (nephrosis) or intestine (protein-losing enteropathy). The latter may occur with subtle intestinal pathology and hence may be more prevalent than commonly appreciated. Clinically, reduced CP appears to be a result rather than a cause of ill-health, and therapy designed to increase CP has limited benefit. The ubiquitous occurrence of

  17. Induced Long-Range Attractive Potentials of Human Serum Albumin by Ligand Binding

    SciTech Connect

    Sato, Takaaki; Komatsu, Teruyuki; Nakagawa, Akito; Tsuchida, Eishun

    2007-05-18

    Small-angle x-ray scattering and dielectric spectroscopy investigation on the solutions of recombinant human serum albumin and its heme hybrid revealed that heme incorporation induces a specific long-range attractive potential between protein molecules. This is evidenced by the enhanced forward intensity upon heme binding, despite no hindrance to rotatory Brownian motion, unbiased colloid osmotic pressure, and discontiguous nearest-neighbor distance, confirming monodispersity of the proteins. The heme-induced potential may play a trigger role in recognition of the ligand-filled human serum albumins in the circulatory system.

  18. Alteration of human serum albumin tertiary structure induced by glycation. Spectroscopic study

    NASA Astrophysics Data System (ADS)

    Szkudlarek, A.; Maciążek-Jurczyk, M.; Chudzik, M.; Równicka-Zubik, J.; Sułkowska, A.

    2016-01-01

    The modification of human serum albumin (HSA) structure by non-enzymatic glycation is one of the underlying factors that contribute to the development of complications of diabetes and neurodegenerative diseases. The aim of the present work was to estimate how glycation of HSA altered its tertiary structure. Changes of albumin conformation were investigated by comparison of glycated (gHSA) and non-glycated human serum albumin (HSA) absorption spectra, red edge excitation shift (REES) and synchronous spectra. Effect of glycation on human serum albumin tertiary structure was also investigated by 1H NMR spectroscopy. Formation of gHSA Advanced Glycation End-products (AGEs) caused absorption of UV-VIS light between 310 nm and 400 nm while for non-glycated HSA in this region no absorbance has been registered. Analysis of red edge excitation shift effect allowed for observation of structural changes of gHSA in the hydrophobic pocket containing the tryptophanyl residue. Moreover changes in the microenvironment of tryptophanyl and tyrosyl residues brought about AGEs on the basis of synchronous fluorescence spectroscopy have been confirmed. The influence of glycation process on serum albumin binding to 5-dimethylaminonaphthalene-1-sulfonamide (DNSA), 2-(p-toluidino) naphthalene-6-sulfonic acid (TNS), has been studied. Fluorescence analysis showed that environment of both binding site I and II is modified by galactose glycation.

  19. Effects of non-enzymatic glycation in human serum albumin. Spectroscopic analysis

    NASA Astrophysics Data System (ADS)

    Szkudlarek, A.; Sułkowska, A.; Maciążek-Jurczyk, M.; Chudzik, M.; Równicka-Zubik, J.

    2016-01-01

    Human serum albumin (HSA), transporting protein, is exposed during its life to numerous factors that cause its functions become impaired. One of the basic factors - glycation of HSA - occurs in diabetes and may affect HSA-drug binding. Accumulation of advanced glycation end-products (AGEs) leads to diseases e.g. diabetic and non-diabetic cardiovascular diseases, Alzheimer disease, renal disfunction and in normal aging. The aim of the present work was to estimate how non-enzymatic glycation of human serum albumin altered its tertiary structure using fluorescence technique. We compared glycated human serum albumin by glucose (gHSAGLC) with HSA glycated by fructose (gHSAFRC). We focused on presenting the differences between gHSAFRC and nonglycated (HSA) albumin used acrylamide (Ac), potassium iodide (KI) and 2-(p-toluidino)naphthalene-6-sulfonic acid (TNS). Changes of the microenvironment around the tryptophan residue (Trp-214) of non-glycated and glycated proteins was investigated by the red-edge excitation shift method. Effect of glycation on ligand binding was examined by the binding of phenylbutazone (PHB) and ketoprofen (KP), which a primary high affinity binding site in serum albumin is subdomain IIA and IIIA, respectively. At an excitation and an emission wavelength of λex 335 nm and λem 420 nm, respectively the increase of fluorescence intensity and the blue-shift of maximum fluorescence was observed. It indicates that the glycation products decreases the polarity microenvironment around the fluorophores. Analysis of red-edge excitation shift method showed that the red-shift for gHSAFRC is higher than for HSA. Non-enzymatic glycation also caused, that the Trp residue of gHSAFRC becomes less accessible for the negatively charged quencher (I-), KSV value is smaller for gHSAFRC than for HSA. TNS fluorescent measurement demonstrated the decrease of hydrophobicity in the glycated albumin. KSV constants for gHSA-PHB systems are higher than for the unmodified serum

  20. Effects of non-enzymatic glycation in human serum albumin. Spectroscopic analysis.

    PubMed

    Szkudlarek, A; Sułkowska, A; Maciążek-Jurczyk, M; Chudzik, M; Równicka-Zubik, J

    2016-01-01

    Human serum albumin (HSA), transporting protein, is exposed during its life to numerous factors that cause its functions become impaired. One of the basic factors --glycation of HSA--occurs in diabetes and may affect HSA-drug binding. Accumulation of advanced glycation end-products (AGEs) leads to diseases e.g. diabetic and non-diabetic cardiovascular diseases, Alzheimer disease, renal disfunction and in normal aging. The aim of the present work was to estimate how non-enzymatic glycation of human serum albumin altered its tertiary structure using fluorescence technique. We compared glycated human serum albumin by glucose (gHSA(GLC)) with HSA glycated by fructose (gHSA(FRC)). We focused on presenting the differences between gHSA(FRC) and nonglycated (HSA) albumin used acrylamide (Ac), potassium iodide (KI) and 2-(p-toluidino)naphthalene-6-sulfonic acid (TNS). Changes of the microenvironment around the tryptophan residue (Trp-214) of non-glycated and glycated proteins was investigated by the red-edge excitation shift method. Effect of glycation on ligand binding was examined by the binding of phenylbutazone (PHB) and ketoprofen (KP), which a primary high affinity binding site in serum albumin is subdomain IIA and IIIA, respectively. At an excitation and an emission wavelength of λex 335nm and λem 420nm, respectively the increase of fluorescence intensity and the blue-shift of maximum fluorescence was observed. It indicates that the glycation products decreases the polarity microenvironment around the fluorophores. Analysis of red-edge excitation shift method showed that the red-shift for gHSA(FRC) is higher than for HSA. Non-enzymatic glycation also caused, that the Trp residue of gHSA(FRC) becomes less accessible for the negatively charged quencher (I(-)), KSV value is smaller for gHSA(FRC) than for HSA. TNS fluorescent measurement demonstrated the decrease of hydrophobicity in the glycated albumin. KSV constants for gHSA-PHB systems are higher than for the

  1. Structure of Serum Albumin

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C.; Ho, Joseph X.

    1994-01-01

    Because of its availability, low cost, stability, and unusual ligand-binding properties, serum albumin has been one of the mst extensively studied and applied proteins in biochemistry. However, as a protein, albumin is far from typical, and the widespread interest in and application of albumin have not been balanced by an understanding of its molecular structure. Indeed, for more than 30 years structural information was surmised based solely on techniques such as hydrodynamics, low-angle X-ray scattering, and predictive methods.

  2. Determination of human albumin in serum and urine samples by constant-energy synchronous fluorescence method.

    PubMed

    Madrakian, Tayyebeh; Bagheri, Habibollah; Afkhami, Abbas

    2015-08-01

    A sensitive spectrofluorimetric method using constant-energy synchronous fluorescence technique is proposed for the determination of human albumin without separation. In this method, no reagent was used for enhancement of the fluorescence signal of albumin in the solution. Effects of some parameters, such as energy difference between excitation and emission monochromators (ΔE), emission and excitation slit widths and scan rate of wavelength were studied and the optimum conditions were established. For this purpose factorial design and response surface method were employed for optimization of the effective parameters on the fluorescence signal. The results showed that the scan rate of the wavelength has no significant effect on the analytical signal. The calibration curve was linear in the range 0.1-220.0 µg mL(-1) of albumin with a detection limit of 7.0 × 10(-3)  µg mL(-1). The relative standard deviations (RSD) for six replicate measurements of albumin were calculated as 2.2%, 1.7% and 1.3% for 0.5, 10.0 and 100.0 µg mL(-1) albumin, respectively. Furthermore the proposed method has been employed for the determination of albumin in human serum and urine samples.

  3. Nanoencapsulation of ultra-small superparamagnetic particles of iron oxide into human serum albumin nanoparticles

    PubMed Central

    Altinok, Mahmut; Urfels, Stephan; Bauer, Johann

    2014-01-01

    Summary Human serum albumin nanoparticles have been utilized as drug delivery systems for a variety of medical applications. Since ultra-small superparamagnetic particles of iron oxide (USPIO) are used as contrast agents in magnetic resonance imaging, their encapsulation into the protein matrix enables the synthesis of diagnostic and theranostic agents by surface modification and co-encapsulation of active pharmaceutical ingredients. The present investigation deals with the surface modification and nanoencapsulation of USPIO into an albumin matrix by using ethanolic desolvation. Particles of narrow size distribution and with a defined particle structure have been achieved. PMID:25551054

  4. Interactions Between Sirolimus and Anti-Inflammatory Drugs: Competitive Binding for Human Serum Albumin

    PubMed Central

    Khodaei, Arash; Bolandnazar, Soheila; Valizadeh, Hadi; Hasani, Leila; Zakeri-Milani, Parvin

    2016-01-01

    Purpose: The aim of the present study was investigating the effects of three anti-inflammatory drugs, on Sirolimus protein biding. The binding site of Sirolimus on human serum albumin (HSA) was also determined. Methods: Six different concentrations of Sirolimus were separately exposed to HSA at pH 7.4 and 37°C. Ultrafiltration method was used for separating free drug; then free drug concentrations were measured by HPLC. Finally, Sirolimus protein binding parameters was calculated using Scatchard plots. The same processes were conducted in the presence of NSAIDs at lower concentration of albumin and different pH conditions. To characterize the binding site of Sirolimus on albumin, the free concentration of warfarin sodium and Diazepam, site I and II specific probes, bound to albumin were measured upon the addition of increasing Sirolimus concentrations. Results: Based on the obtained results presence of Diclofenac, Piroxicam and Naproxen, could significantly decrease the percentage of Sirolimus protein binding. The Binding reduction was the most in the presence of Piroxicam. Sirolimus-NSAIDs interactions were increased in higher pH values and also in lower albumin concentrations. Probe displacement study showed that Sirolimus may mainly bind to site I on albumin molecule. Conclusion: More considerations in co-administration of NSAIDs and Sirolimus is recommended. PMID:27478785

  5. Effect of the conditions of isolation on the physicochemical properties of human serum albumin in the norm and with pathology

    NASA Astrophysics Data System (ADS)

    Ivanov, A. I.; Zhbankov, R. G.; Korolenko, E. A.; Korolik, E. V.; Meleshchenko, L. A.; Sarnatskaya, V. V.; Nikolaev, V. G.; Nikolaichik, V. V.; Yushko, L. A.

    1997-01-01

    Differential scanning calorimetry and IR spectrosocopy were used to investigate the effect of the procedure of isolation of human serum albumin on its physicochemical characteristics. It is shown that fractionation of blood plasma with ethylene glycol followed by ion exchange chromatography can be used to obtain albumin of normal donors that is similar to the albumin in the nonfractionated plasma according to melting thermograms. Endotherms of human serum albumin samples that were obtained by affinity chromatography and preparative electrophoresis are bimodal, unlike the monophasic for albumin obtained by polyethylene glycol precipitation. These changes result from a higher content of nonetherified fatty acids in the albumin samples obtained by affinity chromatography and from modification of the secondary protein structure in the samples obtained by electrophoresis. Analysis of melting thermograms of serum albumin from patients with uremia, chronic hepatitis, and peritonitis shows that fractionation of blood with polyethylene glycol preserves the thermodynamic characteristics of the various pathological serum albumins to the greatest extent. The present results demonstrate the advantage of polyethylene glycol fractionation for isolation of native preparations of normal and “pathological” human serum albumin.

  6. Spectral Fluorescence Properties of an Anionic Oxacarbocyanine Dye in Complexes with Human Serum Albumin

    NASA Astrophysics Data System (ADS)

    Pronkin, P. G.; Tatikolov, A. S.

    2015-07-01

    The spectral fluorescence properties of the anionic oxacarbocyanine dye 3,3'-di-(γ-sulfopropyl)-5,5'-diphenyl-9-ethyloxacarbocyanine betaine (OCC) were studied in solutions and in complexes with human serum albumin (HSA). Interaction with HSA leads to a significant increase in the fluorescence of the dye. We studied quenching of the fluorescence of OCC in a complex with HSA by ibuprofen and warfarin. Data on quenching of fluorescence by ibuprofen indicate binding of the dye to binding site II of subdomain IIIA in the HSA molecule. Synchronous fluorescence spectra of human serum albumin in the presence of OCC showed that complexation with OCC does not lead to appreciable rearrangement of the protein molecule at the binding site.

  7. Forster resonance energy transfer in the system of human serum albumin-xanthene dyes

    NASA Astrophysics Data System (ADS)

    Kochubey, V. I.; Pravdin, A. B.; Melnikov, A. G.; Konstantinova, I.; Alonova, I. V.

    2016-04-01

    The processes of interaction of fluorescent probes: eosin and erythrosine with human serum albumin (HSA) were studied by the methods of absorption and fluorescence spectroscopy. Extinction coefficients of probes were determined. Critical transfer radius and the energy transfer efficiency were defined by fluorescence quenching of HSA. Analysis of the excitation spectra of HSA revealed that the energy transfer process is carried out mainly between tryptophanyl and probes.

  8. Zinc phthalocyanine-conjugated with bovine serum albumin mediated photodynamic therapy of human larynx carcinoma

    NASA Astrophysics Data System (ADS)

    Silva, E. P. O.; Santos, E. D.; Gonçalves, C. S.; Cardoso, M. A. G.; Soares, C. P.; Beltrame, M., Jr.

    2016-10-01

    Phthalocyanines, which are classified as second-generation photosensitizers, have advantageous photophysical properties, and extensive studies have demonstrated their potential applications in photodynamic therapy. The present work describes the preparation of a new zinc phthalocyanine conjugated to bovine serum albumin (compound 4a) and its photodynamic efficiency in human larynx-carcinoma cells (HEp-2 cells). The unconjugated precursor (compound 4) was also studied. Compounds 4 and 4a penetrated efficiently into the cell, exhibiting cytoplasmic localization, and showed no cytotoxicity in the dark. However, high photodynamic activities were observed in HEp-2 cells after treatments with 5 µM photosensitizers and 4.5 J cm-2 light. These conditions were sufficient to decrease the cell viability to 57.93% and 32.75% for compounds 4 and 4a, respectively. The present results demonstrated high photodynamic efficiency of zinc phthalocyanine conjugated with bovine serum albumin in destroying the larynx-carcinoma cells.

  9. Investigation into the interaction of losartan with human serum albumin and glycated human serum albumin by spectroscopic and molecular dynamics simulation techniques: A comparison study.

    PubMed

    Moeinpour, Farid; Mohseni-Shahri, Fatemeh S; Malaekeh-Nikouei, Bizhan; Nassirli, Hooriyeh

    2016-09-25

    The interaction between losartan and human serum albumin (HSA), as well as its glycated form (gHSA) was studied by multiple spectroscopic techniques and molecular dynamics simulation under physiological conditions. The binding information, including the binding constants, effective quenching constant and number of binding sites showed that the binding partiality of losartan to HSA was higher than to gHSA. The findings of three-dimensional fluorescence spectra demonstrated that the binding of losartan to HSA and gHSA would alter the protein conformation. The distances between Trp residue and the binding sites of the drug were evaluated on the basis of the Förster theory, and it was indicated that non-radiative energy transfer from HSA and gHSA to the losartan happened with a high possibility. According to molecular dynamics simulation, the protein secondary and tertiary structure changes were compared in HSA and gHSA for clarifying the obtained results.

  10. Binding of human serum albumin to N-(p-ethoxy-phenyl)-N'-(1-naphthyl)thiourea and synchronous fluorescence determination of human serum albumin.

    PubMed

    Cui, Fengling; Wang, Junli; Cui, Yanrui; Li, Jianping; Lu, Yan; Fan, Jing; Yao, Xiaojun

    2007-06-01

    The binding of N-(p-ethoxy-phenyl)-N'-(1-naphthyl)thiourea (EPNT) to human serum albumin (HSA) was investigated under simulative physiological conditions by fluorescence spectra in combination with UV absorption spectroscopy and a molecular modeling method. A strong fluorescence quenching reaction of EPNT to HSA was observed, and the quenching mechanism was suggested to be static quenching according to the Stern-Volmer equation. The binding constants (K) at different temperatures as well as thermodynamic parameters, enthalpy change (DeltaH) and entropy change (DeltaS), were calculated according to relevant fluorescent data and the vant' Hoff equation. This indicated that a hydrophobic interaction was a predominant intermolecular force for stabilizing the complex, which is in agreement with the results of molecule modeling study. The effects of energy transfer and other ions on the binding constant were considered. In addition, synchronous fluorescence technology was successfully applied to the determination of HSA added into the EPNT solution. PMID:17575357

  11. Human Serum Versus Human Serum Albumin Supplementation in Human Islet Pretransplantation Culture: In Vitro and In Vivo Assessment.

    PubMed

    Nacher, Montserrat; Estil Les, Elisabet; Garcia, Ainhoa; Nadal, Belen; Pairó, Mar; Garcia, Cristofer; Secanella, Lluís; Novials, Anna; Montanya, Eduard

    2016-01-01

    There is conflicting evidence favoring both the use of human serum (HS) and of human serum albumin (HSA) in human islet culture. We evaluated the effects of HS versus HSA supplementation on 1) in vitro β-cell viability and function and 2) in vivo islet graft revascularization, islet viability, β-cell death, and metabolic outcome after transplantation. Islets isolated from 14 cadaveric organ donors were cultured for 3 days in CMRL 1066 medium supplemented with HS or HSA. After 3 days in culture, β-cell apoptosis was lower in HS group (1.41 ± 0.27 vs. 2.38 ± 0.39%, p = 0.029), and the recovery of islets was 77 ± 11% and 54 ± 1% in HS- and HSA-cultured groups, respectively. Glucose-stimulated insulin secretion (GSIS) was higher in HS group (29.4, range 10.4-99.9, vs. 22.3, range 8.7-70.6, p = 0.031). In vivo viability and revascularization was determined in HS- and HSA-cultured islets transplanted into the anterior chamber of the eye of Balb/c mice (n = 14), and β-cell apoptosis in paraffin-embedded mouse eyes. Islet viability and β-cell apoptosis were similar in both groups. Revascularization was observed in one graft (HS group) on day 10 after transplantation. Islet function was determined in streptozotocin (STZ)-diabetic nude mice (n = 33) transplanted with 2,000 IEQs cultured with HS or HSA that showed similar blood glucose levels and percentage of normoglycemic animals over time. In conclusion, human islets cultured in medium supplemented with HS showed higher survival in vitro, as well as islet viability and function. The higher in vitro survival increased the number of islets available for transplantation. However, the beneficial effect on viability and function did not translate into an improved metabolic evolution when a similar number of HSA- and HS-cultured islets was transplanted. PMID:25955150

  12. Immunotoxicity Assessment of Rice-Derived Recombinant Human Serum Albumin Using Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Fu, Kai; Cheng, Qin; Liu, Zhenwei; Chen, Zhen; Wang, Yan; Ruan, Honggang; Zhou, Lu; Xiong, Jie; Xiao, Ruijing; Liu, Shengwu; Zhang, Qiuping; Yang, Daichang

    2014-01-01

    Human serum albumin (HSA) is extensively used in clinics to treat a variety of diseases, such as hypoproteinemia, hemorrhagic shock, serious burn injuries, cirrhotic ascites and fetal erythroblastosis. To address supply shortages and high safety risks from limited human donors, we recently developed recombinant technology to produce HSA from rice endosperm. To assess the risk potential of HSA derived from Oryza sativa (OsrHSA) before a First-in-human (FIH) trial, we compared OsrHSA and plasma-derived HSA (pHSA), evaluating the potential for an immune reaction and toxicity using human peripheral blood mononuclear cells (PBMCs). The results indicated that neither OsrHSA nor pHSA stimulated T cell proliferation at 1x and 5x dosages. We also found no significant differences in the profiles of the CD4+ and CD8+ T cell subsets between OsrHSA- and pHSA-treated cells. Furthermore, the results showed that there were no significant differences between OsrHSA and pHSA in the production of cytokines such as interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-10 and IL-4. Our results demonstrated that OsrHSA has equivalent immunotoxicity to pHSA when using the PBMC model. Moreover, this ex vivo system could provide an alternative approach to predict potential risks in novel biopharmaceutical development. PMID:25099245

  13. PEGylated Human Serum Albumin: Review of PEGylation, Purification and Characterization Methods

    PubMed Central

    Akbarzadehlaleh, Parvin; Mirzaei, Mona; Mashahdi-Keshtiban, Mahdiyeh; Shamsasenjan, Karim; Heydari, Hamidreza

    2016-01-01

    Human serum albumin (HSA) is a non-glycosylated, negatively charged protein (Mw: about 65-kDa) that has one free cystein residue (Cys 34), and 17 disulfide bridges that these bridges have main role in its stability and longer biological life-time (15 to 19 days). As HSA is a multifunctional protein, it can also bind to other molecules and ions in addition to its role in maintaining colloidal osmotic pressure (COP) in various diseases. In critical illnesses changes in the level of albumin between the intravascular and extravascular compartments and the decrease in its serum concentration need to be compensated using exogenous albumin; but as the size of HSA is an important parameter in retention within the circulation, therefore increasing its molecular size and hydrodynamic radius of HSA by covalent attachment of poly ethylene glycol (PEG), that is known as PEGylation, provides HSA as a superior volume expander that not only can prevent the interstitial edema but also can reduce the infusion frequency. This review focuses on various PEGylation methods of HSA (solid phase and liquid phase), and compares various methods to purifiy and characterize the pegylated form. PMID:27766215

  14. Imatinib binding to human serum albumin modulates heme association and reactivity.

    PubMed

    Di Muzio, Elena; Polticelli, Fabio; Trezza, Viviana; Fanali, Gabriella; Fasano, Mauro; Ascenzi, Paolo

    2014-10-15

    Imatinib, an inhibitor of the Bcr-Abl tyrosine kinase, is approximately 95% bound to plasma proteins, α1-acid glycoprotein (AGP) being the primary carrier. However, human serum albumin (HSA) may represent the secondary carrier of imatinib in pathological states characterized by low AGP levels, such as pancreatic cancer, hepatic cirrhosis, hepatitis, hyperthyroidism, nephrotic syndrome, malnutrition, and cachexia. Here, thermodynamics of imatinib binding to full-length HSA and its recombinant Asp1-Glu382 truncated form (containing only the FA1, FA2, FA6, and FA7 binding sites; trHSA), in the absence and presence of ferric heme (heme-Fe(III)), and the thermodynamics of heme-Fe(III) binding to HSA and trHSA, in the absence and presence of imatinib, has been investigated. Moreover, the effect of imatinib on kinetics of peroxynitrite detoxification by ferric human serum heme-albumin (HSA-heme-Fe(III)) and ferric truncated human serum heme-albumin (trHSA-heme-Fe(III)) has been explored. All data were obtained at pH 7.0, and 20.0 °C and 37.0 °C. Imatinib binding to the FA7 site of HSA and trHSA inhibits allosterically heme-Fe(III) association to the FA1 site and vice versa, according to linked functions. Moreover, imatinib binding to the secondary FA2 site of HSA-heme-Fe(III) inhibits allosterically peroxynitrite detoxification. Docking simulations and local structural comparison with other imatinib-binding proteins support functional data indicating the preferential binding of imatinib to the FA1 and FA7 sites of HSA, and to the FA2 and FA7 sites of HSA-heme-Fe(III). Present results highlight the allosteric coupling of the FA1, FA2, and FA7 sites of HSA, and may be relevant in modulating ligand binding and reactivity properties of HSA in vivo. PMID:25057771

  15. On the interaction of luminol with human serum albumin: Nature and thermodynamics of ligand binding

    NASA Astrophysics Data System (ADS)

    Moyon, N. Shaemningwar; Mitra, Sivaprasad

    2010-09-01

    The mechanism and thermodynamic parameters for the binding of luminol (LH 2) with human serum albumin was explored by steady state and picosecond time-resolved fluorescence spectroscopy. It was shown that out of two possible LH 2 conformers present is solution, only one is accessible for binding with HSA. The thermodynamic parameters like enthalpy (Δ H) and entropy (Δ S) change corresponding to the ligand binding process were also estimated by performing the experiment at different temperatures. The ligand replacement experiment with bilirubin confirms that LH 2 binds into the sub-domain IIA of the protein.

  16. Orientation of the water molecules of hydration of human serum albumin.

    PubMed

    van Oss, C J; Good, R J

    1988-04-01

    Through contact-angle measurements with a number of liquids, on layers of hydrated human serum albumin (HSA), built on anisotropic ultrafilter membranes, the apolar, Lifshitz-van der Waals surface tension component, as well as the polar, electron-acceptor and electron-donor parameters of the hydrated layers could be determined. From these data, it was found that the degree of orientation of the water molecules of hydration of HSA is approximately 98% in the first layer of hydration and approximately 30% of the second layer. The water molecules of hydration are oriented with the H atoms closest to, and the O atoms farthest from, the protein surface.

  17. Time-insensitive fluorescent sensor for human serum albumin and its unusual red shift.

    PubMed

    Smith, Sara E; Williams, Jessica M; Ando, Shin; Koide, Kazunori

    2014-03-01

    The concentration of human serum albumin (HSA) indicates the health state of individuals and is routinely measured by UV spectroscopy with bromocresol. However, this method tends to overestimate HSA, and more critically, depends highly on the timing, in seconds, of the measurements. Here, we report an analog of 2',7'-dichlorofluorescein that can be used as a fluorescent sensor to quantify HSA in human sera. The accuracy of this new method proved superior to that of bromocresol when an international standard serum sample was analyzed. This method is more convenient than the bromocresol method because it allows for fluorescence measurements during a >15 min period. Colorimetric analysis was also performed to further investigate the effects of the binding of the sensor to HSA. These spectroscopic studies suggest that absorption and emission changes upon HSA binding may be due to the dehydration of the dye and/or stabilization of the tritylic cation species.

  18. Effects of Fenton Reaction on Human Serum Albumin: An In Vitro Study

    PubMed Central

    Khosravifarsani, Meysam; Monfared, Ali Shabestani; Pouramir, Mahdi; Zabihi, Ebrahim

    2016-01-01

    Introduction Human serum albumin (HSA) is a critical protein in human blood plasma, which can be highly damaged by oxidative stress. The aim of this study was to analyze modifications of this protein after oxidation using a Fenton system. Methods In this 2015 experiment, different ratios of Fenton reagent (Fe2+/H2O2) was incubated with one concentration of human serum albumin (1mg/ml). Hence, HSA was incubated 30 min with various combinations of a Fenton system and quantified oxidation products such as carbonyl groups, fragmentations, degradations, and oxidized free thiol group using reliable techniques. Image and data analysis were carried out using ImageJ software and Excel (version 2007), respectively. Results An SDS-PAGE profile showed no cross link and aggregation. However, protein band intensity has decreased to 50% in the highest ratio of H2O2/Fe. Carbonylation assay indicated carbonyl/protein (molc/molp) ratio increased linearly in lower ratios and the values plateau at higher levels of H2O2/Fe 2+. The only free sulfhydryl group on HSA was oxidized in all ratios of the Fenton system. Conclusion To sum, the structure of HSA has been changed following treatment with Hydroxyl Radical as the main product of Fenton reaction. These data confirm the antioxidant activity of HSA. PMID:27790352

  19. Preliminary crystallographic studies of four crystal forms of serum albumin

    NASA Technical Reports Server (NTRS)

    Carter, D. C.; Chang, B.; Ho, J. X.; Keeling, K.; Krishnasami, Z.

    1994-01-01

    Several crystal forms of serum albumin suitable for three-dimensional structure determination have been grown. These forms include crystals of recombinant and wild-type human serum albumin, baboon serum albumin, and canine serum albumin. The intrinsic limits of X-ray diffraction for these crystals are in the range 0.28-0.22 nm. Two of the crystal forms produced from human and canine albumin include incorporated long-chain fatty acids. Molecular replacement experiments have been successfully conducted on each crystal form using the previously determined atomic coordinates of human serum albumin illustrating the conserved tertiary structure.

  20. Human Serum Albumin (HSA) Suppresses the Effects of Glycerol Monolaurate (GML) on Human T Cell Activation and Function

    PubMed Central

    Zhang, Michael S.; Houtman, Jon C. D.

    2016-01-01

    Glycerol monolaurate (GML) is a monoglyceride with well characterized anti-microbial properties. Because of these properties, GML is widely used in food, cosmetics, and personal care products and currently being tested as a therapeutic for menstrual associated toxic shock syndrome, superficial wound infections, and HIV transmission. Recently, we have described that GML potently suppresses select T cell receptor (TCR)-induced signaling events, leading to reduced human T cell effector functions. However, how soluble host factors present in the blood and at sites of infection affect GML-mediated human T cell suppression is unknown. In this study, we have characterized how human serum albumin (HSA) affects GML-induced inhibition of human T cells. We found that HSA and other serum albumins bind to 12 carbon acyl side chain of GML at low micromolar affinities and restores the TCR-induced formation of LAT, PLC-γ1, and AKT microclusters at the plasma membrane. Additionally, HSA reverses GML mediated inhibition of AKT phosphorylation and partially restores cytokine production in GML treated cells. Our data reveal that HSA, one of the most abundant proteins in the human serum and at sites of infections, potently reverses the suppression of human T cells by GML. This suggests that GML-driven human T cell suppression depends upon the local tissue environment, with albumin concentration being a major determinant of GML function. PMID:27764189

  1. Effects of fluorinated and hydrogenated surfactants on human serum albumin at different pHs.

    PubMed

    Sabín, Juan; Prieto, Gerardo; González-Pérez, Alfredo; Ruso, Juan M; Sarmiento, Félix

    2006-01-01

    Complexation between human serum albumin (HSA) and two different surfactants, one fully fluorinated (sodium perfluorooctanoate, SPFO) and one fully hydrogenated (sodium caprylate, SO), was studied using zeta-potential measurements and difference spectroscopy. The study was carried out at three different pHs, 3.2, 6.7, and 10.0. The spectroscopy study was performed at pHs 6.7 and 10.0, given that at pH 3.2 high turbidity was observed in the wide range of surfactant concentrations. The results were interpreted in terms of the electrostatic and hydrophobic contributions to the stability of the different phases formed in the water-surfactant-HSA system. Solutions and precipitates were observed in the concentration range investigated in more detail. Using Pace methods, the thermodynamic values of the surfactant-induced conformational changes in HSA were determined for sodium perfluorooctanoate in the concentration range 2-12 mmol dm(-3) at pH 6.7 and 5-22 mmol dm(-3) at pH 10.0. Electrophoretic measurements were used to characterize surfactant adsorption by determining the number of molecules adsorbed on the surface of HSA and the Gibbs energy of adsorption. Finally, the interactions between human serum albumin and other anionic surfactants studied by other authors were compared with those observed in the present work.

  2. A study on human serum albumin influence on glycation of fibrinogen

    SciTech Connect

    Kielmas, Martyna; Szewczuk, Zbigniew; Stefanowicz, Piotr

    2013-09-13

    Highlights: •The glycation of fibrinogen was investigated by isotopic labeling method. •The potential glycation sites in fibrinogen were identified. •Human serum albumin (HSA) inhibits the glycation of fibrinogen. •The effect of HSA on fibrinogen glycation is sequence-dependent. -- Abstract: Although in vivo glycation proceeds in complex mixture of proteins, previous studies did not take in consideration the influence of protein–protein interaction on Maillard reaction. The aim of our study was to test the influence of human serum albumin (HSA) on glycation of fibrinogen. The isotopic labeling using [{sup 13}C{sub 6}] glucose combined with LC-MS were applied as tool for identification possible glycation sites in fibrinogen and for evaluation the effect of HSA on the glycation level of selected amino acids in fibrinogen. The obtained data indicate that the addition of HSA protects the fibrinogen from glycation. The level of glycation in presence of HSA is reduced by 30–60% and depends on the location of glycated residue in sequence of protein.

  3. Interaction of cyproheptadine hydrochloride with human serum albumin using spectroscopy and molecular modeling methods.

    PubMed

    Jiang, Hua; Chen, Rongrong; Wang, Hongcui; Pu, Hanlin

    2013-01-01

    The interaction between cyproheptadine hydrochloride (CYP) and human serum albumin (HSA) was investigated by fluorescence spectroscopy, UV-vis absorption spectroscopy, Fourier transform infrared spectroscopy (FT-IR) and molecular modeling at a physiological pH (7.40). Fluorescence of HSA was quenched remarkably by CYP and the quenching mechanism was considered as static quenching since it formed a complex. The association constants Ka and number of binding sites n were calculated at different temperatures. According to Förster's theory of non-radiation energy transfer, the distance r between donor (human serum albumin) and acceptor (cyproheptadine hydrochloride) was obtained. The effect of common ions on the binding constant was also investigated. The effect of CYP on the conformation of HSA was analyzed using FT-IR, synchronous fluorescence spectroscopy and 3D fluorescence spectra. The thermodynamic parameters ΔH and ΔS were calculated to be -14.37 kJ mol(-1) and 38.03 J mol(-1) K(-1), respectively, which suggested that hydrophobic forces played a major role in stabilizing the HSA-CYP complex. In addition, examination of molecular modeling indicated that CYP could bind to site I of HSA and that hydrophobic interaction was the major acting force, which was in agreement with binding mode studies.

  4. Surface modification of PLGA nanoparticles via human serum albumin conjugation for controlled delivery of docetaxel

    PubMed Central

    2013-01-01

    Background Poly lactic-co-glycolic acid (PLGA) based nanoparticles are considered to be a promising drug carrier in tumor targeting but suffer from the high level of opsonization by reticuloendothelial system due to their hydrophobic structure. As a result surface modification of these nanoparticles has been widely studied as an essential step in their development. Among various surface modifications, human serum albumin (HSA) possesses advantages including small size, hydrophilic surface and accumulation in leaky vasculature of tumors through passive targeting and a probable active transport into tumor tissues. Methods PLGA nanoparticles of docetaxel were prepared by emulsification evaporation method and were surface conjugated with human serum albumin. Fourier transform infrared spectrum was used to confirm the conjugation reaction where nuclear magnetic resonance was utilized for conjugation ratio determination. In addition, transmission electron microscopy showed two different contrast media in conjugated nanoparticles. Furthermore, cytotoxicity of free docetaxel, unconjugated and conjugated PLGA nanoparticles was studied in HepG2 cells. Results Size, zeta potential and drug loading of PLGA nanoparticles were about 199 nm, −11.07 mV, and 4%, respectively where size, zeta potential and drug loading of conjugated nanoparticles were found to be 204 nm, −5.6 mV and 3.6% respectively. Conjugated nanoparticles represented a three-phasic release pattern with a 20% burst effect for docetaxel on the first day. Cytotoxicity experiment showed that the IC50 of HSA conjugated PLGA nanoparticles (5.4 μg) was significantly lower than both free docetaxel (20.2 μg) and unconjugated PLGA nanoparticles (6.2 μg). Conclusion In conclusion surface modification of PLGA nanoparticles through HSA conjugation results in more cytotoxicity against tumor cell lines compared with free docetaxel and unconjugated PLGA nanoparticles. Albumin conjugated PLGA nanoparticles may

  5. Interaction of weakly bound antibiotics neomycin and lincomycin with bovine and human serum albumin: biophysical approach.

    PubMed

    Keswani, Neelam; Choudhary, Sinjan; Kishore, Nand

    2010-07-01

    The thermodynamics of interaction of neomycin and lincomycin with bovine serum albumin (BSA) and human serum albumin (HSA) has been studied using isothermal titration calorimetry (ITC), in combination with UV-visible, steady state and time resolved fluorescence spectroscopic measurements. Neomycin is observed to bind weakly to BSA and HSA whereas lincomycin did not show any evidence for binding with the native state of these proteins, rather it interacts in the presence of surfactants. The ITC results suggest 1 : 1 binding stoichiometry for neomycin in the studied temperature range. The values of the van't Hoff enthalpy do not agree with the calorimetric enthalpy in the case of neomycin, suggesting conformational changes in the protein upon ligand binding, as well as with the rise in the temperature. Experiments at different ionic strengths, and in the presence of tetrabutyl ammonium bromide and surfactants suggest the predominant involvement of electrostatic interactions in the complexation process of neomycin with BSA and HSA, and non-specific interaction behaviour of lincomycin with these proteins.

  6. Research of the interaction between kangai injection and human serum albumin by fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Ye, Changbin; Lin, Xiaogang; Zhu, Hao; Li, Wenchao; Wu, Jie

    2015-10-01

    The interaction between drugs and serum albumin is the theoretical basis of pharmacology research. Kangai injection with invigorating Qi, enhancing the immune function, is widely used for a variety of malignant tumor treatment. Fluorescence spectroscopy was adopted due to its high sensitivity and other advantages. The interaction between kangai injection and human serum albumin (HSA) in physiological buffer (pH 7.4) was investigated by fluorescence spectroscopy and UV-Vis absorption spectroscopy. The results of fluorescence spectrum at three temperature (296K, 303K and 310K) showed the degree of binding at 310K is the highest. Also, the maximum emission peak has a slight blue shift, which indicates that the interaction between kangai injection and HSA has an effect on the conformation of HSA. That is, the microenvironment of tryptophan increase hydrophobic due to the increase of the concentration of kangai injection. Results obtained from analysis of fluorescence spectrum and fluorescence intensity indicated that kangai injection has a strong ability to quench the intrinsic fluorescence of HSA. And according to the Stern-Volume equation, the quenching mechanism is static quenching, which is further proved by the UV-Vis absorption spectroscopy.

  7. A Fluorescence Quenching Study of the Interaction of Nebivolol Hydrochloride with Bovine and Human Serum Albumin

    NASA Astrophysics Data System (ADS)

    Abdel-Aziz, L.; Abdel-Fattah, L.; El-Kosasy, A.; Gaied, M.

    2015-09-01

    The interaction of nebivolol hydrochloride (NH), a β1-blocker, with bovine serum albumin (BSA) has been investigated at different pH values using the fluorescence quenching technique. The effect of different temperatures was studied at physiological pH 7.4. The binding constants of NH with BSA at 288, 298, and 309 K were found to be 2.691 × 1011, 1.38 × 1010, and 6.27 × 108 M-1, respectively. From the Arrhenius plot, the thermodynamic parameters, ΔH0 and ΔS0, were estimated to be -204.48 kJ/mol and -491.42 J/mol × K, respectively. This indicates that Van der Waals interactions and hydrogen bonds play a major role in the reaction. The effect of some inorganic divalent cations (Cu2+, Ni2+, and Zn2+) on binding of NH to BSA was also studied at physiological pH 7.4. Conformational investigation of BSA was done using synchronous fluorescence, showing the change in the microenvironment of the tryptophan residues. Fluorescence quenching reactions of NH to human serum albumin (HSA) and to γ-globulins were investigated and the binding parameters were obtained.

  8. Interaction of chlorogenic acids and quinides from coffee with human serum albumin.

    PubMed

    Sinisi, Valentina; Forzato, Cristina; Cefarin, Nicola; Navarini, Luciano; Berti, Federico

    2015-02-01

    Chlorogenic acids and their derivatives are abundant in coffee and their composition changes between coffee species. Human serum albumin (HSA) interacts with this family of compounds with high affinity. We have studied by fluorescence spectroscopy the specific binding of HSA with eight compounds that belong to the coffee polyphenols family, four acids (caffeic acid, ferulic acid, 5-O-caffeoyl quinic acid, and 3,4-dimethoxycinnamic acid) and four lactones (3,4-O-dicaffeoyl-1,5-γ-quinide, 3-O-[3,4-(dimethoxy)cinnamoyl]-1,5-γ-quinide, 3,4-O-bis[3,4-(dimethoxy)cinnamoyl]-1,5-γ-quinide, and 1,3,4-O-tris[3,4-(dimethoxy)cinnamoyl]-1,5-γ-quinide), finding dissociation constants of the albumin-chlorogenic acids and albumin-quinides complexes in the micromolar range, between 2 and 30μM. Such values are comparable with those of the most powerful binders of albumin, and more favourable than the values obtained for the majority of drugs. Interestingly in the case of 3,4-O-dicaffeoyl-1,5-γ-quinide, we have observed the entrance of two ligand molecules in the same binding site, leading up to a first dissociation constant even in the hundred nanomolar range, which is to our knowledge the highest affinity ever observed for HSA and its ligands. The displacement of warfarin, a reference drug binding to HSA, by the quinide has also been demonstrated.

  9. Interaction of chlorogenic acids and quinides from coffee with human serum albumin.

    PubMed

    Sinisi, Valentina; Forzato, Cristina; Cefarin, Nicola; Navarini, Luciano; Berti, Federico

    2015-02-01

    Chlorogenic acids and their derivatives are abundant in coffee and their composition changes between coffee species. Human serum albumin (HSA) interacts with this family of compounds with high affinity. We have studied by fluorescence spectroscopy the specific binding of HSA with eight compounds that belong to the coffee polyphenols family, four acids (caffeic acid, ferulic acid, 5-O-caffeoyl quinic acid, and 3,4-dimethoxycinnamic acid) and four lactones (3,4-O-dicaffeoyl-1,5-γ-quinide, 3-O-[3,4-(dimethoxy)cinnamoyl]-1,5-γ-quinide, 3,4-O-bis[3,4-(dimethoxy)cinnamoyl]-1,5-γ-quinide, and 1,3,4-O-tris[3,4-(dimethoxy)cinnamoyl]-1,5-γ-quinide), finding dissociation constants of the albumin-chlorogenic acids and albumin-quinides complexes in the micromolar range, between 2 and 30μM. Such values are comparable with those of the most powerful binders of albumin, and more favourable than the values obtained for the majority of drugs. Interestingly in the case of 3,4-O-dicaffeoyl-1,5-γ-quinide, we have observed the entrance of two ligand molecules in the same binding site, leading up to a first dissociation constant even in the hundred nanomolar range, which is to our knowledge the highest affinity ever observed for HSA and its ligands. The displacement of warfarin, a reference drug binding to HSA, by the quinide has also been demonstrated. PMID:25172718

  10. O{sub 2}-mediated oxidation of ferrous nitrosylated human serum heme-albumin is limited by nitrogen monoxide dissociation

    SciTech Connect

    Ascenzi, Paolo; Gullotta, Francesca; Gioia, Magda; Coletta, Massimo; Fasano, Mauro

    2011-03-04

    Research highlights: {yields} Human serum heme-albumin displays globin-like properties. {yields} O{sub 2}-mediated oxidation of ferrous nitrosylated human serum heme-albumin. {yields} Allosteric modulation of human serum heme-albumin reactivity. {yields} Rifampicin is an allosteric effector of human serum heme-albumin. {yields} Human serum heme-albumin is a ROS and NOS scavenger. -- Abstract: Human serum heme-albumin (HSA-heme-Fe) displays globin-like properties. Here, kinetics of O{sub 2}-mediated oxidation of ferrous nitrosylated HSA-heme-Fe (HSA-heme-Fe(II)-NO) is reported. Values of the first-order rate constants for O{sub 2}-mediated oxidation of HSA-heme-Fe(II)-NO (i.e., for ferric HSA-heme-Fe formation) and for NO dissociation from HSA-heme-Fe(II)-NO (i.e., for NO replacement by CO) are k = 9.8 x 10{sup -5} and 8.3 x 10{sup -4} s{sup -1}, and h = 1.3 x 10{sup -4} and 8.5 x 10{sup -4} s{sup -1}, in the absence and presence of rifampicin, respectively, at pH = 7.0 and T = 20.0 {sup o}C. The coincidence of values of k and h indicates that NO dissociation represents the rate limiting step of O{sub 2}-mediated oxidation of HSA-heme-Fe(II)-NO. Mixing HSA-heme-Fe(II)-NO with O{sub 2} does not lead to the formation of the transient adduct(s), but leads to the final ferric HSA-heme-Fe derivative. These results reflect the fast O{sub 2}-mediated oxidation of ferrous HSA-heme-Fe and highlight the role of drugs in modulating allosterically the heme-Fe-atom reactivity.

  11. Binding of Citreoviridin to Human Serum Albumin: Multispectroscopic and Molecular Docking

    PubMed Central

    Hou, Haifeng; Qu, Xiaolan; Li, Yuqin; Kong, Yueyue; Jia, Baoxiu; Yao, Xiaojun; Jiang, Baofa

    2015-01-01

    Citreoviridin (CIT), a mycotoxin produced by Penicillium citreonigrum, is a common contaminant of wide range of agriproducts and detrimental to human and animal health. In this study, the interaction of CIT with human serum albumin (HSA) is researched by steady-state fluorescence, ultraviolet-visible (UV-Vis) absorption, circular dichroism (CD) methods, and molecular modeling. The association constants, binding site numbers, and corresponding thermodynamic parameters are used to investigate the quenching mechanism. The alternations of HSA secondary structure in the presence of CIT are demonstrated with UV-Vis, synchronous fluorescence, and CD spectra. The molecular modeling results reveal that CIT can bind with hydrophobic pocket of HSA with hydrophobic and hydrogen bond force. Moreover, an apparent distance of 3.25 nm between Trp214 and CIT is obtained via fluorescence resonance energy transfer method. PMID:25977915

  12. Amino acid substitutions in genetic variants of human serum albumin and in sequences inferred from molecular cloning

    SciTech Connect

    Takahashi, N.; Takahashi, Y.; Blumberg, B.S.; Putnam, F.W.

    1987-07-01

    The structural changes in four genetic variants of human serum albumin were analyzed by tandem high-pressure liquid chromatography (HPLC) of the tryptic peptides, HPLC mapping and isoelectric focusing of the CNBr fragments, and amino acid sequence analysis of the purified peptides. Lysine-372 of normal (common) albumin A was changed to glutamic acid both in albumin Naskapi, a widespread polymorphic variant of North American Indians, and in albumin Mersin found in Eti Turks. The two variants also exhibited anomalous migration in NaDodSO/sub 4//PAGE, which is attributed to a conformational change. The identity of albumins Naskapi and Mersin may have originated through descent from a common mid-Asiatic founder of the two migrating ethnic groups, or it may represent identical but independent mutations of the albumin gene. In albumin Adana, from Eti Turks, the substitution site was not identified but was localized to the region from positions 447 through 548. The substitution of aspartic acid-550 by glycine was found in albumin Mexico-2 from four individuals of the Pima tribe. Although only single-point substitutions have been found in these and in certain other genetic variants of human albumin, five differences exist in the amino acid sequences inferred from cDNA sequences by workers in three other laboratories. However, our results on albumin A and on 14 different genetic variants accord with the amino acid sequence of albumin deduced from the genomic sequence. The apparent amino acid substitutions inferred from comparison of individual cDNA sequences probably reflect artifacts in cloning or in cDNA sequence analysis rather than polymorphism of the coding sections of the albumin gene.

  13. Amino acid substitutions in genetic variants of human serum albumin and in sequences inferred from molecular cloning.

    PubMed

    Takahashi, N; Takahashi, Y; Blumberg, B S; Putnam, F W

    1987-07-01

    The structural changes in four genetic variants of human serum albumin were analyzed by tandem high-pressure liquid chromatography (HPLC) of the tryptic peptides, HPLC mapping and isoelectric focusing of the CNBr fragments, and amino acid sequence analysis of the purified peptides. Lysine-372 of normal (common) albumin A was changed to glutamic acid both in albumin Naskapi, a widespread polymorphic variant of North American Indians, and in albumin Mersin found in Eti Turks. The two variants also exhibited anomalous migration in NaDodSO4/PAGE, which is attributed to a conformational change. The identity of albumins Naskapi and Mersin may have originated through descent from a common mid-Asiatic founder of the two migrating ethnic groups, or it may represent identical but independent mutations of the albumin gene. In albumin Adana, from Eti Turks, the substitution site was not identified but was localized to the region from positions 447 through 548. The substitution of aspartic acid-550 by glycine was found in albumin Mexico-2 from four individuals of the Pima tribe. Although only single-point substitutions have been found in these and in certain other genetic variants of human albumin, five differences exist in the amino acid sequences inferred from cDNA sequences by workers in three other laboratories. However, our results on albumin A and on 14 different genetic variants accord with the amino acid sequence of albumin deduced from the genomic sequence. The apparent amino acid substitutions inferred from comparison of individual cDNA sequences probably reflect artifacts in cloning or in cDNA sequence analysis rather than polymorphism of the coding sections of the albumin gene.

  14. Protein Crystal Serum Albumin

    NASA Technical Reports Server (NTRS)

    1998-01-01

    As the most abundant protein in the circulatory system albumin contributes 80% to colloid osmotic blood pressure. Albumin is also chiefly responsible for the maintenance of blood pH. It is located in every tissue and bodily secretion, with extracellular protein comprising 60% of total albumin. Perhaps the most outstanding property of albumin is its ability to bind reversibly to an incredible variety of ligands. It is widely accepted in the pharmaceutical industry that the overall distribution, metabolism, and efficiency of many drugs are rendered ineffective because of their unusually high affinity for this abundant protein. An understanding of the chemistry of the various classes of pharmaceutical interactions with albumin can suggest new approaches to drug therapy and design. Principal Investigator: Dan Carter/New Century Pharmaceuticals

  15. Fluorescence study on the interaction of human serum albumin with Butein in liposomes

    NASA Astrophysics Data System (ADS)

    Toprak, Mahmut

    2016-02-01

    The interaction of Butein with human serum albumin in L-egg lecithin phosphatidycholine (PC) liposome has been investigated by fluorescence and absorption spectroscopy. The results of the fluorescence measurement indicated that Butein effectively quenched the intrinsic fluorescence of HSA via static quenching. The Stern-Volmer plots in all the liposome solutions showed a positive deviation from the linearity. According to the thermodynamic parameters, the hydrophobic interactions appeared be the major interaction forces between Butein and HSA. The effect of Butein on the conformation of HSA was also investigated by the synchronous fluorescence under the same experimental conditions. In addition, the partition coefficient of the Butein in the PC liposomes was also determined by using the fluorescence quenching process. The obtained results can be of biological significance in pharmacology and clinical medicine.

  16. Auto-oxidation of Isoniazid Leads to Isonicotinic-Lysine Adducts on Human Serum Albumin.

    PubMed

    Meng, Xiaoli; Maggs, James L; Usui, Toru; Whitaker, Paul; French, Neil S; Naisbitt, Dean J; Park, B Kevin

    2015-01-20

    Isoniazid (INH), a widely used antituberculosis drug, has been associated with serious drug-induced liver injury (DILI). INH-modified proteins have been proposed to play important roles in INH DILI; however, it remains to be determined whether INH or reactive metabolites bind irreversibly to proteins. In this study, mass spectrometry was used to define protein modifications by INH in vitro and in patients taking INH therapy. When INH was incubated with N-acetyl lysine (NAL), the same isonicotinic-NAL (IN-NAL) adducts were detected irrespective of the presence or absence of any oxidative enzymes, indicating auto-oxidation may have been involved. In addition, we found that INH could also bind to human serum albumin (HSA) via an auto-oxidation pathway, forming isonicotinic amide adducts with lysine residues in HSA. Similar adducts were detected in plasma samples isolated from patients taking INH therapy. Our results show that INH forms protein adducts in the absence of metabolism.

  17. Curcumin promotes fibril formation in F isomer of human serum albumin via amorphous aggregation.

    PubMed

    Mothi, Nivin; Muthu, Shivani A; Kale, Avinash; Ahmad, Basir

    2015-12-01

    We here describe the amyloid fibrils promoting behavior of curcumin, which ability to inhibit amyloid fibrillization of several globular proteins is well documented. Transmission electron microscopy (TEM), 90° light scattering (RLS), thioflavine T (ThT) and Congo red (CR) binding studies demonstrated that both F (pH3.4) and E (pH1.8) isomers of human serum albumin (HSA) in the absence and presence of curcumin initially converted into amorphous aggregates. Interestingly, only the sample containing F isomer preincubated with curcumin formed fibrils on incubation for longer period. We also found that curcumin strongly bind to the F isomer, alter its secondary, tertiary structures and thermal stability. We conclude that the conversion of intermediate states into amorphous aggregate to fibrils is dictated by its conformation. This study provides unique insights into ligand-controlled HSA aggregation pathway and should provide a useful model system to study both amorphous and the fibrillar aggregation of multidomain proteins.

  18. Affinity of rosmarinic acid to human serum albumin and its effect on protein conformation stability.

    PubMed

    Peng, Xin; Wang, Xiangchao; Qi, Wei; Su, Rongxin; He, Zhimin

    2016-02-01

    Rosmarinic acid (RA) is a natural polyphenol contained in many aromatic plants with promising biological activities. The interaction between RA and human serum albumin (HSA) was investigated by multi-spectroscopic, electrochemistry, molecular docking and molecular dynamics simulation methods. The fluorescence emission of HSA was quenched by RA through a combined static and dynamic quenching mechanism, but the static quenching was the major constituent. Fluorescence experiments suggested that RA was bound to HSA with moderately strong binding affinity through hydrophobic interaction. The probable binding location of RA was located near site I of HSA. Additionally, as shown by the Fourier transform infrared (FT-IR) and circular dichroism (CD) spectra, RA can result in conformational and structural alterations of HSA. Furthermore, the molecular dynamics studies were used to investigate the stability of the HSA and HSA-RA system. Altogether, the results can provide an important insight for the applications of RA in the food industry.

  19. Influence of the galloyl moiety in tea catechins on binding affinity for human serum albumin.

    PubMed

    Minoda, Kanako; Ichikawa, Tatsuya; Katsumata, Tomoharu; Onobori, Ken-ichi; Mori, Taiki; Suzuki, Yukiko; Ishii, Takeshi; Nakayama, Tsutomu

    2010-01-01

    The major catechins of green tea extract are (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg). Recent research has indicated that catechins form complexes with human serum albumin (HSA) in blood, and differences in their binding affinity toward HSA are believed to modulate their bioavailability. In this study, we kinetically investigated the interaction between the catechins and HSA immobilized on a quartz-crystal microbalance (QCM). The association constants obtained from the frequency changes of QCM revealed interactions of ECg and EGCg with HSA that are 100 times stronger than those of EC and EGC. Furthermore, comparisons of these catechins by native-gel electrophoresis/blotting with redox-cycling staining revealed that, in a phosphate buffer, ECg and EGCg have a higher binding affinity toward HSA than EC and EGC. These observations indicate that catechins with a galloyl moiety have higher binding affinities toward HSA than catechins lacking a galloyl moiety.

  20. [Influence of direct electric current on hydrodynamic diameter of human serum albumin].

    PubMed

    Korpan, M I; Gorchev, V F; Chekman, I S; Gun'ko, V M; Fialko-Moser, V

    2008-01-01

    The effect of the weak electric current (2 mA/cm2) on structural characteristics (hydrodynamic diameter and molecular weight) of the human serum albumin (HSA) was studied using photon correlation spectroscopy (PCS). The average diameter of initial HSA globule is approximately 7 nm (66.8 kDa). After electric current treatment during 2-5 min the diameter of HSA monomer increases to 7.5 nm. The duration of electric current treatment being increased to 20 min the size of HSA monomers decreases to 6.4 nm. The behaviour of HSA oligomers is close to that of monomers. Consequently, changes in the sizes of monomers and oligomers of HSA under the electric current treatment are caused by the change in the charge density stimulating change of tertiary structure of molecules and possible addition of ions from the buffer solution to them.

  1. Interaction of diuron to human serum albumin: Insights from spectroscopic and molecular docking studies.

    PubMed

    Chen, Huilun; Rao, Honghao; Yang, Jian; Qiao, Yongxiang; Wang, Fei; Yao, Jun

    2016-01-01

    This investigation was undertaken to determine the interaction of diuron with human serum albumin (HSA) was studied by monitoring the spectral behavior of diuron-HSA system. The fluorescence of HSA at 340 nm excited at 230 nm was obviously quenched by diuron due to dynamic collision and the quenching constant was of the order of 10(4) L mol(-1) at 310 K. However, no fluorescence quenching was observed when excited at 280 nm. Thermodynamic investigations revealed that the combination between diuron and HSA was entropy driven by predominantly hydrophobic interactions. The binding of diuron induced the drastic reduction in α-helix conformation and the significant enhancement in β-turn conformation of HSA. In addition, both sites marker competition study and molecular modeling simulation evidenced the binding of diuron to HSA primarily took place in subdomain IIIA (Sudlow's site II). PMID:26671830

  2. A calorimetric study on interactions of colchicine with human serum albumin

    NASA Astrophysics Data System (ADS)

    Zhao, Qiang; Xu, Xiang-Yu; Sun, Xiang-Jun; Liu, Min; Sun, De-Zhi; Li, Lin-Wei

    2009-08-01

    Interaction of colchicine (COL) with human serum albumin (HSA) in buffer solutions (pH 7.2) has been investigated by isothermal titration calorimetry (ITC) combined with circular dichroism (CD) and UV-vis spectra. Heats of the interactions have been determined at 298.15 K. Based on the calorimetric data and reasonable suppositions for the bio-macromolecule - ligand binding process, the equilibrium constants, standard changes of enthalpy, entropy and Gibbs free energy of the processes are obtained. The results show that there are two classes of ligand binding sites. The first-class binding is mainly driven by entropy, while the second-class binding is synergistically driven by entropy and enthalpy. Circular dichroism (CD) and UV-vis spectra show that COL can change the secondary structure of HSA molecule.

  3. Affinity of rosmarinic acid to human serum albumin and its effect on protein conformation stability.

    PubMed

    Peng, Xin; Wang, Xiangchao; Qi, Wei; Su, Rongxin; He, Zhimin

    2016-02-01

    Rosmarinic acid (RA) is a natural polyphenol contained in many aromatic plants with promising biological activities. The interaction between RA and human serum albumin (HSA) was investigated by multi-spectroscopic, electrochemistry, molecular docking and molecular dynamics simulation methods. The fluorescence emission of HSA was quenched by RA through a combined static and dynamic quenching mechanism, but the static quenching was the major constituent. Fluorescence experiments suggested that RA was bound to HSA with moderately strong binding affinity through hydrophobic interaction. The probable binding location of RA was located near site I of HSA. Additionally, as shown by the Fourier transform infrared (FT-IR) and circular dichroism (CD) spectra, RA can result in conformational and structural alterations of HSA. Furthermore, the molecular dynamics studies were used to investigate the stability of the HSA and HSA-RA system. Altogether, the results can provide an important insight for the applications of RA in the food industry. PMID:26304336

  4. Binding of caffeine, theophylline, and theobromine with human serum albumin: A spectroscopic study

    NASA Astrophysics Data System (ADS)

    Zhang, Hong-Mei; Chen, Ting-Ting; Zhou, Qiu-Hua; Wang, Yan-Qing

    2009-12-01

    The interaction between three purine alkaloids (caffeine, theophylline, and theobromine) and human serum albumin (HSA) was investigated using UV/vis absorption, circular dichroism (CD), fluorescence, synchronous fluorescence, and three-dimensional fluorescence spectra techniques. The results revealed that three alkaloids caused the fluorescence quenching of HSA by the formation of alkaloid-HSA complex. The binding site number n and apparent binding constant KA, corresponding thermodynamic parameters the free energy change (Δ G), enthalpy change (Δ H), and entropy change (Δ S) at different temperatures were calculated. The hydrophobic interaction plays a major role in stabilizing the complex. The distance r between donor (HSA) and acceptor (alkaloids) was obtained according to fluorescence resonance energy transfer. The effect of alkaloids on the conformation of HSA was analyzed using circular dichroism (CD), UV/vis absorption, synchronous fluorescence and three-dimensional fluorescence spectra techniques.

  5. Investigation of ketoprofen binding to human serum albumin by spectral methods

    NASA Astrophysics Data System (ADS)

    Bi, Shuyun; Yan, Lili; Sun, Yantao; Zhang, Hanqi

    2011-01-01

    The binding of ketoprofen with human serum albumin (HSA) was studied by fluorescence and absorption spectroscopic methods. Quenching of fluorescence of HSA was found to be a static quenching process. At 288.15, 298.15, 308.15 and 318.15 K, the binding constants and binding sites were obtained. The effects of Cu 2+, Al 3+, Ca 2+, Pb 2+ and K + on the binding at 288.15 K were also studied. The thermodynamic parameters, Δ H, Δ G and Δ S were got and the main sort of acting force between ketoprofen and HSA was studied. Based on the Förster's theory of non-radiation energy transfer, the binding average distance, r, between the acceptor (ketoprofen) and the donor (HSA) was calculated.

  6. Effect of dendrimer generation on the interactions between human serum albumin and dendrigraft polylysines.

    PubMed

    Sisavath, Nicolas; Le Saux, Thomas; Leclercq, Laurent; Cottet, Hervé

    2014-04-22

    This work aims at studying the interaction between human serum albumin and different generations of dendrigraft poly-L-lysine (DGL) in physiological conditions. The binding constants and stoichiometry of the interaction were successfully determined using frontal analysis continuous capillary electrophoresis. The effect of generation on the interaction was evaluated for the five first generations of DGL. An increase of the binding constant accompanied with a decrease of the HSA:DGL (1:n) stoichiometry and a decrease of the cooperativity with dendrimer generation was observed. These findings were in good agreement with the increase of ligand (DGL) size, the increase of electrostatic ligand-ligand repulsion, and the localization of two negatively charged interaction sites on the HSA. The effect of the ligand topology (linear vs dendrigraft) on the HSA interaction revealed that linear poly(L-lysine) leads to much lower stoichiometry compared to DGL of similar molar mass due to much higher flexibility and contour length.

  7. Synthesis of imidazole derivatives and the spectral characterization of the binding properties towards human serum albumin

    NASA Astrophysics Data System (ADS)

    Yue, Yuanyuan; Dong, Qiao; Zhang, Yajie; Li, Xiaoge; Yan, Xuyang; Sun, Yahui; Liu, Jianming

    2016-01-01

    Small molecular drugs that can combine with target proteins specifically, and then block relative signal pathway, finally obtain the purpose of treatment. For this reason, the synthesis of novel imidazole derivatives was described and this study explored the details of imidazole derivatives binding to human serum albumin (HSA). The data of steady-state and time-resolved fluorescence showed that the conjugation of imidazole derivatives with HSA yielded quenching by a static mechanism. Meanwhile, the number of binding sites, the binding constants, and the thermodynamic parameters were also measured; the raw data indicated that imidazole derivatives could spontaneously bind with HSA through hydrophobic interactions and hydrogen bonds which agreed well with the results from the molecular modeling study. Competitive binding experiments confirmed the location of binding. Furthermore, alteration of the secondary structure of HSA in the presence of the imidazole derivatives was tested.

  8. Structural basis of non-steroidal anti-inflammatory drug diclofenac binding to human serum albumin.

    PubMed

    Zhang, Yao; Lee, Philbert; Liang, Shichu; Zhou, Zuping; Wu, Xiaoyang; Yang, Feng; Liang, Hong

    2015-11-01

    Human serum albumin (HSA) is the most abundant protein in plasma, which plays a central role in drug pharmacokinetics because most compounds bound to HSA in blood circulation. To understand binding characterization of non-steroidal anti-inflammatory drugs to HSA, we resolved the structure of diclofenac and HSA complex by X-ray crystallography. HSA-palmitic acid-diclofenac structure reveals two distinct binding sites for three diclofenac in HSA. One diclofenac is located at the IB subdomain, and its carboxylate group projects toward polar environment, forming hydrogen bond with one water molecule. The other two diclofenac molecules cobind in big hydrophobic cavity of the IIA subdomain without interactive association. Among them, one binds in main chamber of big hydrophobic cavity, and its carboxylate group forms hydrogen bonds with Lys199 and Arg218, as well as one water molecule, whereas another diclofenac binds in side chamber, its carboxylate group projects out cavity, forming hydrogen bond with Ser480.

  9. [Interaction between ambroxol hydrochloride and human serum albumin studied by spectroscopic and molecular modeling methods].

    PubMed

    Liang, Jing; Feng, Su-Ling

    2011-04-01

    In the present paper, the interaction between ambroxol hydrochloride (ABX) and human serum albumin (HSA) was studied under simulative physiological condition by spectroscopy and molecular modeling method. Stern-Volmer curvers at different temperatures and UV-Vis absorption spectroscopy showed that ABX quenched the fluorescence of HSA mainly through dynamic quenching mode. On the basis of the thermodynamic data, the main binding force between them is hydrophobic interaction. According to the theory of Forster non-radiation energy transfer, the binding distance between the donor and the acceptor was 3.01 nm. The effect of ABX on the conformation of HSA was analyzed by the synchronous and three-dimensional fluorescence spectroscopy. Furthermore, using the molecular modeling method, the interaction between them was predicted from molecular angle: ABX might locate in the subdomain III A of HSA. PMID:21714251

  10. Binding properties of drospirenone with human serum albumin and lysozyme in vitro

    NASA Astrophysics Data System (ADS)

    Wang, Qing; Ma, Xiangling; He, Jiawei; Sun, Qiaomei; Li, Yuanzhi; Li, Hui

    2016-01-01

    The interaction of drospirenone (DP) with human serum albumin (HSA)/lysozyme (LYZ) was investigated using different optical techniques and molecular models. Results from the emission and time resolved fluorescence studies revealed that HSA/LYZ emission quenching with DP was initiated by static quenching mechanism. The LYZ-DP system was more easily influenced by temperature than the HSA-DP system. Displacement experiments demonstrated that the DP binding site was mainly located in site 1 of HSA. Based on the docking methods, DP was mainly bound in the active site hinge region where Trp-62 and Trp-63 are located. Conformation study showed that DP had different effects on the local conformation of HSA and LYZ molecules.

  11. Photoinduced covalent binding of frusemide and frusemide glucuronide to human serum albumin

    PubMed Central

    Mizuma, Takashi; McDonagh, Antony F; Lin, Emil T; Benet, Leslie Z

    1999-01-01

    Aims To study reaction of photoactivated frusemide (F) and F glucuronide (Fgnd metabolite) with human serum albumin in order to find a clue to clarify a mechanism of phototoxic blisters from high frusemide dosage. Methods F was exposed to light in the presence of human serum albumin (HSA). HSA treated with this method (TR-HSA) was characterized by fluorescence spectroscopic experiment, alkali treatment and reversible binding experiment. Results Less 4-hydroxyl-N-furfuryl-5-sulphamoylanthranilic acid (4HFSA, a photodegradation product of F) was formed in the presence of HSA than in the absence of HSA. A new fluorescence spectrum excited at 320 nm was observed for TR-HSA. Alkali treatment of TR-HSA released 4HFSA. Quenching of the fluorescence due to the lone tryptophan near the warfarin-binding site of HSA was observed in TR-HSA. The reversible binding of F or naproxen to the warfarin-binding site of TR-HSA was less than to that of native HSA. These results indicate the photoactivated F was covalently bound to the warfarin-binding site of HSA. The covalent binding of Fgnd, which is also reversibly bound to the wafarin-binding site of HSA, was also induced by exposure to sunlight. Fgnd was more photoactive than F, indicating that F could be activated by glucuronidation to become a more photoactive compound. Conclusions The reactivity of photoactivated F and Fgnd to HSA and/or to other endogenous compounds may cause the phototoxic blisters that result at high F dosage. PMID:10383564

  12. Mapping the interactions between the Alzheimer's Aβ-peptide and human serum albumin beyond domain resolution.

    PubMed

    Algamal, Moustafa; Milojevic, Julijana; Jafari, Naeimeh; Zhang, William; Melacini, Giuseppe

    2013-10-01

    Human serum albumin (HSA) is a potent inhibitor of Aβ self-association and this novel, to our knowledge, function of HSA is of potential therapeutic interest for the treatment of Alzheimer's disease. It is known that HSA interacts with Aβ oligomers through binding sites evenly partitioned across the three albumin domains and with comparable affinities. However, as of this writing, no information is available on the HSA-Aβ interactions beyond domain resolution. Here, we map the HSA-Aβ interactions at subdomain and peptide resolution. We show that each separate subdomain of HSA domain 3 inhibits Aβ self-association. We also show that fatty acids (FAs) compete with Aβ oligomers for binding to domain 3, but the determinant of the HSA/Aβ oligomer interactions are markedly distinct from those of FAs. Although salt bridges with the FA carboxylate determine the FA binding affinities, hydrophobic contacts are pivotal for Aβ oligomer recognition. Specifically, we identified a site of Aβ oligomer recognition that spans the HSA (494-515) region and aligns with the central hydrophobic core of Aβ. The HSA (495-515) segment includes residues affected by FA binding and this segment is prone to self-associate into β-amyloids, suggesting that sites involved in fibrilization may provide a lead to develop inhibitors of Aβ self-association. PMID:24094411

  13. Reviewing the binding of a series of parabens to human serum albumin.

    PubMed

    Greige-Gerges, Hélène; Kaissi, Rana; Magdalou, Jacques; Jraij, Alia

    2013-04-01

    To better understand the factors that contribute to the accumulation of unmetabolized parabens (p-hydroxybenzoic acid esters) in breast cancer tissue, the binding of a series of parabens (methyl-, ethyl-, butyl-, benzyl-paraben) to human serum albumin (HSA) was investigated by fluorescence spectroscopy and also their ability to modify the binding parameters of albumin site markers. Emission spectra of HSA upon fluorescence excitation of Trp 214 residue at 295 nm were recorded at different molar ratios of PB/HSA and data were corrected for the inner-filter effect. A significant inner-filter effect was obtained for molar ratios of 2.0 and above. For lower molar ratios, a slight increase in fluorescence of HSA was detected. p-Hydroxybenzoic acid, the main metabolite of parabens, did not modify the fluorescence of HSA whatever the molar ratio used. Binding parameters for compounds that are markers of site I, bilirubin and warfarin, were determined in the absence and presence of methyl, butyl and benzyl paraben at molar ratios of PB/HSA of 0, 1 and 2. No variation of the binding constants of these markers was observed. The results indicate that parabens weakly interact with HSA thus suggesting that they are in a free form in blood and therefore more available to reach tissues.

  14. Crystals of Human Serum Albumin for Use in Genetic Engineering and Rational Drug Design

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C. (Inventor)

    1994-01-01

    This invention pertains to crystals of serum albumin and processes for growing them. The purpose of the invention is to provide crystals of serum albumin which can be studied to determine binding sites for drugs. Form 2 crystals grow in the monoclinic space P2(sub 1), and possesses the following unit cell constraints: a = 58.9 +/- 7, b = 88.3 +/- 7, c = 60.7 +/- 7, Beta = 101.0 +/- 2 degrees. One advantage of the invention is that it will allow rational drug design

  15. On-site preparation of technetium-99m labeled human serum albumin for clinical application.

    PubMed

    Wang, Yuh-Feng; Chuang, Mei-Hua; Chiu, Jainn-Shiun; Cham, Thau-Ming; Chung, Mei-Ing

    2007-04-01

    Technetium-99m labeled human serum albumin (Tc-99m HSA) is an important radiopharmaceutical for clinical applications, such as cardiac function tests or protein-losing gastroenteropathy assessment. However, because of transfusion-induced infectious diseases, the safety of serum products is a serious concern. In this context, serum products acquired from patients themselves are the most ideal tracer. However, the development of rapid separation and easy clinical labeling methods is not yet well established. Under such situation, products from the same ethnic group or country are now recommended by the World Health Organization as an alternative preparation. This article describes the on-site preparation of Tc-99m HSA from locally supplied serum products. Different formulations were prepared and the labeling efficiency and stability were examined. Radio-labeling efficiencies were more than 90% in all preparation protocols, except for one that omitted the stannous solution. The most cost-effective protocol contained HSA 0.1 mg, treated with stannous fluoride 0.2 mg, and mixed with Tc-99m pertechnetate 30 mCi. A biodistribution study was performed in rats using a gamma camera immediately after intravenous administration of radiolabeled HSA. Tissue/organ uptake was obtained by measuring the radioactivity in organs after sacrificing the rats at timed intervals. The biologic half-life was about 32 min, determined from sequential venous blood collections. These data indicate that our preparation of Tc-99m HSA is useful and potentially applicable clinically. In addition, this on-site preparation provides the possibility of labeling a patient's own serum for subsequent clinical application.

  16. Albumin - blood (serum) test

    MedlinePlus

    ... conditions for which the test may be performed: Burns (widespread) Wilson disease If you are receiving large amounts of intravenous fluids, the result of this test may be inaccurate. Albumin will be decreased during pregnancy.

  17. Characterization of the interaction between 8-bromoadenosine with human serum albumin and its analytical application

    NASA Astrophysics Data System (ADS)

    Cui, Fengling; Yan, Yinghua; Zhang, Qiangzhai; Yao, Xiaojun; Qu, Guirong; Lu, Yan

    2009-11-01

    This study was designed to examine the interaction of 8-bromoadenosine with human serum albumin (HSA) by fluorescence spectroscopy in combination with molecular modeling under simulative physiological conditions. The results of fluorescence measurements indicate that 8-bromoadenosine has a strong ability to quench the intrinsic fluorescence of HSA through static quenching procedure. The binding constants ( K) at different temperatures and thermodynamic parameters, enthalpy changes (Δ H) and entropy changes (Δ S) were calculated according to the fluorescence data. The results showed that the hydrophobic force played the major role in the binding of 8-bromoadenosine to HSA. The fluorescence experimental results were in agreement with the results obtained by molecular modeling study. The effects of some normal positive and negative ions on the binding constants were also discussed. Moreover, the synchronous fluorescence technique was used to characterize the interaction of 8-bromoadenosine to HSA and successfully applied to determine the total proteins in human serum, urine and saliva samples at room temperature under the optimum conditions with a wide linear range and satisfactory results.

  18. Interaction of prometryn to human serum albumin: insights from spectroscopic and molecular docking studies.

    PubMed

    Wang, Yaping; Zhang, Guowen; Wang, Langhong

    2014-01-01

    Prometryn possesses much potential hazard to environment because of its chemical stability and biological toxicity. Here, the binding properties of prometryn with human serum albumin (HSA) and the protein structural changes were determined under simulative physiological conditions (pH 7.4) by multispectroscopic methods including fluorescence, UV-vis absorption, Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopy, coupled with molecular modeling technique. The result of fluorescence titration suggested that the fluorescence quenching of HSA by prometryn was considered as a static quenching procedure. The negative enthalpy change (ΔH(○)) and positive entropy change (ΔS(○)) values indicated that the binding process was governed mainly by hydrophobic interactions and hydrogen bonds. The site marker displacement experiments suggested the location of prometryn binding to HSA was Sudlow's site I in subdomain IIA. Furthermore, molecular docking studies revealed prometryn can bind in the large hydrophobic activity of subdomain IIA. Analysis of UV-vis absorption, synchronous fluorescence, CD and FT-IR spectra demonstrated that the addition of prometryn resulted in rearrangement and conformational alteration of HSA with reduction in α-helix and increases in β-sheet, β-turn and random coil structures. This work provided reasonable model helping us further understand the transportation, distribution and toxicity effect of prometryn when it spreads into human blood serum. PMID:24485317

  19. Locating the Binding Sites of Pb(II) Ion with Human and Bovine Serum Albumins

    PubMed Central

    Belatik, Ahmed; Hotchandani, Surat; Carpentier, Robert; Tajmir-Riahi, Heidar-Ali

    2012-01-01

    Lead is a potent environmental toxin that has accumulated above its natural level as a result of human activity. Pb cation shows major affinity towards protein complexation and it has been used as modulator of protein-membrane interactions. We located the binding sites of Pb(II) with human serum (HSA) and bovine serum albumins (BSA) at physiological conditions, using constant protein concentration and various Pb contents. FTIR, UV-visible, CD, fluorescence and X-ray photoelectron spectroscopic (XPS) methods were used to analyse Pb binding sites, the binding constant and the effect of metal ion complexation on HSA and BSA stability and conformations. Structural analysis showed that Pb binds strongly to HSA and BSA via hydrophilic contacts with overall binding constants of KPb-HSA = 8.2 (±0.8)×104 M−1 and KPb-BSA = 7.5 (±0.7)×104 M−1. The number of bound Pb cation per protein is 0.7 per HSA and BSA complexes. XPS located the binding sites of Pb cation with protein N and O atoms. Pb complexation alters protein conformation by a major reduction of α-helix from 57% (free HSA) to 48% (metal-complex) and 63% (free BSA) to 52% (metal-complex) inducing a partial protein destabilization. PMID:22574219

  20. Human serum albumin-TRAIL conjugate for the treatment of rheumatoid arthritis.

    PubMed

    Byeon, Hyeong Jun; Min, Sun Young; Kim, Insoo; Lee, Eun Seong; Oh, Kyung Taek; Shin, Beom Soo; Lee, Kang Choon; Youn, Yu Seok

    2014-12-17

    Albumin conjugation is viewed as an effective means of protracting short in vivo lifespans of proteins and targeting rheumatoid arthritis (RA). In this study, we present a human serum albumin (HSA) conjugate linked with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) via a bifunctional PEG derivative (HSA-TRAIL). Prepared HSA-TRAIL was found to have a larger molecular size (∼240 kDa, 15.4 nm) than TRAIL (∼66 kDa, 6.2 nm), and its bioactivity (apoptosis, cytotoxicity, and antiproliferation) was well preserved in Mia Paca-2 cells and mouse splenocytes. The enhanced therapeutic efficacy of HSA-TRAIL was demonstrated in collagen-induced arthritis (CIA) mice. The incidence and clinical scores, expressed as degree of erythema and swelling in HSA-TRAIL-treated mice, were remarkably lower than those of TRAIL-treated mice. The serum levels of pro-inflammatory cytokines IFN-γ, TNF-α, IL-1β, and IL-2 in HSA-TRAIL-treated mice were significantly lower than those of TRAIL-treated mice. Furthermore, HSA-TRAIL accumulated in the hind paws of CIA mice, not in naïve TRAIL mice. Pharmacokinetic profiles of HSA-TRAIL were greatly improved in comparison to those of TRAIL (AUCinf: 844.1 ± 130.0 vs 36.0 ± 1.2 ng·h/mL; t1/2: 6.20 ± 0.72 vs 0.23 ± 0.01 h, respectively). The HSA-TRAIL conjugate, which presents clear advantages of targeting RA and long systemic circulation by HSA and unique anti-inflammatory efficacy by TRAIL, has potential as a novel treatment for rheumatoid arthritis. PMID:25387356

  1. Evalution of capillary electrophoresis-frontal analysis for the study of low molecular weight drug-human serum albumin interactions.

    PubMed

    Østergaard, Jesper; Schou, Christian; Larsen, Claus; Heegaard, Niels H H

    2002-09-01

    Capillary electrophoresis frontal analysis was applied to 12 low molecular weight compounds including 8 drug substances displaying a range of different properties with respect to binding affinity, binding location, structure, lipophilicity, charge at physiological pH, and electrophoretic mobility. It was found that capillary electrophoresis frontal analysis can be used as a general method to study and quantify drug-human serum albumin interactions. The binding parameters obtained were consistent with literature values. Dextran was in some cases added to the run buffer to improve separation of the drug and human serum albumin plateau peaks. Results indicate that mobility differences between free and complexed human serum albumin give rise to only minor errors. Capillary electrophoresis frontal analysis was also found applicable to the study of human serum albumin drug displacement reactions. Low sensitivity of the UV-detection system was found to be the major limitation of capillary electrophoresis frontal analysis. The method is simple, and minimal effort has to be put into method development, which makes it well suited for screening in early drug development.

  2. Human islet purification: a prospective comparison of Euro-Ficoll and bovine serum albumin density gradients.

    PubMed

    Chadwick, D R; Robertson, G S; Contractor, H; Swift, S; Rose, S; Thirdborough, S T; Chamberlain, R; James, R F; Bell, P R; London, N J

    1993-01-01

    Euro-Ficoll (EF) and bovine serum albumin (BSA) are the two most commonly used media for the density gradient purification of human pancreatic islets. The aim of this study was to compare these two media with respect to the efficiency of human islet isolation. Ten human pancreata were collagenase-digested, and samples of digest were separated on either a continuous linear density gradient of BSA or a discontinuous gradient of EF (1.108/1.096/1.037/Euro-Collins). Efficiency of islet purification was assessed by insulin and amylase assay of aliquots aspirated from the BSA gradients, and from the interfaces of the EF gradients. Islets were obtained from two interfaces in the EF gradients. Islet yield from the upper interface was generally poor (median 28% of total insulin; range 2-71%), but purity was better than for an equivalent yield using BSA [1% (0-3%) amylase contamination for EF versus 6% (0-37%) for BSA; P = 0.013]. Pooling both EF interfaces increased yield to 66% (17-81%) but markedly reduced purity [46% (0-50%) amylase for EF versus 31% (0-52%) for BSA]. In conclusion, the efficiency of human islet purification is similar, though disappointingly low, with BSA and with EF. Considerable scope exists, therefore, for improvement in the density gradient purification of human islets. PMID:8329732

  3. Influence of MLS laser radiation on erythrocyte membrane fluidity and secondary structure of human serum albumin.

    PubMed

    Pasternak, Kamila; Nowacka, Olga; Wróbel, Dominika; Pieszyński, Ireneusz; Bryszewska, Maria; Kujawa, Jolanta

    2014-03-01

    The biostimulating activity of low level laser radiation of various wavelengths and energy doses is widely documented in the literature, but the mechanisms of the intracellular reactions involved are not precisely known. The aim of this paper is to evaluate the influence of low level laser radiation from an multiwave locked system (MLS) of two wavelengths (wavelength = 808 nm in continuous emission and 905 nm in pulsed emission) on the human erythrocyte membrane and on the secondary structure of human serum albumin (HSA). Human erythrocytes membranes and HSA were irradiated with laser light of low intensity with surface energy density ranging from 0.46 to 4.9 J cm(-2) and surface energy power density 195 mW cm(-2) (1,000 Hz) and 230 mW cm(-2) (2,000 Hz). Structural and functional changes in the erythrocyte membrane were characterized by its fluidity, while changes in the protein were monitored by its secondary structure. Dose-dependent changes in erythrocyte membrane fluidity were induced by near-infrared laser radiation. Slight changes in the secondary structure of HSA were also noted. MLS laser radiation influences the structure and function of the human erythrocyte membrane resulting in a change in fluidity.

  4. Cabazitaxel-loaded human serum albumin nanoparticles as a therapeutic agent against prostate cancer.

    PubMed

    Qu, Na; Lee, Robert J; Sun, Yating; Cai, Guangsheng; Wang, Junyang; Wang, Mengqiao; Lu, Jiahui; Meng, Qingfan; Teng, Lirong; Wang, Di; Teng, Lesheng

    2016-01-01

    Cabazitaxel-loaded human serum albumin nanoparticles (Cbz-NPs) were synthesized to overcome vehicle-related toxicity of current clinical formulation of the drug based on Tween-80 (Cbz-Tween). A salting-out method was used for NP synthesis that avoids the use of chlorinated organic solvent and is simpler compared to the methods based on emulsion-solvent evaporation. Cbz-NPs had a narrow particle size distribution, suitable drug loading content (4.9%), and superior blood biocompatibility based on in vitro hemolysis assay. Blood circulation, tumor uptake, and antitumor activity of Cbz-NPs were assessed in prostatic cancer xenograft-bearing nude mice. Cbz-NPs exhibited prolonged blood circulation and greater accumulation of Cbz in tumors along with reduced toxicity compared to Cbz-Tween. Moreover, hematoxylin and eosin histopathological staining of organs revealed consistent results. The levels of blood urea nitrogen and serum creatinine in drug-treated mice showed that Cbz-NPs were less toxic than Cbz-Tween to the kidneys. In conclusion, Cbz-NPs provide a promising therapeutic for prostate cancer. PMID:27555767

  5. Cabazitaxel-loaded human serum albumin nanoparticles as a therapeutic agent against prostate cancer

    PubMed Central

    Qu, Na; Lee, Robert J; Sun, Yating; Cai, Guangsheng; Wang, Junyang; Wang, Mengqiao; Lu, Jiahui; Meng, Qingfan; Teng, Lirong; Wang, Di; Teng, Lesheng

    2016-01-01

    Cabazitaxel-loaded human serum albumin nanoparticles (Cbz-NPs) were synthesized to overcome vehicle-related toxicity of current clinical formulation of the drug based on Tween-80 (Cbz-Tween). A salting-out method was used for NP synthesis that avoids the use of chlorinated organic solvent and is simpler compared to the methods based on emulsion-solvent evaporation. Cbz-NPs had a narrow particle size distribution, suitable drug loading content (4.9%), and superior blood biocompatibility based on in vitro hemolysis assay. Blood circulation, tumor uptake, and antitumor activity of Cbz-NPs were assessed in prostatic cancer xenograft-bearing nude mice. Cbz-NPs exhibited prolonged blood circulation and greater accumulation of Cbz in tumors along with reduced toxicity compared to Cbz-Tween. Moreover, hematoxylin and eosin histopathological staining of organs revealed consistent results. The levels of blood urea nitrogen and serum creatinine in drug-treated mice showed that Cbz-NPs were less toxic than Cbz-Tween to the kidneys. In conclusion, Cbz-NPs provide a promising therapeutic for prostate cancer. PMID:27555767

  6. Quantification of total content of non-esterified fatty acids bound to human serum albumin.

    PubMed

    Pavićević, Ivan D; Jovanović, Vesna B; Takić, Marija M; Aćimović, Jelena M; Penezić, Ana Z; Mandić, Ljuba M

    2016-09-10

    Non-esterified fatty acids bound to the human serum albumin (HSA) contribute to several HSAs properties of special concern in pathologies, for instance to the reactivity of the free HSA-Cys34 thiol group (important antioxidative thiol pool in plasma), and to the affinity for binding of molecules and ions (for example cobalt as a prominent biomarker in heart ischemia). Therefore, the method for determination of FAs bound to HSA was developed. FAs were released from HSA (previously isolated from serum by ammonium sulfate precipitation) using acidic copper(II) sulfate in phosphoric acid, extracted by n-heptane-chloroform (4:1, v/v) mixture, spotted on TL silica-gel and then developed with n-heptane-chloroform-acetic acid (5:3:0.3, v/v/v). Common office flatbed scanner and software solution for densitometric image analysis, developed in R, were used. The linearity of calibration curve in concentration range from 0.1 to 5.0mmol/L stearic acid was achieved. The method was proved to be precise (with RSD of 1.4-4.7%) and accurate. Accuracy was examined by standard addition method (recoveries 97.2-102.5%) and by comparison to results of GC. The method is sample saving, technically less demanding, and cheap, and therefore suitable for determination of FAs/HSA ratio when elevated concentrations of free FAs are reliable diagnostic/risk parameter of pathological states.

  7. Quantification of total content of non-esterified fatty acids bound to human serum albumin.

    PubMed

    Pavićević, Ivan D; Jovanović, Vesna B; Takić, Marija M; Aćimović, Jelena M; Penezić, Ana Z; Mandić, Ljuba M

    2016-09-10

    Non-esterified fatty acids bound to the human serum albumin (HSA) contribute to several HSAs properties of special concern in pathologies, for instance to the reactivity of the free HSA-Cys34 thiol group (important antioxidative thiol pool in plasma), and to the affinity for binding of molecules and ions (for example cobalt as a prominent biomarker in heart ischemia). Therefore, the method for determination of FAs bound to HSA was developed. FAs were released from HSA (previously isolated from serum by ammonium sulfate precipitation) using acidic copper(II) sulfate in phosphoric acid, extracted by n-heptane-chloroform (4:1, v/v) mixture, spotted on TL silica-gel and then developed with n-heptane-chloroform-acetic acid (5:3:0.3, v/v/v). Common office flatbed scanner and software solution for densitometric image analysis, developed in R, were used. The linearity of calibration curve in concentration range from 0.1 to 5.0mmol/L stearic acid was achieved. The method was proved to be precise (with RSD of 1.4-4.7%) and accurate. Accuracy was examined by standard addition method (recoveries 97.2-102.5%) and by comparison to results of GC. The method is sample saving, technically less demanding, and cheap, and therefore suitable for determination of FAs/HSA ratio when elevated concentrations of free FAs are reliable diagnostic/risk parameter of pathological states. PMID:27394177

  8. A donor splice mutation and a single-base deletion produce two carboxyl-terminal variants of human serum albumin

    SciTech Connect

    Watkins, S.; Madison, J.; Davis, E.; Sakamoto, Yasushi; Putnam, F.W. ); Galliano, M.; Minchiotti, L. )

    1991-07-15

    At least 35 allelic variants of human serum albumin have been sequenced at the protein level. All except two COOH-terminal variants, Catania and Venezia, are readily explainable as single-point substitutions. The two chain-termination variants are clustered in certain locations in Italy and are found in numerous unrelated individuals. In order to correlate the protein change in these variants with the corresponding DNA mutation, the two variant albumin genes have been cloned, sequenced, and compared to normal albumin genomic DNA. In the Catania variant, a single base deletion and subsequent frameshift leads to a shortened and altered COOH terminus. Albumin Venezia is caused by a mutation that alters the first consensus nucleotide of the 5{prime} donor splice junction of intron 14 and the 3{prime} end of exon 14, which is shortened from 68 to 43 base pairs. This change leads to an exon skipping event resulting in direct splicing of exon 13 to exon 15. The predicted Venezia albumin product has a truncated amino acid sequence (580 residues instead of 585), and the COOH-terminal sequence is altered after Glu-571. The variant COOH terminus ends with the dibasic sequence Arg-Lys that is apparently removed through stepwise cleavage by serum carboxypeptidase B to yield several forms of circulating albumin.

  9. Methyl-triclosan binding to human serum albumin: multi-spectroscopic study and visualized molecular simulation.

    PubMed

    Lv, Wenjuan; Chen, Yonglei; Li, Dayong; Chen, Xingguo; Leszczynski, Jerzy

    2013-10-01

    Methyl-triclosan (MTCS), a transformation product and metabolite of triclosan, has been widely spread in environment through the daily use of triclosan which is a commonly used anti-bacterial and anti-fungal substance in consumer products. Once entering human body, MTCS could affect the conformation of human serum albumin (HSA) by forming MTCS-HSA complex and alter function of protein and endocrine in human body. To evaluate the potential toxicity of MTCS, the binding mechanism of HSA with MTCS was investigated by UV-vis absorption, circular dichroism and Fourier transform infrared spectroscopy. Binding constants, thermodynamic parameters, the binding forces and the specific binding site were studied in detail. Binding constant at room tempreture (T = 298K) is 6.32 × 10(3)L mol(-1); ΔH(0), ΔS(0) and ΔG(0) were 22.48 kJ mol(-1), 148.16 J mol(-1)K(-1) and -21.68 kJ mol(-1), respectively. The results showed that the interactions between MTCS and HSA are mainly hydrophobic forces. The effects of MTCS on HSA conformation were also discussed. The binding distance (r = 1.2 nm) for MTCS-HSA system was calculated by the efficiency of fluorescence resonance energy transfer. The visualized binding details were also exhibited by molecular modeling method and the results could agree well with that from the experimental study.

  10. Probing the interaction of a new synthesized CdTe quantum dots with human serum albumin and bovine serum albumin by spectroscopic methods.

    PubMed

    Bardajee, Ghasem Rezanejade; Hooshyar, Zari

    2016-05-01

    A novel CdTe quantum dots (QDs) were prepared in aqueous phase via a facile method. At first, poly (acrylic amide) grafted onto sodium alginate (PAAm-g-SA) were successfully synthesized and then TGA capped CdTe QDs (CdTe-TGA QDs) were embed into it. The prepared CdTe-PAAm-g-SA QDs were optimized and characterized by transmission electron microscopy (TEM), thermo-gravimetric (TG) analysis, Fourier transform infrared (FT-IR), UV-vis and fluorescence spectroscopy. The characterization results indicated that CdTe-TGA QDs, with particles size of 2.90 nm, were uniformly dispersed on the chains of PAAm-g-SA biopolymer. CdTe-PAAm-g-SA QDs also exhibited excellent UV-vis absorption and high fluorescence intensity. To explore biological behavior of CdTe-PAAm-g-SA QDs, the interactions between CdTe-PAAm-g-SA QDs and human serum albumin (HSA) (or bovine serum albumin (BSA)) were investigated by cyclic voltammetry, FT-IR, UV-vis, and fluorescence spectroscopic. The results confirmed the formation of CdTe-PAAm-g-SA QDs-HSA (or BSA) complex with high binding affinities. The thermodynamic parameters (ΔG<0, ΔH<0 and ΔS<0) were indicated that binding reaction was spontaneous and van der Waals interactions and hydrogen-bond interactions played a major role in stabilizing the CdTe-PAAm-g-SA QDs-HSA (or BSA) complexes. The binding distance between CdTe-PAAm-g-SA QDs and HSA (or BSA)) was calculated about 1.37 nm and 1.27 nm, respectively, according to Forster non-radiative energy transfer theory (FRET). Analyzing FT-IR spectra showed that the formation of QDs-HSA and QDs-BSA complexes led to conformational changes of the HSA and BSA proteins. All these experimental results clarified the effective transportation and elimination of CdTe-PAAm-g-SA QDs in the body by binding to HSA and BSA, which could be a useful guideline for the estimation of QDs as a drug carrier.

  11. Spontaneous transfer of stearic acids between human serum albumin and PEG:2000-grafted DPPC membranes.

    PubMed

    Pantusa, Manuela; Stirpe, Andrea; Sportelli, Luigi; Bartucci, Rosa

    2010-05-01

    Electron spin resonance (ESR) spectroscopy is used to study the transfer of stearic acids between human serum albumin (HSA) and sterically stabilized liposomes (SSL) composed of dipalmitoylphosphatidylcholine (DPPC) and of submicellar content of poly(ethylene glycol:2000)-dipalmitoylphosphatidylethanolamine (PEG:2000-DPPE). Protein/lipid dispersions are considered in which spin-labelled stearic acids at the 16th carbon atom along the acyl chain (16-SASL) are inserted either in the protein or in the SSL. Two component ESR spectra with different rotational mobility are obtained over a broad range of temperature and membrane composition. Indeed, superimposed to an anisotropic protein-signal, appears a more isotropic lipid-signal. Since in the samples only one matrix (protein or membranes) is spin-labelled, the other component accounts for the transfer of 16-SASL between albumin and membranes. The two components have been resolved and quantified by spectral subtractions, and the fraction, f (p) (16-SASL), of spin labels bound non-covalently to the protein has been used to monitor the transfer. It is found that it depends on the type of donor and acceptor matrix, on the physical state of the membranes and on the grafting density of the polymer-lipids. Indeed, it is favoured from SSL to HSA and the fraction of stearic acids transferred increases with temperature in both directions of transfer. Moreover, in the presence of polymer-lipids, the transfer from HSA to SSL is slightly attenuated, especially in the brush regime of the polymer-chains. Instead, the transfer from SSL to HSA is favoured by the polymer-lipids much more in the mushroom than in the brush regime. PMID:19350232

  12. Fluorescence and Docking Studies of the Interaction between Human Serum Albumin and Pheophytin.

    PubMed

    Chaves, Otávio Augusto; Amorim, Ana Paula de O; Castro, Larissa H E; Sant'Anna, Carlos Mauricio R; de Oliveira, Márcia C C; Cesarin-Sobrinho, Dari; Netto-Ferreira, José Carlos; Ferreira, Aurélio B B

    2015-01-01

    In the North of Brazil (Pará and Amazonas states) the leaves of the plant Talinum triangulare (popular: cariru) replace spinach as food. From a phytochemical point of view, they are rich in compounds of the group of pheophytins. These substances, related to chlorophyll, have photophysical properties that give them potential application in photodynamic therapy. Human serum albumin (HSA) is one of the main endogenous vehicles for biodistribution of molecules by blood plasma. Association constants and thermodynamic parameters for the interaction of HSA with pheophytin from Talinum triangulare were studied by UV-Vis absorption, fluorescence techniques, and molecular modeling (docking). Fluorescence quenching of the HSA's internal fluorophore (tryptophan) at temperatures 296 K, 303 K, and 310 K, resulted in values for the association constants of the order of 10⁴ L∙mol(-1), indicating a moderate interaction between the compound and the albumin. The negative values of ΔG° indicate a spontaneous process; ΔH° = 15.5 kJ∙mol(-1) indicates an endothermic process of association and ΔS° = 0.145 kJ∙mol(-1)∙K(-1) shows that the interaction between HSA and pheophytin occurs mainly by hydrophobic factors. The observed Trp fluorescence quenching is static: there is initial non-fluorescent association, in the ground state, HSA:Pheophytin. Possible solution obtained by a molecular docking study suggests that pheophytin is able to interact with HSA by means of hydrogen bonds with three lysine and one arginine residues, whereas the phytyl group is inserted in a hydrophobic pocket, close to Trp-214. PMID:26516829

  13. PEGylated human serum albumin (HSA) nanoparticles: preparation, characterization and quantification of the PEGylation extent

    NASA Astrophysics Data System (ADS)

    Fahrländer, E.; Schelhaas, S.; Jacobs, A. H.; Langer, K.

    2015-04-01

    Modification with poly(ethylene glycol) (PEG) is a widely used method for the prolongation of plasma half-life of colloidal carrier systems such as nanoparticles prepared from human serum albumin (HSA). However, the quantification of the PEGylation extent is still challenging. Moreover, the influence of different PEG derivatives, which are commonly used for nanoparticle conjugation, has not been investigated so far. The objective of the present study is to develop a method for the quantification of PEG and to monitor the influence of diverse PEG reagents on the amount of PEG linked to the surface of HSA nanoparticles. A size exclusion chromatography method with refractive index detection was established which enabled the quantification of unreacted PEG in the supernatant. The achieved results were confirmed using a fluorescent PEG derivative, which was detected by photometry and fluorimetry. Additionally, PEGylated HSA nanoparticles were enzymatically digested and the linked amount of fluorescently active PEG was directly determined. All the analytical methods confirmed that under optimized PEGylation conditions a PEGylation efficiency of up to 0.5 mg PEG per mg nanoparticle could be achieved. Model calculations made a ‘brush’ conformation of the PEG chains on the particle surface very likely. By incubating the nanoparticles with fetal bovine serum the reduced adsorption of serum proteins on PEGylated HSA nanoparticles compared to non-PEGylated HSA nanoparticles was demonstrated using sodium dodecylsulfate polyacrylamide gel electrophoresis. Finally, the positive effect of PEGylation on plasma half-life was demonstrated in an in vivo study in mice. Compared to unmodified nanoparticles the PEGylation led to a four times larger plasma half-life.

  14. Biophysical studies of interaction between hydrolysable tannins isolated from Oenothera gigas and Geranium sanguineum with human serum albumin.

    PubMed

    Sekowski, Szymon; Ionov, Maksim; Kaszuba, Mateusz; Mavlyanov, Saidmukhtar; Bryszewska, Maria; Zamaraeva, Maria

    2014-11-01

    Tannins, secondary plant metabolites, possess diverse biological activities and can interact with biopolymers such as lipids or proteins. Interactions between tannins and proteins depend on the structures of both and can result in changes in protein structure and activity. Because human serum albumin is the most abundant protein in plasma and responsible for interactions with important biological compounds (e.g. bilirubin) and proper blood pressure, therefore, it is very important to investigate reactions between HSA and tannins. This paper describes the interaction between human serum albumin (HSA) and two tannins: bihexahydroxydiphenoyl-trigalloylglucose (BDTG) and 1-O-galloyl-4,6-hexahydroxydiphenoyl-β-d-glucose (OGβDG), isolated from Geranium sanguineum and Oenothera gigas leafs, respectively. Optical (spectrofluorimetric) and chiral optical (circular dichroism) methods were used in this study. Fluorescence analysis demonstrated that OGβDG quenched HSA fluorescence more strongly than BDTG. Both OGβDG and BDTG formed complexes with albumin and caused a red shift of the fluorescence spectra but did not significantly change the protein secondary structure. Our studies clearly demonstrate that the tested tannins interact very strongly with human serum albumin (quenching constant K=88,277.26±407.04 M(-1) and K=55,552.67±583.07 M(-1) respectively for OGβDG and BDTG) in a manner depending on their chemical structure.

  15. Anion binding properties of human serum albumin from halide ion quadrupole relaxation.

    PubMed

    Norne, J E; Hjalmarsson, S G; Lindman, B; Zeppezauer, M

    1975-07-29

    The nuclear magnetic quadrupole relaxation enhancement of 35Cl-, 81Br-, and 12I- anions on binding to human serum albumin has been studied under conditions of variable protein and anion concentration and also in the presence of simple inorganic, amphiphilic, and complex anions which compete with the halide ions for the protein anion binding sites. Two classes of anion binding sites with greatly different binding constans were identified. Experiments at variable halide ion concentration were employed to determin the Cl- and I- binding constants. By means of 35 Cl nuclear magnetic resonance (NMR) the relative affinity for different anions was determined by competition experiments for both the strong and the weak anion binding sites. Anion binding follows the sequence SO42- smaller than F- smaller than CH3COO- smaller than Ci- smaller Br- smaller than NO3- smaller than I- smaller than ClO4- smaller than SCN- smaller than Pt(CN)42- smaller than Au(CN)2- smaller than CH3(CH2)11OSO3- for the high affinity sites, and the sequence SO42- congruent to F- congruent to Cl- smaller CH3COO- smaller than NO3- smaller than Br- smaller than I- smaller than ClO4- smaller than SCN- for the low affinity sites. These series are nearly identical with the well-known lyotropic series. Consequently, those effects of anions on proteins described by the lyotropic series can be correlated with the affinities of the anions for binding to the protein. The data suggest that the physical nature of the interaction is the same for both types of biding sites, and that the differences in affinity between different binding sites must be explained in terms of tertiary structure. Analogous experiments performed using 127I- quadrupole relaxation gave results very similar to those obtained with 35Cl-. A comparison between the Cl-, Br- and I- ions revealed that, as a result of the increasing affinity for the weak anion binding sites in the series Cl- smaller than Br- smaller than I-, Cl- is much more

  16. Ibuprofen Impairs Allosterically Peroxynitrite Isomerization by Ferric Human Serum Heme-Albumin*

    PubMed Central

    Ascenzi, Paolo; di Masi, Alessandra; Coletta, Massimo; Ciaccio, Chiara; Fanali, Gabriella; Nicoletti, Francesco P.; Smulevich, Giulietta; Fasano, Mauro

    2009-01-01

    Human serum albumin (HSA) participates in heme scavenging; in turn, heme endows HSA with myoglobin-like reactivity and spectroscopic properties. Here, the allosteric effect of ibuprofen on peroxynitrite isomerization to NO3− catalyzed by ferric human serum heme-albumin (HSA-heme-Fe(III)) is reported. Data were obtained at 22.0 °C. HSA-heme-Fe(III) catalyzes peroxynitrite isomerization in the absence and presence of CO2; the values of the second order catalytic rate constant (kon) are 4.1 × 105 and 4.5 × 105 m−1 s−1, respectively. Moreover, HSA-heme-Fe(III) prevents peroxynitrite-mediated nitration of free added l-tyrosine. The pH dependence of kon (pKa = 6.9) suggests that peroxynitrous acid reacts preferentially with the heme-Fe(III) atom, in the absence and presence of CO2. The HSA-heme-Fe(III)-catalyzed isomerization of peroxynitrite has been ascribed to the reactive pentacoordinated heme-Fe(III) atom. In the absence and presence of CO2, ibuprofen impairs dose-dependently peroxynitrite isomerization by HSA-heme-Fe(III) and facilitates the nitration of free added l-tyrosine; the value of the dissociation equilibrium constant for ibuprofen binding to HSA-heme-Fe(III) (L) ranges between 7.7 × 10−4 and 9.7 × 10−4 m. Under conditions where [ibuprofen] is ≫L, the kinetics of HSA-heme-Fe(III)-catalyzed isomerization of peroxynitrite is superimposable to that obtained in the absence of HSA-heme-Fe(III) or in the presence of non-catalytic HSA-heme-Fe(III)-cyanide complex and HSA. Ibuprofen binding impairs allosterically peroxynitrite isomerization by HSA-heme-Fe(III), inducing the hexacoordination of the heme-Fe(III) atom. These results represent the first evidence for peroxynitrite isomerization by HSA-heme-Fe(III), highlighting the allosteric modulation of HSA-heme-Fe(III) reactivity by heterotropic interaction(s), and outlining the role of drugs in modulating HSA functions. The present results could be relevant for the drug-dependent protective role

  17. Studies on the interaction between vincamine and human serum albumin: a spectroscopic approach.

    PubMed

    Pu, Hanlin; Jiang, Hua; Chen, Rongrong; Wang, Hongcui

    2014-08-01

    The interaction between vincamine (VCM) and human serum albumin (HSA) has been studied using a fluorescence quenching technique in combination with UV/vis absorption spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, circular dichroism (CD) spectroscopy and molecular modeling under conditions similar to human physiological conditions. VCM effectively quenched the intrinsic fluorescence of HSA via static quenching. The binding constants were calculated from the fluorescence data. Thermodynamic analysis by Van't Hoff equation revealed enthalpy change (ΔH) and entropy change (ΔS) were -4.57 kJ/mol and 76.26 J/mol/K, respectively, which indicated that the binding process was spontaneous and the hydrophobic interaction was the predominant force. The distance r between the donor (HSA) and acceptor (VCM) was obtained according to the Förster's theory of non-radiative energy transfer and found to be 4.41 nm. Metal ions, viz., Na(+), K(+), Li(+), Ni(2+), Ca(2+), Zn(2+) and Al(3+) were found to influence binding of the drug to protein. The 3D fluorescence, FT-IR and CD spectral results revealed changes in the secondary structure of the protein upon interaction with VCM. Furthermore, molecular modeling indicated that VCM could bind to the subdomain IIA (site I) of HSA.

  18. Potential toxicity of sulfanilamide antibiotic: binding of sulfamethazine to human serum albumin.

    PubMed

    Chen, Jiabin; Zhou, Xuefei; Zhang, Yalei; Gao, Haiping

    2012-08-15

    Antibiotics are widely used in daily life but their abuse has posed a potential threat to human health. The interaction between human serum albumin (HSA) and sulfamethazine (SMZ) was investigated by capillary electrophoresis, fluorescence spectrometry, and circular dichroism. The binding constant and site were determined to be 1.09×10(4) M(-1) and 1.14 at 309.5 K. The thermodynamic determination indicated that the interaction was driven by enthalpy change, where the electrostatic interaction and hydrogen bond were the dominant binding force. The binding distance between SMZ and tryptophan residue of HSA was obtained to be 3.07 nm according to Fǒrster non-radioactive energy transfer theory. The site marker competition revealed that SMZ bound into subdomain IIA of HSA. The binding of SMZ induced the unfolding of the polypeptides of HSA and transferred the secondary conformation of HSA. The equilibrium dialysis showed that only 0.13 mM SMZ decreased vitamin B(2) by 38% transported on the HSA. This work provides a new quantitative evaluation method for antibiotics to cause the protein damage. PMID:22750172

  19. HPLC separation of human serum albumin isoforms based on their isoelectric points.

    PubMed

    Turell, Lucía; Botti, Horacio; Bonilla, Lucía; Torres, María José; Schopfer, Francisco; Freeman, Bruce A; Armas, Larissa; Ricciardi, Alejandro; Alvarez, Beatriz; Radi, Rafael

    2014-01-01

    Human serum albumin (HSA) is the most abundant protein in plasma. Cys34, the only free Cys residue, is the predominant plasma thiol and a relevant sacrificial antioxidant. Both in vivo circulating HSA and pharmaceutical preparations are heterogeneous with respect to the oxidation state of Cys34. In this work, we developed an external pH gradient chromatofocusing procedure that allows the analysis of the oxidation status of HSA in human plasma and biopharmaceutical products based on the different apparent isoelectric points and chemical properties of the redox isoforms. Specifically, reduced-mercury blocked HSA (HSA-SHg(+)), HSA with Cys34 oxidized to sulfenic acid (HSA-SOH) and HSA oxidized to sulfinate anion (HSA-SO2(-)) can be separated with resolutions of 1.4 and 3.1 (first and last pair) and hence quantified and purified. In addition, an N-terminally degraded isoform (HSA3-585) in different redox states can be resolved as well. Confirmation of the identity of the chromatofocusing isolated isoforms was achieved by high resolution whole protein MS. It is proposed that the chromatofocusing procedure can be used to produce more exact and complete descriptions of the redox status of HSA in vivo and in vitro. Finally, the scalability capabilities of the chromatofocusing procedure allow for the preparation of highly pure standards of several redox isoforms of HSA.

  20. Binding of fullerol to human serum albumin: spectroscopic and electrochemical approach.

    PubMed

    Zhang, Mei-Fang; Xu, Zi-Qiang; Ge, Yu-Shu; Jiang, Feng-Lei; Liu, Yi

    2012-03-01

    The potential impact of human exposure to carbonaceous nanomaterials in the environment becomes a concerning issue. Here we report on the interaction of fullerol with human serum albumin (HSA) using spectroscopic and electrochemical methods. The water-soluble fullerene derivative (fullerol) was synthesized and characterized by IR, (1)H NMR, TG-DSC, XRD, HR-TEM, etc. The spectroscopic methods show that the fluorescence quenching of HSA by fullerol is the result of the formation of an HSA-fullerol complex. Binding parameters such as ΔG, ΔH and ΔS were calculated, and the quenching constant K(a) at different temperatures was determined using the modified Stern-Volmer equation. The electrochemical experiments further confirmed the conclusions. In addition, the influences of coexisting heavy metal ions have also been studied in the present system. The circular dichroism spectra (CD), 3D fluorescence spectra and FT-IR spectra results suggest that the secondary structure of HSA was changed by fullerol. Based on the site marker competitive experiments, we can predict the possible binding position of fullerol on the HSA was located at the site of sub domain II A. Furthermore, the distance r between donor (HSA) and acceptor (fullerol) was obtained according to the famous fluorescence resonance energy transfer (FRET) mechanism.

  1. HPLC separation of human serum albumin isoforms based on their isoelectric points.

    PubMed

    Turell, Lucía; Botti, Horacio; Bonilla, Lucía; Torres, María José; Schopfer, Francisco; Freeman, Bruce A; Armas, Larissa; Ricciardi, Alejandro; Alvarez, Beatriz; Radi, Rafael

    2014-01-01

    Human serum albumin (HSA) is the most abundant protein in plasma. Cys34, the only free Cys residue, is the predominant plasma thiol and a relevant sacrificial antioxidant. Both in vivo circulating HSA and pharmaceutical preparations are heterogeneous with respect to the oxidation state of Cys34. In this work, we developed an external pH gradient chromatofocusing procedure that allows the analysis of the oxidation status of HSA in human plasma and biopharmaceutical products based on the different apparent isoelectric points and chemical properties of the redox isoforms. Specifically, reduced-mercury blocked HSA (HSA-SHg(+)), HSA with Cys34 oxidized to sulfenic acid (HSA-SOH) and HSA oxidized to sulfinate anion (HSA-SO2(-)) can be separated with resolutions of 1.4 and 3.1 (first and last pair) and hence quantified and purified. In addition, an N-terminally degraded isoform (HSA3-585) in different redox states can be resolved as well. Confirmation of the identity of the chromatofocusing isolated isoforms was achieved by high resolution whole protein MS. It is proposed that the chromatofocusing procedure can be used to produce more exact and complete descriptions of the redox status of HSA in vivo and in vitro. Finally, the scalability capabilities of the chromatofocusing procedure allow for the preparation of highly pure standards of several redox isoforms of HSA. PMID:24316526

  2. Exploring the interactions of decabrominateddiphenyl ether and tetrabromobisphenol A with human serum albumin.

    PubMed

    Wang, Yan-Qing; Zhang, Hong-Mei; Cao, Jian

    2014-09-01

    Decabrominateddiphenyl ether (deca-BDE) and tetrabromobisphenol A (TBBPA) are known as brominated flame-retardants, which are commonly found in the environment. The binding mechanisms of deca-BDE and TBBPA with human serum albumin (HSA) are still unknown. In this report, the interactions of deca-BDE and TBBPA with HSA were investigated using different spectroscopic methods and molecular modeling. The experimental results indicated the formation of complexes between deca-BDE/TBBPA and HSA with different affinity. These interactions affected the secondary structure of HSA. Thermodynamic investigations revealed that hydrophobic forces mainly drove the binding interactions of deca-BDE/TBBPA with HSA. For TBBPA, hydrogen-bonding interactions were also involved in the binding process of TBBPA with HSA. According to the analysis of experimental and theoretical data, we concluded that the binding site of deca-BDE to HSA located in the subdomain IB, while TBBPA was near to subdomain IIA and Trp-214. The binding interactions of deca-BDE and TBBPA with the most prominent carrier protein in the human circulatory system could influence mechanisms of their biochemical processes. Thus, these binding interactions can play central roles in studying the distribution and toxicity mechanisms of brominated flame-retardants. PMID:25194328

  3. Interaction of oridonin with human serum albumin by isothermal titration calorimetry and spectroscopic techniques.

    PubMed

    Li, Xiangrong; Yang, Zhenhua

    2015-05-01

    Oridonin has been traditionally and widely used for treatment of various human diseases due to its uniquely biological, pharmacological and physiological functions. In this study, the interaction between oridonin and human serum albumin (HSA) was investigated using isothermal titration calorimetry (ITC), in combination with fluorescence spectroscopy and UV-vis absorption spectroscopy. We found that the hydrogen bond and van der Waals force are the major binding forces in the binding of oridonin to HSA. The binding of oridonin to HSA is driven by favorable enthalpy and unfavorable entropy. Oridonin can quench the fluorescence of HSA through a static quenching mechanism. The binding constant between oridonin and HSA is moderate and the equilibrium fraction of unbound oridonin f(u) > 60%. Binding site I is found to be the primary binding site for oridonin. Additionally, oridonin may induce conformational changes of HSA and affect its biological function as the carrier protein. The results of the current study suggest that oridonin can be stored and transported from the circulatory system to reach its target organ to provide its therapeutic effects. But its side-effect in the clinics cannot be overlook. The study provides an accurate and full basic data for clarifying the binding mechanism of oridonin with HSA and is helpful for understanding its effect on protein function during the blood transportation process and its biological activity in vivo.

  4. Study the interactions between human serum albumin and two antifungal drugs: fluconazole and its analogue DTP.

    PubMed

    Zhang, Shao-Lin; Yao, Huankai; Wang, Chenyin; Tam, Kin Y

    2014-11-01

    Binding affinities of fluconazole and its analogue 2-(2,4-dichlorophenyl)-1,3-di(1H-1,2,4-triazol-yl)-2-propanol (DTP) to human serum albumin (HSA) were investigated under approximately human physiological conditions. The obtained result indicated that HSA could generate fluorescent quenching by fluconazole and DTP because of the formation of non-fluorescent ground-state complexes. Binding parameters calculated from the Stern-Volmer and the Scatchard equations showed that fluconazole and DTP bind to HSA with binding affinities of the order 10(4)L/mol. The thermodynamic parameters revealed that the binding was characterized by negative enthalpy and positive entropy changes, suggesting that the binding reaction was exothermic. Hydrogen bonds and hydrophobic interaction were found to be the predominant intermolecular forces stabilizing the drug-protein. The effect of metal ions on the binding constants of fluconazole-HSA complex suggested that the presence of Mg(2+) and Zn(2+) ions could decrease the free drug level and extend the half-life in the systematic circulation. Docking experiments revealed that fluconazole and DTP binds in HSA mainly by hydrophobic interaction with the possibility of hydrogen bonds formation between the drugs and the residues Arg 222, Lys 199 and Lys 195 in HSA.

  5. Co-amoxiclav Effects on the Structural and Binding Properties of Human Serum Albumin

    PubMed Central

    Hesami Takallu, Saeed; Rezaei Tavirani, Mostafa; Kalantari, Shiva; Amir Bakhtiarvand, Mahrooz; Mahdavi, Sayed Mohammad

    2010-01-01

    Human serum albumin (HSA) is the most abundant plasma protein in the human body. HSA plays an important role in drug transport and metabolism. This protein has a high affinity to a very wide range of materials, including metals such as Cu2+ and Zn2+, fatty acids, amino acids and metabolites such as bilirubin and many drug compounds. In this study, we investigated the effects of co-amoxiclav, as a drug which could be carried by this protein, on HSA structure and binding properties via spectroscopy and electrochemistry techniques. Based on this study, it was found that a therapeutic dose of co-amoxiclav as well as doses 4 to 8 folds higher than the therapeutic dose has no considerable effect on the HSA tertiary structure at 37oC. However, a dose 2 folds that of the therapeutic dose has a slight effect, but higher doses of the drug has a mild effect in pathological temperature (42oC). In addition, charge density of HSA surface is decreased at 42oC, compared to 37oC. Hence, this finding suggests a reduced role of HSA in regulation of osmotic pressure in the fever conditions, compared to the physiological conditions. Co-amoxiclav reduces the charge surface density of HSA at physiological and pathological temperatures and therefore alters its binding properties, which could be important in drug interference and complications. PMID:24363734

  6. Human Serum Albumin Conjugates of 7-Ethyl-10-hydroxycamptothecin (SN38) for Cancer Treatment

    PubMed Central

    Sepehri, Nima; Rouhani, Hasti; Gharghabi, Mehdi; Tavassolian, Faranak; Amini, Mohsen; Ostad, Seyed Nasser

    2014-01-01

    SN38 (7-ethyl-10-hydroxy-comptothecin) is a potent metabolite of irinotecan, which has been approved for treatment of metastatic colorectal cancer. Considering the notable potency of SN38, it has been introduced as an anticancer candidate. In this study, human serum albumin (HSA) conjugates of SN38 were formulated to overcome the solubility problem beside improving the active form stability and tumor tissue targeting. In this target, two different molar ratios of conjugates (SN38 : HSA 15 : 1 and 60 : 1) were prepared by derivatization of 20-hydroxyl group of SN38 with glycine, followed by addition of succinyl group to glycine through which HSA was covalently attached. The conjugates with particle size of about 100 nm revealed enhanced water solubility and were relatively stable in neutral and acidic solutions. For SN38-HSA-15 and SN38-HSA-60 IC50 values were compared with irinotecan in HT-29 human colon cancer cells. Furthermore, biodistribution studies of SN38-HSA conjugate resulted in proper blood concentration level within 4 h. Moreover, blood cytotoxicity assay revealed no toxicity effect on liver and spleen. Collectively, our present investigation offers a water-soluble form of SN38 attached to HSA and suggests using favorable properties as a promising anticancer agent for further preclinical and clinical investigations. PMID:24895635

  7. Effect of (-)-epigallocatechin-3-gallate on glucose-induced human serum albumin glycation.

    PubMed

    Li, M; Hagerman, A E

    2015-01-01

    (-)-Epigallocatechin-3-gallate (EGCg) is a naturally occurring polyphenol found in plant-based foods and beverages such as green tea. Although EGCg can eliminate carbonyl species produced by glucose autoxidation and thus can inhibit protein glycation, it is also reported to be a pro-oxidant that stimulates protein glycation in vitro. To better understand the balance between antioxidant and pro-oxidant features of EGCg, we evaluated EGCg-mediated bioactivities in a human serum albumin (HSA)/glucose model by varying three different parameters (glucose level, EGCg concentration, and time of exposure to EGCg). Measurements of glycation-induced fluorescence, protein carbonyls, and electrophoretic mobility showed that the level of HSA glycation was positively related to the glucose level over the range 10-100 mM during a 21-day incubation at 37°C and pH: 7.4. Under mild glycemic pressure (10 mM), long exposure to EGCg enhanced HSA glycation, while brief exposure to low concentrations of EGCg did not. Under high glycemic pressure (100 mM glucose), long exposure to EGCg inhibited glycation. For the first time we showed that brief exposure to EGCg reversed glycation-induced fluorescence, indicating a restorative effect. In conclusion, our research identified glucose level, EGCg concentration, and time of exposure as critical factors dictating EGCg bioactivities in HSA glycation. EGCg did not affect HSA glycation under normal physiological conditions but had a potential therapeutic effect on HSA severely damaged by glycation.

  8. Conformational changes in human serum albumin induced by sodium perfluorooctanoate in aqueous solutions.

    PubMed

    Messina, Paula V; Prieto, Gerardo; Ruso, Juan M; Sarmiento, Félix

    2005-08-18

    Conformational changes in the bulk solution and at the air-aqueous interface of human serum albumin (HSA) induced by changes in concentration of sodium perfluorooctanoate (C(7)F(15)COO(-)Na(+)) were studied by difference spectroscopy, zeta-potential data, and axisymmetric drop shape analysis. zeta-potential was used to monitor the formation of the HSA-C(7)F(15)COO(-)Na(+) complex and the surface charge of the complex. The conformational transition of HSA in the bulk solution was followed as a function of denaturant concentration by absorbance measurements at 280 nm. The data were analyzed to obtain values for the Gibbs energies of the transition in water (DeltaG(0)(W)) and in a hydrophobic environment (DeltaG(0)(hc)) pertaining to saturated protein-surfactant complexes. The conformational changes that surfactants induce in HSA molecules alter its absorption behavior at the air-water interface. Dynamic surface measurements were used to evaluate this behavior. At low [C(7)F(15)COO(-)Na(+)], proteins present three adsorption regimes: induction time, monolayer saturation, and interfacial gelation. When surfactant concentration increases and conformational changes in the bulk solution occur, the adsorption regimes disappear. HSA molecules in an intermediate conformational state migrate to the air-water interface and form a unique monolayer. At high [C(7)F(15)COO(-)Na(+)], the adsorption of denatured molecules exhibits a behavior analogous to that of dilute solutions.

  9. Adsorption of an amphiphilic penicillin onto human serum albumin: characterisation of the complex.

    PubMed

    Ruso, J M; Taboada, P; Varela, L M; Attwood, D; Mosquera, V

    2001-08-30

    The complex formed by the interaction of the amphiphilic penicillin drug nafcillin and human serum albumin (HSA) in water at 25 degrees C has been characterised using a range of physicochemical techniques. Measurements of the solution conductivity and the electrophoretic mobility of the complexes have shown an ionic adsorption of the drug on the protein surface leading to a surface saturation at a nafcillin concentration of 0.012 mmol kg(-1) and subsequent formation of drug micelles in solutions of higher nafcillin concentration. Measurements of the size of the complex and the thickness of the adsorbed layer by static and dynamic light scattering have shown a gradual change in hydrodynamic radius of the complex with increasing drug concentration typical of a saturation rather than a denaturation process, the magnitude of the change being insufficient to account for any appreciable extension or unfolding of the HSA molecule. The interaction potential between the HSA/nafcillin complexes, and the stability of the complexes were determined from the dependence of diffusion coefficients on protein concentration by application of the DLVO colloidal stability theory. The results indicate decreasing stability of the colloidal dispersion of the drug/protein complexes with an increase in the concentration of added drug.

  10. Investigation the interaction of Daphnin with human serum albumin using optical spectroscopy and molecular modeling methods

    NASA Astrophysics Data System (ADS)

    Zhu, Jinhua; Wu, Liye; Zhang, Qingyou; Chen, Xingguo; Liu, Xiuhua

    2012-09-01

    The interaction between Daphnin with human serum albumin has been studied for the first time by spectroscopic methods including fluorescence quenching technology, circular dichroism (CD) spectroscopy and Fourier transform infrared (FT-IR) spectroscopy under simulative physiological conditions. The results of fluorescence titration revealed that Daphnin can quench the intrinsic fluorescence of HSA by static quenching and there is a single class of binding site on HSA. In addition, the studies of CD spectroscopy and FT-IR spectroscopy showed that the protein secondary structure changed with increases of α-helices at the drug to protein molar ratio of 2. Furthermore, the thermodynamic functions ΔH0 and ΔS0 for the reaction were calculated to be 11.626 kJ mol-1 and 118.843 J mol-1 K-1 according to Van't Hoff equation. The thermodynamic parameters (ΔH0 and ΔS0) and the molecular modeling study indicated that hydrophobic force played an important role to stabilize the Daphnin-HSA complex, and Daphnin could bind within the subdomain IIA of the HSA.

  11. Binding and relaxometric properties of heme complexes with cyanogen bromide fragments of human serum albumin.

    PubMed Central

    Monzani, Enrico; Curto, Maria; Galliano, Monica; Minchiotti, Lorenzo; Aime, Silvio; Baroni, Simona; Fasano, Mauro; Amoresano, Angela; Salzano, Anna Maria; Pucci, Piero; Casella, Luigi

    2002-01-01

    The spectroscopic and reactivity properties of hemin complexes formed with cyanogen bromide fragments B (residues 1-123), C (124-298), A (299-585), and D (1-298) of human serum albumin (HSA) have been investigated. The complex hemin-D exhibits binding, spectral, circular dichroism, and reactivity characteristics very similar to those of hemin-HSA, indicating that fragment D contains the entire HSA domain involved in heme binding. The characteristics of the other hemin complexes are different, and a detailed investigation of the properties of hemin-C has been carried out because this fragment contains the HSA binding region of several important drugs. Hemin-C contains a low-spin Fe(III) center, with two imidazole ligands, but the complex undergoes a reversible structural transition at basic pH leading to a high-spin, five-coordinated Fe(III) species. This change determines a marked increase in the relaxation rate of water protons. Limited proteolysis experiments and mass spectral analysis carried out on fragment C and hemin-C show that the region encompassing residues Glu-208 to Trp-214 is protected from activity of proteases in the complex and, therefore, is involved in the interaction with hemin. A structural model of fragment C enables us to propose that His-242 and His-288 are the axial ligands for the Fe(III) center. PMID:12324442

  12. (99m)Tc-human serum albumin nanocolloids: particle sizing and radioactivity distribution.

    PubMed

    Persico, Marco G; Lodola, Lorenzo; Buroni, Federica E; Morandotti, Marco; Pallavicini, Piersandro; Aprile, Carlo

    2015-07-01

    Several parameters affect the biodistribution of administered nanocolloids (NC) for Sentinel Lymph Node (SLN) detection: particle size distribution, number of Tc atoms per particle and specific activity (SA). Relatively few data are available with frequently conflicting results. (99m)Tc-NC-human serum albumin (HSA) Nanocoll®, Nanoalbumon® and Nanotop® were analysed for particles' dimensional and radioactivity distribution, and a mathematical model was elaborated to estimate the number of particles involved. Commercially available kits were reconstituted at maximal SA of 11 MBq/µg HSA. Particles size distribution was evaluated by Dynamic Light Scattering. These data were related to the radioactivity distribution analysis passing labelled NC through three polycarbonate filters (15-30-50-nm pore size) under vacuum. Highest radioactivity was carried by 30-50 nm particles. The smallest ones, even though most numerous, carried only the 10% of (99m)Tc atoms. Nanocoll and Nanotop are not significantly different, while Nanoalbumon is characterized by largest particles (>30 nm) that carried the most of radioactivity (80%). Smallest particles could saturate the clearing capacity of macrophages; therefore, if the tracer is used for SLN detection, more node tiers could be visualized, reducing accuracy of SLN mapping. Manufacturers could implement technical leaflets with particle size distribution and could improve the labelling protocol to provide clinicians useful information.

  13. [The interaction of vitamin B6 with the human serum albumin].

    PubMed

    Wang, Jian-lin; Fu, Lian-chun; Zhou, Shi-wu; Chen, Zhi-jian; Lu, Wen-bo; Ye, Xue-min; Meng, Guang-zheng; Song, Zeng-fu

    2005-06-01

    The Human Serum Albumin (HSA) can emit fluorescence (lamda(em) = 350 nm) under irradiation of ultraviolet light (lamda(ex) = 296 nm). After the vitamin B6 (B6) was added into HSA solution the fluorescence of HSA was quenched partially. This quenching was static quenching owing to the fact that ro (fluorescence life time (life time of the molecule excitation state) of HSA without B6) equaled to tau(i) (fluorescence life time of HSA with B6). The formation constant K between HSA and B6 was observed from the experiment, and then the binding distance R0 was determined (R0 = 1.872 nm). The CD spectra of samples (HSA, [HSA] = 1 x 10(-5) mol x L(-1), HSA +B6, [B6] = 5 x 10(-8), 1.5 x 10(-7), 2.5 x 10(-7), 3.5x 10(-7), 4.5x 10(-7) mol x L(-1)) were measured, and all CD spectra were almost the same. From [theta] values, the contents (percent content) of four structure(alpha-Helix, beta-Pleated, Beta-corner, and Randon wind) of samples can be calculated, and we find that all samples contain almost the same structure contents. PMID:16201370

  14. Spectroscopy and Molecular Modeling Study on Binding of Nickel Phthalocyanine to Human Serum Albumin.

    PubMed

    Dezhampanah, Hamid; Firouzi, Roghaye; Hasani, Leila

    2016-01-01

    The interaction of nickel tetra sulfunated phthalocyanine( NiTSPc) with human serum albumin (HSA), in 20 mM phosphate buffer pH 7.4 was investigated using advanced techniques including fluorescence, synchronous fluorescence, Fourier transform infrared (FT-IR), circular dichroism (CD) spectroscopy and molecular docking. The fluorescence quenching measurements showed a single binding site on HSA for NiTSPc with the binding constant (Kb) value equals to 1.26×106 at 25°C. The results showed that quenching mechanism of HSA by NiTSPc was of dynamic type. The results from FTIR and CD spectroscopies demonstrated that NiTSPc binds to amino acid residues of the main polypeptide chain in protein destroying the hydrogen bonding network. The corresponding thermodynamic parameters were then calculated by analysis of fluorescence data using van't Hoff plot. These data indicated that driving force for interaction was mainly hydrophobic in nature and the process was entropy driven. The information obtained from CD, FT-IR and synchronous fluorescence spectroscopies revealed that both microenvironment and conformation of HSA was changed. Molecular docking study confirmed the binding mode obtained by experimental data. PMID:27449940

  15. [First pass effect by infusing 99mTc-human serum albumin into the hepatic artery].

    PubMed

    Ozawa, T; Kimura, K; Koyanagi, Y; Aoki, T; Kusama, M; Takagi, S; Kakuta, T; Yoshimatsu, A; Ishikawa, M; Yasuda, D

    1988-08-01

    The fundamental principles of intra-arterial infusion chemotherapy are thought to be increased local drug concentration and the "first-pass" effect. The concentration in the rest of the body can only be decreased if there is local elimination of the infused drug before reaching the systemic circulation. This is referred to as the "first-pass" effect. In the evaluation of "first-pass" effect, the uptake of liver after infusing 99mTc-human serum albumin (99mTc-HSA) in the hepatic artery by injecting the subcutaneously implanted silicon reservoir was compared with that obtained after intravenous administration of 99mTc-HSA. In order to remove the factor of portal infusion, each count of liver up take had been continued for only 24 seconds after starting the liver uptake. The results are as follows: for 24 cases excepting 6 cases with catheter obstruction, the mean i.a./i.v. ratio was 7.92 +/- 3.34 (range 3.25 to 17.25). Although the elimination rate of drugs in the liver varies with each drug, the infusion of intraarterial chemotherapy should be about 8 times more concentrative than intravenous administration on the "first-pass" effect.

  16. Cys34-PEGylated Human Serum Albumin for Drug Binding and Delivery

    PubMed Central

    Mehtala, Jonathan G.; Kulczar, Chris; Lavan, Monika; Knipp, Gregory; Wei, Alexander

    2015-01-01

    Polyethylene glycol (PEG) derivatives were conjugated onto the Cys-34 residue of human serum albumin (HSA) to determine their effects on the solubilization, permeation, and cytotoxic activity of hydrophobic drugs such as paclitaxel (PTX). PEG(C34)HSA conjugates were prepared on a multigram scale by treating native HSA (n-HSA) with 5- or 20-kDa mPEG-maleimide, resulting in up to 77% conversion of the mono-PEGylated adduct. Nanoparticle tracking analysis of PEG(C34)HSA formulations in phosphate buffer revealed an increase in nanosized aggregates relative to n-HSA, both in the absence and presence of PTX. Cell viability studies conducted with MCF-7 breast cancer cells indicated that PTX cytotoxicity was enhanced by PEG(C34)HSA when mixed at 10:1 mole ratios, up to a two-fold increase in potency relative to n-HSA. The PEG(C34)HSA conjugates were also evaluated as PTX carriers across monolayers of HUVEC and hCMEC/D3 cells, and found to have nearly identical permeation profiles as n-HSA. PMID:25918947

  17. A Comprehensive Computational Study of the Interaction between Human Serum Albumin and Fullerenes.

    PubMed

    Leonis, Georgios; Avramopoulos, Aggelos; Papavasileiou, Konstantinos D; Reis, Heribert; Steinbrecher, Thomas; Papadopoulos, Manthos G

    2015-12-01

    Human serum albumin (HSA) is the most abundant blood plasma protein, which transports fatty acids, hormones, and drugs. We consider nanoparticle-HSA interactions by investigating the binding of HSA with three fullerene analogs. Long MD simulations, quantum mechanical (fragment molecular orbital, energy decomposition analysis, atoms-in-molecules), and free energy methods elucidated the binding mechanism in these complexes. Such a systematic study is valuable due to the lack of comprehensive theoretical approaches to date. The main elements of the mechanism include the following: binding to IIA site results in allosteric modulation of the IIIA and heme binding sites with an increase in α-helical structure of IIIA. Fullerenes displayed high binding affinities for HSA; therefore, HSA can be used as a fullerene carrier, facilitating any toxic function the fullerene may exert. Complex formation is driven by hydrogen bonding, van der Waals, nonpolar, charge transfer, and dispersion energy contributions. Proper functionalization of C60 has enhanced its binding to HSA by more than an order of magnitude. This feature may be important for biological applications (e.g., photodynamic therapy of cancer). Satisfactory agreement with relevant experimental and theoretical data has been obtained.

  18. Computational studies of the binding mechanisms of fullerenes to human serum albumin.

    PubMed

    Li, Jinyu; Jiang, Lizhi; Zhu, Xiaolei

    2015-07-01

    Fullerene and its derivatives show promising prospects for applications in a vast array of biological systems. A key aspect concerning their biomedical applications is how they interact with proteins from molecular levels, which is still poorly understood. In the current study, we investigated the structural and thermodynamic basis of the interactions between two pharmacologically relevant fullerene derivatives and human serum albumin (HSA) using molecular docking, molecular dynamics simulations, and binding free energy calculations. Our results demonstrate that fullerenes steadily bind with HSA at the interfacial cavity formed by subdomains IIA and IIIA. In agreement with available experimental data, our simulations show that the global structure of HSA becomes more compact in the presence of fullerene, while local structural dynamics of the binding cavity behaves diversely depending on the chemical properties of bound fullerenes. Binding free energy calculations confirmed that the interactions between fullerenes and HSA are dominantly stabilized by van der Waals forces and they further allowed the identification of key residues involved in fullerene binding. The structural and energetic insights obtained from this work may help for the development of fullerene-based drug delivery devices and therapeutic agents with improved biological profile.

  19. Quantitation of the residual DNA from rice-derived recombinant human serum albumin.

    PubMed

    Chen, Zhen; Dai, Huixia; Liu, Zhenwei; Zhang, Liping; Pang, Jianlei; Ou, Jiquan; Yang, Daichang

    2014-04-01

    Residual DNA in recombinant protein pharmaceuticals can potentially cause safety issues in clinical applications; thus, maximum residual limit has been established by drug safety authorities. Assays for residual DNA in Escherichia coli, yeast, and Chinese hamster ovary (CHO) cell expression systems have been established, but no rice residual DNA assay for rice expression systems has been designed. To develop an assay for the quantification of residual DNA that is produced from rice seed, we established a sensitive assay using quantitative real-time polymerase chain reaction (qPCR) based on the 5S ribosomal RNA (rRNA) genes. We found that a 40-cycle qPCR exhibited a linear response when the template concentration was in the range of 2×10(4) to 0.2pg of DNA per reaction in TaqMan and SYBR Green I assays. The amplification efficiency was 103 to 104%, and the amount of residual DNA from recombinant human serum albumin from Oryza sativa (OsrHSA) was less than 3.8ng per dosage, which was lower than that recommended by the World Health Organization (WHO). Our results indicate that the current purification protocol could efficiently remove residual DNA during manufacturing and processing. Furthermore, this protocol could be viable in other cereal crop endosperm expression systems for developing a residual DNA quantitation assay using the highly conserved 5S rRNA gene of the crops.

  20. Novel self-assembly endows human serum albumin nanoparticles with an enhanced antitumor efficacy.

    PubMed

    Ding, Dawei; Tang, Xiaolei; Cao, Xiaoli; Wu, Jinhui; Yuan, Ahu; Qiao, Qian; Pan, Jing; Hu, Yiqiao

    2014-02-01

    Protein-based nanomedicine plays an important role in tumor chemotherapy due to their merits in bioavailability, biocompatibility, biodegradability, and low toxicity. In this study, we developed a novel method of preparing human serum albumin (HSA) nanoparticles for targeted delivery of paclitaxel (PTX) to tumors. HSA-PTX nanoparticles (NPs-PTX) were fabricated via unfolding of HSA in appropriate solution to expose more hydrophobic domains and consequent self-assembling into nanoparticles with added PTX. Via this self-assembly method, a desirable particle size (around 120 nm), a high drug loading (>20%), and a high encapsulation efficiency (near 100%) were obtained. PTX dispersed as an amorphous state in NPs-PTX and the secondary structures of HSA were maintained. In a cytotoxicity study, NPs-PTX displayed an enhanced cytotoxicity in MCF-7 and A549 cells. Confocal microscopy and flow cytometry revealed that the uptake of NPs-PTX was mediated by secreted protein acidic and rich in cysteine and "caveolar" transport. In H22 tumor-bearing mice, NPs-PTX displayed an increasing and everlasting tumor distribution, leading to slower tumor growth and longer mice survival than PTX. Therefore, this novel self-assembly method offers a much easier method to prepare PTX nanoparticles, provides better antitumor efficacy in vitro and in vivo, and more importantly, sets up a delivery platform for other hydrophobic drugs to improve their effectiveness in cancer therapy.

  1. Caffeine and sulfadiazine interact differently with human serum albumin: A combined fluorescence and molecular docking study

    NASA Astrophysics Data System (ADS)

    Islam, Mullah Muhaiminul; Sonu, Vikash K.; Gashnga, Pynsakhiat Miki; Moyon, N. Shaemningwar; Mitra, Sivaprasad

    2016-01-01

    The interaction and binding behavior of the well-known drug sulfadiazine (SDZ) and psychoactive stimulant caffeine (CAF) with human serum albumin (HSA) was monitored by in vitro fluorescence titration and molecular docking calculations under physiological condition. The quenching of protein fluorescence on addition of CAF is due to the formation of protein-drug complex in the ground state; whereas in case of SDZ, the experimental results were explained on the basis of sphere of action model. Although both these compounds bind preferentially in Sudlow's site 1 of the protein, the association constant is approximately two fold higher in case of SDZ (∼4.0 × 104 M-1) in comparison with CAF (∼9.3 × 102 M-1) and correlates well with physico-chemical properties like pKa and lipophilicity of the drugs. Temperature dependent fluorescence study reveals that both SDZ and CAF bind spontaneously with HSA. However, the binding of SDZ with the protein is mainly governed by the hydrophobic forces in contrast with that of CAF; where, the interaction is best explained in terms of electrostatic mechanism. Molecular docking calculation predicts the binding of these drugs in different location of sub-domain IIA in the protein structure.

  2. Exploring binding properties of sertraline with human serum albumin: Combination of spectroscopic and molecular modeling studies.

    PubMed

    Shahlaei, Mohsen; Rahimi, Behnoosh; Nowroozi, Amin; Ashrafi-Kooshk, Mohammad Reza; Sadrjavadi, Komail; Khodarahmi, Reza

    2015-12-01

    Human serum albumin (HSA)-drug binding is an important factor to determine half life and bioavailability of drugs. In the present research, the interaction of sertraline (SER) to HSA was investigated using combination of spectroscopic and molecular modeling techniques. Changes in the UV-Vis, CD and FT-IR spectra as well as a significant degree of tryptophan fluorescence quenching were observed upon SER-HSA interaction. Data obtained by spectroscopic methods along with the computational studies suggest that SER binds to residues located in subdomain IIA of HSA. Analysis of spectroscopic data represented the formation of 1:1 complex, significant binding affinity, negative values of entropy and enthalpy changes and the essential role of hydrophobic interactions in binding of SER to HSA. The binding models were demonstrated in the aspects of SER's conformation, active site interactions, important amino acids and hydrogen bonding. Computational mapping of the possible binding site of SER confirmed that the ligand to be bound in a large hydrophobic cavity of HSA. In accordance with experimental data, computational analyses indicated that SER binding does not alter the secondary structure of the protein. The results not only lead to a better understanding of interaction between SER and HSA but also provide useful data about the influence of SER on the protein conformation. PMID:26471709

  3. Study of interaction between human serum albumin and three phenanthridine derivatives: fluorescence spectroscopy and computational approach.

    PubMed

    Liu, Jianming; Yue, Yuanyuan; Wang, Jing; Yan, Xuyang; Liu, Ren; Sun, Yangyang; Li, Xiaoge

    2015-06-15

    Over the past decades, phenanthridine derivatives have captured the imagination of many chemists due to their wide applications. In the present work, the interaction between phenanthridine derivatives benzo [4,5]imidazo[1,2-a]thieno[2,3-c]quinoline (BTQ), benzo[4,5]imidazo[1,2-a]furo[2,3-c]quinoline (BFQ), 5,6-dimethylbenzo[4,5]imidazo[1,2-a]furo[2,3-c]quinoline (DFQ) and human serum albumin (HSA) were investigated by molecular modeling techniques and spectroscopic methods. The results of molecular modeling simulations revealed that the phenanthridine derivatives could bind on both site I in HSA. Fluorescence data revealed that the fluorescence quenching of HSA by phenanthridine derivatives were the result of the formation of phenanthridine derivatives-HSA complex, and the binding intensity between three phenanthridine derivatives and HSA was BTQ>BFQ>DFQ. Thermodynamics confirmed that the interaction were entropy driven with predominantly hydrophobic forces. The effects of some biological metal ions and toxic ions on the binding affinity between phenanthridine derivatives and HSA were further examined.

  4. Study of interaction between human serum albumin and three phenanthridine derivatives: Fluorescence spectroscopy and computational approach

    NASA Astrophysics Data System (ADS)

    Liu, Jianming; Yue, Yuanyuan; Wang, Jing; Yan, Xuyang; Liu, Ren; Sun, Yangyang; Li, Xiaoge

    2015-06-01

    Over the past decades, phenanthridine derivatives have captured the imagination of many chemists due to their wide applications. In the present work, the interaction between phenanthridine derivatives benzo [4,5]imidazo[1,2-a]thieno[2,3-c]quinoline (BTQ), benzo[4,5]imidazo[1,2-a]furo[2,3-c]quinoline (BFQ), 5,6-dimethylbenzo[4,5]imidazo[1,2-a]furo[2,3-c]quinoline (DFQ) and human serum albumin (HSA) were investigated by molecular modeling techniques and spectroscopic methods. The results of molecular modeling simulations revealed that the phenanthridine derivatives could bind on both site I in HSA. Fluorescence data revealed that the fluorescence quenching of HSA by phenanthridine derivatives were the result of the formation of phenanthridine derivatives-HSA complex, and the binding intensity between three phenanthridine derivatives and HSA was BTQ > BFQ > DFQ. Thermodynamics confirmed that the interaction were entropy driven with predominantly hydrophobic forces. The effects of some biological metal ions and toxic ions on the binding affinity between phenanthridine derivatives and HSA were further examined.

  5. Enhancing the copper(II) complexes cytotoxicity to cancer cells through bound to human serum albumin.

    PubMed

    Gou, Yi; Zhang, Yao; Qi, Jinxu; Zhou, Zuping; Yang, Feng; Liang, Hong

    2015-03-01

    We use Schiff-base salicylaldehyde benzoylhydrazone (HL) as the ligand for copper(II), resulting in the complexes [CuCl(L)]·H2O (C1), [CuNO3(L)]·H2O (C2) and [CuBr(L)]2 (C3). We characterize the Cu(II) compounds' interactions with human serum albumin (HSA) using fluorescence spectroscopy and molecular docking. These studies revealed that Cu(II) compounds propensity bound to IIA subdomain of HSA possible by hydrophobic interactions and hydrogen bond. Cu(II) compounds produce intracellular reactive oxygen species (ROS) in cancer cells. Complexes of HSA and copper(II) compounds enhance about 2-fold cytotoxicity in cancer cells but do not raise cytotoxicity levels in normal cells in vitro. Compared with C3 alone, HSA-C3 complex promotes HepG2 cell apoptosis and has a stronger capacity to promote cell cycle arrest at the G2/M phase of HepG2.

  6. Human serum albumin adsorption study on 62-MHz miniaturized quartz gravimetric sensors.

    PubMed

    Kao, Ping; Patwardhan, Ashish; Allara, David; Tadigadapa, Srinivas

    2008-08-01

    We have designed and fabricated 25-microm-thick quartz resonators operating at a fundamental resonance frequency of approximately 62 MHz. The results show a substantial increase in the mass sensitivity compared to single monolithic commercial resonators operating at lower frequencies in the approximately 5-10-MHz range. The overall performance of the micromachined resonators is demonstrated for the example of human serum albumin protein adsorption from aqueous buffer solutions onto gold electrodes functionalized with self-assembled monolayers. The results show a saturation adsorption frequency change of 6.8 kHz as opposed to 40 Hz for a commercial approximately 5-MHz sensor under identical loading conditions. From the analysis of the adsorption isotherm, the equilibrium adsorption constant of the adsorption of the protein layer was found to be K = 8.03 x 10(6) M(-1), which is in agreement with the values reported in the literature. The high sensitivity of the miniaturized QCM devices can be a significant advantage in both vapor and solution adsorption analyses.

  7. Spectroscopic investigation of the interaction between human serum albumin and three organic acids

    NASA Astrophysics Data System (ADS)

    Liu, Yuan; Xie, Meng-Xia; Jiang, Min; Wang, Ying-Dian

    2005-07-01

    The interactions of human serum albumin (HSA) with sinapic acid (SA), gallic acid (GA) and shikimic acid (SI) were investigated by fluorescence and Fourier transformed infrared spectrometry. Fluorescence results showed that one molecule of protein combined with one molecule of GA at the molar ratio of drug to HSA ranging from 0.1 to 30, and their binding constant ( KA) is 1.1 × 10 4 M -1. While one HSA molecule combined with one or two molecule of SA at the molar ratio of drug to HSA ranging from 0.1 to 4.26 or 4.26 to 30, and their binding affinities ( KA) are 1.92 × 10 3 M -1 and 6.87 × 10 8 M -1, respectively. There is no specific interaction between HSA and SI. Combining the curve-fitting results of infrared amide I and amide III bands, the alterations of protein secondary structures induced by drugs were estimated. The drug-protein combination brought gradual reductions of the protein α-helix structure with increasing the concentrations of SA and GA, but SI did not change the protein secondary structure. From the fluorescence and FT-IR results, the binding mode was discussed in relation to the structures of the organic acids.

  8. Investigation on the interaction between endocrine disruptor triphenyltin with human serum albumin.

    PubMed

    Geng, Bing; Liu, Xue; Tian, Yunlong; Ye, Jing; Li, Hongna; Wu, Jinbao

    2014-01-01

    The interaction between triphenyltin (TPT) and human serum albumin (HSA) in physiological buffer (pH=7.4) was investigated by the fluorescence quenching technique. The results of fluorescence titration revealed that TPT could strongly quench the intrinsic fluorescence of HSA through a static quenching procedure. The apparent binding constants K and number of binding sites n of TPT with HSA were 2.51×10(3) and 0.96 at 298 K which were obtained by the fluorescence quenching method. The thermodynamic parameters enthalpy change (ΔH), entropy change (ΔS) were positive, which indicated that the interaction of TPT with HSA was driven mainly by hydrophobic forces. The process of binding was a spontaneous process in which Gibbs free energy change was negative. The distance r between donor (HSA) and acceptor (TPT) was calculated to be 3.13 nm based on Forster's non-radiative energy transfer theory. The results of synchronous fluorescence, three-dimensional fluorescence and circular dichroism (CD) spectra showed that the triphenyltin induced conformational changes of HSA. PMID:24211812

  9. Pre-denaturing transitions in human serum albumin probed using time-resolved phosphorescence.

    PubMed

    Sagoo, Kulwinder; Hirsch, Richard; Johnston, Pamela; McLoskey, David; Hungerford, Graham

    2014-04-24

    The investigation of protein dynamics has long been of interest, since protein interactions and functions can be determined by their structure and changes in conformation. Although fluorescence, occurring on the nanosecond timescale, from intrinsic fluorescent amino acids has been extensively used, in order to fully access conformational changes longer timescales are required. Phosphorescence enables processes on the microsecond to second timescale to be accessed. However, at room temperature this emission can be weak and non trivial to measure. It requires the removal of oxygen - a common triplet state quencher and appropriate instrumentation. In this work we make use of a chemical deoxygenator to study room temperature phosphorescence from tryptophan in human serum albumin excited using a pulsed UV light emitting diode. This is extended to monitor the phosphorescence emission upon increasing temperature, allowing pre-denaturing transitions to be observed. Time-resolved data are analysed, both as the sum of exponential decays and using a distribution analysis based on non extensive decay kinetics. These results are compared to a fluorescence study and both the average lifetime and contribution of the different emitting components were found to give more dramatic changes on the phosphorescence timescale.

  10. Quantitation of the residual DNA from rice-derived recombinant human serum albumin.

    PubMed

    Chen, Zhen; Dai, Huixia; Liu, Zhenwei; Zhang, Liping; Pang, Jianlei; Ou, Jiquan; Yang, Daichang

    2014-04-01

    Residual DNA in recombinant protein pharmaceuticals can potentially cause safety issues in clinical applications; thus, maximum residual limit has been established by drug safety authorities. Assays for residual DNA in Escherichia coli, yeast, and Chinese hamster ovary (CHO) cell expression systems have been established, but no rice residual DNA assay for rice expression systems has been designed. To develop an assay for the quantification of residual DNA that is produced from rice seed, we established a sensitive assay using quantitative real-time polymerase chain reaction (qPCR) based on the 5S ribosomal RNA (rRNA) genes. We found that a 40-cycle qPCR exhibited a linear response when the template concentration was in the range of 2×10(4) to 0.2pg of DNA per reaction in TaqMan and SYBR Green I assays. The amplification efficiency was 103 to 104%, and the amount of residual DNA from recombinant human serum albumin from Oryza sativa (OsrHSA) was less than 3.8ng per dosage, which was lower than that recommended by the World Health Organization (WHO). Our results indicate that the current purification protocol could efficiently remove residual DNA during manufacturing and processing. Furthermore, this protocol could be viable in other cereal crop endosperm expression systems for developing a residual DNA quantitation assay using the highly conserved 5S rRNA gene of the crops. PMID:24388867

  11. Enhancement of recombinant human serum albumin in transgenic rice cell culture system by cultivation strategy.

    PubMed

    Liu, Yu-Kuo; Li, Yu-Teng; Lu, Ching-Fan; Huang, Li-Fen

    2015-05-25

    Fusion of the sugar-starvation-induced αAmy3 promoter with its signal peptide has enabled secretion of recombinant human serum albumin (rHSA) into the culture medium. To simplify the production process and increase the rHSA yield in rice suspension cells, a one-step strategem without medium change was adopted. The yield of rHSA was increased sixfold by this one-step approach compared with the two-step recombinant protein process, in which a change of the culture medium to sugar-free medium is required. The one-step strategem was applied to check repeated cycle of rHSA production, and the production of rHSA was also higher in each cycle in the one-step, as opposed to the two-step, production process. The use of the one-step process resulted in fewer damaged cells during the cell sugar starvation phase for recombinant protein production. Furthermore, we scaled up the rHSA production in a 2-L airlift and a 2-L stirred tank bioreactor by the one-step approach, and concluded that rHSA can be enriched to 45 mg L(-1) in plant culture commonly used MS medium by the airlift-type bioreactor. Our results suggest that rHSA production can be enriched by this optimized cultivation strategem. PMID:25765580

  12. Differences in Esterase Activity to Aspirin and p-Nitrophenyl Acetate among Human Serum Albumin Preparations.

    PubMed

    Tatsumi, Akitoshi; Okada, Masaya; Inagaki, Yoshihiro; Inoue, Sachiyo; Hamaguchi, Tsuneo; Iwakawa, Seigo

    2016-01-01

    Human serum albumin (HSA) has two major ligand-binding sites, sites I and II, and also hydrolyzes some compounds at both sites. In the present study, we investigated differences in esterase activity among HSA preparations, and also the effects of warfarin, indomethacin, and naproxen on the hydrolytic activities of HSA to aspirin and p-nitrophenyl acetate. The esterase activities of HSA to aspirin or p-nitrophenyl acetate were measured from the pseudo-first-order formation rate constant (kobs) of salicylic acid or p-nitrophenol by HSA. Inter-lot variations were observed in the esterase activities of HSA to aspirin and p-nitrophenyl acetate; however, the esterase activity of HSA to aspirin did not correlate with that to p-nitrophenyl acetate. The inhibitory effects of warfarin and indomethacin on the esterase activity of HSA to aspirin were stronger than that of naproxen. In contrast, the inhibitory effect of naproxen on the esterase activity of HSA to p-nitrophenyl acetate was stronger than those of warfarin and indomethacin. These results suggest that the administration of different commercial HSA preparations and the co-administration with site I or II high-affinity binding drugs may change the pharmacokinetic profiles of drugs that are hydrolyzed by HSA. PMID:27476944

  13. Interaction of human serum albumin with short polyelectrolytes: a study by calorimetry and computer simulations.

    PubMed

    Yu, Shun; Xu, Xiao; Yigit, Cemil; van der Giet, Markus; Zidek, Walter; Jankowski, Joachim; Dzubiella, Joachim; Ballauff, Matthias

    2015-06-21

    We present a comprehensive study of the interaction of human serum albumin (HSA) with poly(acrylic acid) (PAA; number average degree of polymerization: 25) in aqueous solution. The interaction of HSA with PAA is studied in dilute solution as a function of the concentration of added salt (20-100 mM) and temperature (25-37 °C). Isothermal titration calorimetry (ITC) is used to analyze the interaction and to determine the binding constant and related thermodynamic data. It is found that only one PAA chain is bound per HSA molecule. The free energy of binding ΔGb increases with temperature significantly. ΔGb decreases with increasing salt concentration and is dominated by entropic contributions due to the release of bound counterions. Coarse-grained Langevin computer simulations treating the counterions in an explicit manner are used to study the process of binding in detail. These simulations demonstrate that the PAA chains are bound in the Sudlow II site of HSA. Moreover, ΔGb is calculated from the simulations and found to be in very good agreement with the measured data. The simulations demonstrate clearly that the driving force of binding is the release of counterions in full agreement with the ITC-data.

  14. Replica exchange Monte Carlo simulation of human serum albumin-catechin complexes.

    PubMed

    Li, Yunqi; An, Lijia; Huang, Qingrong

    2014-09-01

    Replica exchange Monte Carlo simulation equipped with an orientation-enhanced hydrophobic interaction was utilized to study the impacts of molar ratio and ionic strength on the complex formation of human serum albumin (HSA) and catechin. Only a small amount of catechins was found to act as bridges in the formation of HSA-catechin complexes. Selective binding behavior was observed at low catechin to HSA molar ratio (R). Increase of catechin amount can suppress HSA self-aggregation and diminish the selectivity of protein binding sites. Strong saturation binding with short-range interactions was found to level off at around 4.6 catechins per HSA on average, while this number slowly increased with R when long-range interactions were taken into account. Meanwhile, among the three rings of catechin, the 3,4-dihydroxyphenyl (B-ring) shows the strongest preference to bind HSA. Neither the aggregation nor the binding sites of the HSA-catechin complex was sensitive to ionic strength, suggesting that the electrostatic interaction is not a dominant force in such complexes. These results provide a further molecular level understanding of protein-polyphenol binding, and the strategy employed in this work shows a way to bridge phase behaviors at macroscale and the distribution of binding sites at residue level. PMID:25111890

  15. Probing the binding of procyanidin B3 to human serum albumin by isothermal titration calorimetry

    NASA Astrophysics Data System (ADS)

    Li, Xiangrong; Yan, Yunhui

    2015-02-01

    Proanthocyanidins are a mixture of monomers, oligomers, and polymers of flavan-3-ols that are widely distributed in the plant kingdom. One of the most widely studied proanthocyanidins is procyanidin B3. In this study, the interaction between procyanidin B3 and human serum albumin (HSA) was investigated using isothermal titration calorimetry (ITC). Thermodynamic investigations reveal that the hydrogen bond and van der Waals force are the major binding forces in the binding of procyanidin B3 to HSA. The binding of procyanidin B3 to HSA is driven by favorable enthalpy and unfavorable entropy. The obtained binding constant for procyanidin B3 with HSA is in the intermediate range and the equilibrium fraction of unbound procyanidin B3 fu > 90% at the physiological concentration of HSA shows that procyanidin B3 can be stored and transported from the circulatory system to reach its target site. The stoichiometric binding number n approximately equals to 1, suggesting that one molecule of procyanidin B3 combines with one molecule of HSA and no more procyanidin B3 binding to HSA occurs at the concentration used in this study.

  16. Revealing deposition mechanism of colloid particles on human serum albumin monolayers.

    PubMed

    Nattich-Rak, Małgorzata; Adamczyk, Zbigniew; Kujda, Marta

    2016-01-01

    Colloid particle deposition was applied in order to characterize human serum albumin (HSA) monolayers on mica adsorbed under diffusion transport at pH 3.5. The surface concentration of HSA was determined by a direct AFM imaging of single molecules. The electrokinetic characteristics of the monolayers for various ionic strength were done by in situ streaming potential measurements. In this way the mean-field zeta potential of monolayers was determined. It was shown that the initially negative potential changed its sign for HSA surface concentrations above 2800μm(-2) that was interpreted as overcharging effect. The monolayers were also characterized by the colloid deposition method where negatively charged polystyrene particles, 810nm in diameter were used. The kinetics of particle deposition and their maximum coverage were determined as a function of the HSA monolayer surface concentration. An anomalous deposition of particles on substrates exhibiting a negative zeta potential was observed, which contradicts the mean-field theoretical predictions. This effect was quantitatively interpreted in terms of the random site sequential adsorption model. It was shown that efficient immobilization of particles only occurs at adsorption sites formed by three and more closely adsorbed HSA molecules. These results can be exploited as useful reference data for the analysis of deposition phenomena of bioparticles at protein monolayers that has practical significance for the regulation of the bioadhesive properties of surfaces.

  17. Probing the Sudlow binding site with warfarin: how does gold nanocluster growth alter human serum albumin?

    PubMed

    Russell, B A; Mulheran, P A; Birch, D J S; Chen, Y

    2016-08-17

    The search for new fluorescent molecules is vital to the advancement of molecular imaging and sensing for the benefit of medical and biological studies. One such class of new fluorescent molecule is fluorescent gold nanoclusters encapsulated in Human Serum Albumin (HSA-AuNC). In order to use this new fluorescent molecule as a sensor or fluorescent marker in biological imaging both in vitro and in vivo it is important to understand whether/how the proteins function is changed by the synthesis and presence of the gold nanoclusters inside the protein. Natural HSA acts as the main drug carrier in the blood stream, carrying a multitude of molecules in two major binding sites (Sudlow I and II). To test the effects of gold on the ability of HSA to act as a drug carrier we employed warfarin, an anticoagulant drug, as a fluorescent probe to detect changes between natural HSA and HSA-AuNCs. AuNCs are found to inhibit the take up of warfarin by HSA. Evidence for this is found from fluorescence spectral and lifetime measurements. Interestingly, the presence of warfarin bound to HSA also inhibits the formation of gold nanoclusters within protein. This research provides valuable insight into how protein function can change upon synthesis of AuNCs and how that will affect their use as a fluorescent probe.

  18. A spectroscopic study of the wavelength-dependent photoisomerizations of bilirubins bound to human serum albumin

    NASA Astrophysics Data System (ADS)

    Mazzoni, Marina; Agati, Giovanni; Pratesi, Riccardo; Persico, Maurizio

    2005-12-01

    The wavelength-dependent photoisomerizations of the asymmetric bilirubin BR-IXα and of the symmetric bilirubin-IIIα (BR-III) and mesobilirubin-XIIIα (MBR-XIII) bound to human serum albumin (HSA) in aqueous solution were analysed with the help of an exciton coupling model. The modelling was based on the absorption and circular dichroism (CD) spectra (bisignate Cotton effect). Time-dependent density functional theory (TD-DFT) of the free BR-IX molecule suggested the presence of two main bands of exciton coupling character in the blue region of the spectrum, and other weaker bands of charge transfer character at longer wavelengths. These peculiarities were taken into account to fit the photoisomerization quantum yields in the blue-green region as functions of the wavelength, obtaining the bandshape of the exciton coupling bands from the experimental CD spectra. The other excitons were extracted from the decomposition of the band resulting from the difference between the absorption spectrum and the sum (normalized-to-absorption) of the two CD excitons. We expressed photoisomerization quantum yields in terms of the sum of the contributions to photon absorption deriving from all the exciton states normalized to total absorption. For all the reversible photoprocesses of bilirubins and for the irreversible one of BR-IXα in HSA (i.e. lumirubin formation), we give reliable mean values of the individual state excitation probabilities and photoisomerization efficiencies in the pigment protein complex.

  19. The effect of Berberine on the secondary structure of human serum albumin

    NASA Astrophysics Data System (ADS)

    Li, Ying; He, WenYing; Tian, Jianniao; Tang, Jianghong; Hu, Zhide; Chen, Xingguo

    2005-05-01

    The presence of several high affinity binding sites on human serum albumin (HSA) makes it a possible target for many drugs. This study is designed to examine the effect of Berberine (an ancient Chinese drug used for antimicrobial, antiplasmodial, antidiarrheal and cardiovascular) on the solution structure of HSA using fluorescence, Fourier transform infrared (FT-IR), circular dichroism (CD) spectroscopic methods. The fluorescence spectroscopic results show that the fluorescence intensity of HSA was significantly decreased in the presence of Berberine. The Scatchard's plots indicated that the binding of Berberine to HSA at 296, 303, 318 K is characterized by one binding site with the binding constant is 4.071(±0.128)×10 4, 3.741(±0.089)×10 4, 3.454(±0.110)×10 4 M -1, respectively. The protein conformation is altered (FT-IR and CD data) with reductions of α-helices from 54 to 47% for free HSA to 45-32% and with increases of turn structure5% for free HSA to 18% in the presence of Berberine. The binding process was exothermic, enthalpy driven and spontaneous, as indicated by the thermodynamic analyses, Berberine bound to HSA was mainly based on hydrophobic interaction and electrostatic interaction cannot be excluded from the binding. Furthermore, the displace experiments indicate that Berberine can bind to the subdomain IIA, that is, high affinity site (site II).

  20. Stabilization of Human Serum Albumin against Urea Denaturation by Diazepam and Ketoprofen.

    PubMed

    Manoharan, Pralad; Wong, Yin H; Tayyab, Saad

    2015-01-01

    Stabilizing effect of diazepam and ketoprofen, Sudlow's site II markers on human serum albumin (HSA) against urea denaturation was studied using fluorescence spectroscopy. The two-step, three-state urea transition of HSA was transformed into a single-step, two-state transition with the abolishment of the intermediate state along with a shift of the transition curve towards higher urea concentrations in the presence of diazepam or ketoprofen. Interestingly, a greater shift in the transition curve of HSA was observed in the presence of ketoprofen compared to diazepam. A comparison of the intrinsic fluorescence and three-dimensional fluorescence spectra of HSA and partially-denatured HSAs, obtained in the absence and the presence of diazepam or ketoprofen suggested significant retention of native-like conformation in the partially-denatured states of HSA in the presence of Sudlow's site II markers. Taken together, all these results suggested stabilization of HSA in the presence of diazepam or ketoprofen, being greater in the presence of ketoprofen.

  1. Development of human serum albumin conjugated with near-infrared dye for photoacoustic tumor imaging

    NASA Astrophysics Data System (ADS)

    Kanazaki, Kengo; Sano, Kohei; Makino, Akira; Takahashi, Atsushi; Deguchi, Jun; Ohashi, Manami; Temma, Takashi; Ono, Masahiro; Saji, Hideo

    2014-09-01

    Photoacoustic (PA) imaging has emerged as a noninvasive diagnostic method which detects ultrasonic waves thermoelastically induced by optical absorbers irradiated with laser. For tumor diagnosis, PA contrast agent has been proposed to enhance the PA effect for detecting tumors sensitively. Here, we prepared a human serum albumin (HSA) conjugated with indocyanine green (ICG) as a PA contrast agent allowing enhanced permeability and retention effect for sensitive tumor imaging. The feasibility of PA imaging with HSA-ICG to detect allografted tumors was evaluated in tumor-bearing mice. In vivo fluorescence imaging and radiolabeled biodistribution study showed that the biodistribution dramatically changed as the number of ICG bound to HSA increased, and the maximum accumulation of ICG was achieved when around three ICG molecules were loaded on an HSA. In vivo PA imaging demonstrated a tumor-selective and dose-dependent increase of PA signal intensity in mice injected with HSA-ICG (R2=0.88, 387% increase for HSA-ICG, 104 nmol ICG). In conclusion, HSA-ICG clearly visualized the allografted tumors with high tumor-to-background ratios having high quantitative and spatial resolution for the sensitive PA imaging of tumors. HSA-ICG could be useful as a favorable contrast agent for PA tumor imaging for the management of cancer.

  2. Spread Films of Human Serum Albumin at the Air-Water Interface: Optimization, Morphology, and Durability.

    PubMed

    Campbell, Richard A; Ang, Joo Chuan; Sebastiani, Federica; Tummino, Andrea; White, John W

    2015-12-22

    It has been known for almost one hundred years that a lower surface tension can be achieved at the air-water interface by spreading protein from a concentrated solution than by adsorption from an equivalent total bulk concentration. Nevertheless, the factors that control this nonequilibrium process have not been fully understood. In the present work, we apply ellipsometry, neutron reflectometry, X-ray reflectometry, and Brewster angle microscopy to elaborate the surface loading of human serum albumin in terms of both the macroscopic film morphology and the spreading dynamics. We show that the dominant contribution to the surface loading mechanism is the Marangoni spreading of protein from the bulk of the droplets rather than the direct transfer of their surface films. The films can be spread on a dilute subphase if the concentration of the spreading solution is sufficient; if not, dissolution of the protein occurs, and only a textured adsorbed layer slowly forms. The morphology of the spread protein films comprises an extended network with regions of less textured material or gaps. Further, mechanical cycling of the surface area of the spread films anneals the network into a membrane that approach constant compressibility and has increased durability. Our work provides a new perspective on an old problem in colloid and interface science. The scope for optimization of the surface loading mechanism in a range of systems leading to its exploitation in deposition-based technologies in the future is discussed.

  3. Comparative binding character of two general anaesthetics for sites on human serum albumin.

    PubMed Central

    Liu, Renyu; Meng, Qingcheng; Xi, Jin; Yang, Jinsheng; Ha, Chung-Eun; Bhagavan, Nadhipuram V; Eckenhoff, Roderic G

    2004-01-01

    Propofol and halothane are clinically used general anaesthetics, which are transported primarily by HSA (human serum albumin) in the blood. Binding characteristics are therefore of interest for both the pharmacokinetics and pharmacodynamics of these drugs. We characterized anaesthetic-HSA interactions in solution using elution chromatography, ITC (isothermal titration calorimetry), hydrogen-exchange experiments and geometric analyses of high-resolution structures. Binding affinity of propofol to HSA was determined to have a K(d) of 65 microM and a stoichiometry of approx. 2, whereas the binding of halothane to HSA showed a K(d) of 1.6 mM and a stoichiometry of approx. 7. Anaesthetic-HSA interactions are exothermic, with propofol having a larger negative enthalpy change relative to halothane. Hydrogen-exchange studies in isolated recombinant domains of HSA showed that propofol-binding sites are primarily found in domain III, whereas halothane sites are more widely distributed. Both location and stoichiometry from these solution studies agree with data derived from X-ray crystal-structure studies, and further analyses of the architecture of sites from these structures suggested that greater hydrophobic contacts, van der Waals interactions and hydrogen-bond formation account for the stronger binding of propofol as compared with the less potent anaesthetic, halothane. PMID:14759223

  4. Molecular interaction and energy transfer between human serum albumin and bioactive component Aloe dihydrocoumarin

    NASA Astrophysics Data System (ADS)

    Zhang, Xiu-Feng; Xie, Ling; Liu, Yang; Xiang, Jun-Feng; Li, Lin; Tang, Ya-Lin

    2008-10-01

    Aloe dihydrocoumarin is an antioxidant and a candidate of immunomodulatory drug on the immune system and can balance physiological reactive oxygen species (ROS) levels which may be useful to maintain homeostasis. In order to explore the mechanism of drug action at a molecular level, the binding of Aloe dihydrocoumarin with human serum albumin (HSA) has been investigated by fluorescence, ultraviolet (UV), circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy, fluorescence dynamics, and molecular dynamic docking for the first time. We observed a quenching of fluorescence of HSA in the presence of Aloe dihydrocoumarin and also analyzed the quenching results using the Stern-Volmer equation and obtained high affinity binding to HSA. A Förster type fluorescence resonance energy transfer mechanism is involved in this quenching of Trp fluorescence by Aloe dihydrocoumarin. From the CD and FT-IR results, it is apparent that the interaction of Aloe dihydrocoumarin with HSA causes a conformational change of the protein, with the loss of α-helix stability and the gain of β-sheet and β-turn content. Data obtained by fluorescence spectroscopy, fluorescence dynamics, CD, and FT-IR experiments along with the docking studies suggest that Aloe dihydrocoumarin binds to residues located in subdomain IIA of HSA.

  5. Ethanol or/and captopril-induced precipitation and secondary conformational changes of human serum albumin

    NASA Astrophysics Data System (ADS)

    Lin, Shan-Yang; Li, Mei-Jane; Wei, Yen-Shan

    2004-11-01

    We determined the secondary structure of solid-state native human serum albumin (HSA) and its precipitates induced by ethanol, captopril, or a captopril/ethanol mixture. A transmission Fourier transform infrared (FT-IR) microspectroscopy equipped with a thermal analyzer was used. The secondary structural composition of solid-state native HSA was 54% α-helices (1655 cm -1), 22% β-turns (1679 cm -1), and 23% β-sheets (1633 cm -1). After ethanol treatment, a new peak was observed at 1690 cm -1, and the peak at 1633 cm -1 was more apparent in the HSA precipitates. The corresponding compositions consisted of 59% α-helices, 17% β-turns, and 24% β-sheets. After treatment with captopril with or without ethanol, the percentage of α-helices and β-turns decreased in both HSA precipitates, but the percentage of β-sheets increased. The temperature-dependent structural transformation from α-helices/random coils to β-sheets for the solid-state HSA samples occurred at markedly different onset temperatures. The onset temperature for native HSA was 85 °C, and that for HSA precipitates obtained from ethanol, captopril, or captopril/ethanol was 100, 48 or 57 °C, respectively. The thermal-induced structural transformation from α-helices/random coils to β-sheets implies a partial unfolding structure in these HSA samples.

  6. The role of acetoacetate in Amadori product formation of human serum albumin.

    PubMed

    Bohlooli, Mousa; Ghaffari-Moghaddam, Mansour; Khajeh, Mostafa; Shahraki-Fallah, Gholamreza; Haghighi-Kekhaiye, Batool; Sheibani, Nader

    2016-10-01

    Amadori product is an important and stable intermediate, which is produced during glycation process. It is a marker of hyperglycemia in diabetes mellitus, and its accumulation in the body contributes to microvascular complication of diabetes including diabetic nephropathy and retinopathy. In this study, the effect of acetoacetate on the formation of Amadori products and biophysical properties of human serum albumin (HSA), after incubation with glucose, was investigated using various methods. These included circular dichroism (CD), Fourier transform infrared (FTIR) spectroscopy, and UV-visible and fluorescence spectroscopy. Our results indicated that the production of Amadori products in HSA incubated with glucose (GHSA) was increased in the presence of acetoacetate. We also detected alterations in the secondary and tertiary structure of GHSA, which was increased in the presence of acetoacetate. These changes were attributed to the formation of covalent bonds between the carbonyl group of acetoacetate and the nucleophilic groups (lysine residues) of HSA. Thus, acetoacetate can enhance the production of Amadori products through formation of covalent bonds with biomaterials. PMID:27614245

  7. ANALYSIS OF DRUG-PROTEIN BINDING BY ULTRAFAST AFFINITY CHROMATOGRAPHY USING IMMOBILIZED HUMAN SERUM ALBUMIN

    PubMed Central

    Mallik, Rangan; Yoo, Michelle J.; Briscoe, Chad J.; Hage, David S.

    2010-01-01

    Human serum albumin (HSA) was explored for use as a stationary phase and ligand in affinity microcolumns for the ultrafast extraction of free drug fractions and the use of this information for the analysis of drug-protein binding. Warfarin, imipramine, and ibuprofen were used as model analytes in this study. It was found that greater than 95% extraction of all these drugs could be achieved in as little as 250 ms on HSA microcolumns. The retained drug fraction was then eluted from the same column under isocratic conditions, giving elution in less than 40 s when working at 4.5 mL/min. The chromatographic behavior of this system gave a good fit with that predicted by computer simulations based on a reversible, saturable model for the binding of an injected drug with immobilized HSA. The free fractions measured by this method were found to be comparable to those determined by ultrafiltration, and equilibrium constants estimated by this approach gave good agreement with literature values. Advantages of this method include its speed and the relatively low cost of microcolumns that contain HSA. The ability of HSA to bind many types of drugs also creates the possibility of using the same affinity microcolumn to study and measure the free fractions for a variety of pharmaceutical agents. These properties make this technique appealing for use in drug binding studies and in the high-throughput screening of new drug candidates. PMID:20227701

  8. Studies of drug interactions with glycated human serum albumin by high-performance affinity chromatography

    PubMed Central

    Matsuda, Ryan; Kye, So-Hwang; Anguizola, Jeanethe; Hage, David S.

    2015-01-01

    Diabetes is a health condition associated with elevated levels of glucose in the bloodstream and affects 366 million people worldwide. Type II diabetes is often treated with sulfonylurea drugs, which are known to bind tightly in blood to the transport protein human serum albumin (HSA). One consequence of the elevated levels of glucose in diabetes is the non-enzymatic glycation of proteins such as HSA. Several areas of HSA are now known to be affected by glycation-related modifications, which may in turn affect the binding of sulfonylurea drugs and other solutes to this protein. This review discusses some recent studies that have examined these changes in drug-protein binding by employing high-performance affinity chromatography (HPAC). A description of the theoretical and experimental techniques that were used in these studies is given. The information on drug interactions with glycated HSA, as obtained through this method, is also summarized. In addition, the potential advantages of this approach in the areas of biointeraction analysis and personalized medicine are considered. PMID:26526139

  9. Studies of drug interactions with glycated human serum albumin by high-performance affinity chromatography.

    PubMed

    Matsuda, Ryan; Kye, So-Hwang; Anguizola, Jeanethe; Hage, David S

    2014-08-01

    Diabetes is a health condition associated with elevated levels of glucose in the bloodstream and affects 366 million people worldwide. Type II diabetes is often treated with sulfonylurea drugs, which are known to bind tightly in blood to the transport protein human serum albumin (HSA). One consequence of the elevated levels of glucose in diabetes is the non-enzymatic glycation of proteins such as HSA. Several areas of HSA are now known to be affected by glycation-related modifications, which may in turn affect the binding of sulfonylurea drugs and other solutes to this protein. This review discusses some recent studies that have examined these changes in drug-protein binding by employing high-performance affinity chromatography (HPAC). A description of the theoretical and experimental techniques that were used in these studies is given. The information on drug interactions with glycated HSA, as obtained through this method, is also summarized. In addition, the potential advantages of this approach in the areas of biointeraction analysis and personalized medicine are considered. PMID:26526139

  10. Interactions of acidic pharmaceuticals with human serum albumin: insights into the molecular toxicity of emerging pollutants.

    PubMed

    Chen, Jiabin; Zhou, Xuefei; Zhang, Yalei; Qian, Yajie; Gao, Haiping

    2012-10-01

    Acidic pharmaceuticals such as diclofenac (DCF), clofibric acid (CA) and ketoprofen (KTP) have been detected frequently in environmental media. In order to reveal the toxicity of such emerging pollutants, their interactions with human serum albumin (HSA) were investigated by capillary electrophoresis, molecular spectrometry, and equilibrium dialysis. The binding constants and sites of these acidic pharmaceuticals with HSA were obtained. The thermodynamic parameters, e.g. enthalpy change and entropy change of these interactions were calculated to characterize that all the reactions resulted from hydrophobic and electrostatic interactions. The static quenching of the fluorescence of HSA was observed when interacted with acidic pharmaceuticals, indicating acidic pharmaceuticals bound to Tryptophan residue of HSA. The 3D fluorescence and circular dichroism confirmed that the secondary conformation of HSA changed after the interactions with the pharmaceuticals. At physiological condition, only 0.12 mM acidic pharmaceuticals reduced the binding of vitamin B(2) to HSA by 37, 30 and 21% for DCF, KTP and CA, respectively. This work provides an insight into non-covalent interactions between emerging contaminants and biomolecule, and is helpful for clarifying the toxic mechanism of such emerging contaminants. PMID:22307229

  11. In vitro imaging and human serum albumin responsive dimeric lanthanide DO3A complex.

    PubMed

    Fung, Yuen On; Wu, Wanqing; Yeung, Chi-Tung; Kong, Hoi-Kuan; Wong, Kenny Kam-Cheng; Lo, Wai-Sum; Law, Ga-Lai; Wong, Ka-Leung; Lau, Chi-Kong; Lee, Chi-Sing; Wong, Wing-Tak

    2011-06-20

    Two series of dimeric DO3A (1,4,7,10-tetraazacyclodecane-1,4,7-triacetate) lanthanide complexes (LnL(1)-LnL(2), Ln = Eu, Gd, and Tb) have been synthesized with two different bridged chromophores. The X-ray structures of dimeric LnL(1) (Ln = Gd and Tb) complexes show that each metal ion has nine coordination numbers with eight directly bound donor atoms of the ligand and one oxygen donor from the water molecule. Photophysical measurements indicate that the bridged antenna in LnL(2) gives a higher efficiency than that of LnL(1) and is responsive to the protein Human Serum Albumin (HSA), giving an f-f luminescence signal enhancement with a binding constant log K = 4.84. In vitro imaging of EuL(1) and EuL(2) in HeLa cells has been recorded, and EuL(2) has demonstrated a higher rate of cellular uptake and low cytotoxicity (IC(50) = 3 mM).

  12. Binding of the bioactive component Aloe dihydroisocoumarin with human serum albumin

    NASA Astrophysics Data System (ADS)

    Zhang, Xiu-Feng; Xie, Ling; Liu, Yang; Xiang, Jun-Feng; Tang, Ya-Lin

    2008-11-01

    Aloe dihydroisocoumarin, one of new components isolated from Aloe vera, can scavenge reactive oxygen species. In order to explore the mechanism of drug action at a molecular level, the binding of Aloe dihydroisocoumarin with human serum albumin (HSA) has been investigated by using fluorescence, ultraviolet (UV), circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy, fluorescence dynamics, and molecular dynamic docking for the first time. We observed a quenching of fluorescence of HSA in the presence of Aloe dihydroisocoumarin and also analyzed the quenching results using the Stern-Volmer equation and obtained high affinity binding to HSA. An isoemissive point at 414 nm is seen, indicating that the quenching of HSA fluorescence depends on the formation of Aloe dihydroisocoumarin-HSA complex, which is further confirmed by fluorescence dynamic result. From the CD and FT-IR results, it is apparent that the interaction of Aloe dihydroisocoumarin with HSA causes a conformational change of the protein, with the gain of α-helix, β-sheet and random coil stability and the loss of β-turn content. Data obtained by fluorescence spectroscopy, fluorescence dynamics, CD, and FTIR experiments along with the docking studies suggest that Aloe dihydroisocoumarin binds to residues located in subdomain IIA of HSA.

  13. Study on the interaction of levocetirizine dihydrochloride with human serum albumin by molecular spectroscopy

    NASA Astrophysics Data System (ADS)

    Liu, Xiangping; Du, Yingxiang; Sun, Wen; Kou, Junping; Yu, Boyang

    2009-12-01

    The interaction between cetirizine dihydrochloride and human serum albumin (HSA) has been examined by the spectroscopic techniques first. According to Stern-Volmer equation at different temperatures and the UV-vis spectra examination it was demonstrated that HSA fluorescence quenching initiated by levocetirizine was static. The values of binding constant ( KA) and the number of binding sites ( n) for levocetirizine and HSA were smaller than those for cetirizine and HSA, which meant that the transport of drug was regulated by the stereoselectivity of HSA to the enantiomer. The effect of the non-enzymatic glycosylation (NEG) on the interaction between levocetirizine and HSA signified that the administration of levocetirizine for diabetes should be different from the normal. The positive Δ S° and negative Δ H° indicated that ionic interaction played a major role between levocetirizine and HSA. Circular dichroism (CD) measurement showed that the secondary structure of HSA has changed in the presence of levocetirizine, and α-helical content decreased from 63.1% for free HSA to 54.9% for combined HSA, and accordingly the other secondary structure (β-strand, β-turns and others) contents increased to some extent. Finally, by the competitive binding experiments it was deduced that levocetirizine specifically bound to HSA in the region of site II, which meant the curative effect of levocetirizine should be reconsidered when it was administrated together with other site II drugs.

  14. Formaldehyde adduct to human serum albumin with reference to aspartame intake.

    PubMed

    Gilli, Giorgio; Schilirò, Tiziana; Traversi, Deborah; Pignata, Cristina; Cordara, Sara; Carraro, Elisabetta

    2008-01-01

    A preliminary study was performed to evaluate the role of formaldehyde (F) deriving from aspartame intake in the production of the adduct F-human serum albumin (F-HSA) by mean of a sera-epidemiological investigation. A blood-donors population (68 subjects) was analysed for the presence of anti-F-HSA IgG by an indirect competitive immunoenzymatic assay (displacement assay). Only the 41% of the subjects were aspartame consumer and with a low daily intake (0.96mg/(kgday)). A 50% sera-prevalence of IgG anti-F-HSA was observed in the population, but no association between this biomarker and aspartame intake was pointed out. A significant association was found between the IgG anti-F-HSA presence and exogenous F exposure sources (cigarette active smoke and occupational exposure). Considering the low number of the investigated subjects and the low doses of aspartame consumption, the results of this preliminary study seems to suggest that aspartame low intake does not influence the formation of F adducts.

  15. Anesthetic 2,2,2-trifluoroethanol induces amyloidogenesis and cytotoxicity in human serum albumin.

    PubMed

    Naeem, Aabgeena; Iram, Afshin; Bhat, Sheraz Ahmed

    2015-08-01

    Trifluoroethanol (TFE) mimics the membrane environments as it simulates the hydrophobic environment and better stabilizes the secondary structures in peptides owing to its hydrophobicity and hydrogen bond-forming properties. Its dielectric constant approximates that of the interior of proteins and is one-third of that of water. Human serum albumin (HSA) is a biological transporter. The effect of TFE on HSA gives the clue about the conformational changes taking place in HSA on transport of ligands across the biological membranes. At 25% (v/v) and 60% (v/v) TFE, HSA exhibits non-native β-sheet, altered tryptophan fluorescence, exposed hydrophobic clusters, increased thioflavin T fluorescence and prominent red shifted Congo red absorbance, and large hydrodynamic radii suggesting the aggregate formation. Isothermal titration calorimetric results indicate weak binding of TFE and HSA. This suggests that solvent-mediated effects dominate the interaction of TFE and HSA. TEM confirmed prefibrillar at 25% (v/v) and fibrillar aggregates at 60% (v/v) TFE. Comet assay of prefibrillar aggregates showed DNA damage causing cell necrosis hence confirming cytotoxic nature. On increasing concentration of TFE to 80% (v/v), HSA showed retention of native-like secondary structure, increased Trp and ANS fluorescence, a transition from β-sheet to α-helix. Thus, TFE at high concentration possess anti- aggregating potency.

  16. Interplay of Multiple Interaction Forces: Binding of Norfloxacin to Human Serum Albumin.

    PubMed

    Paul, Bijan K; Ghosh, Narayani; Mukherjee, Saptarshi

    2015-10-15

    Herein, the binding interaction of a potential chemotherapeutic antibacterial drug norfloxacin (NOF) with a serum transport protein, human serum albumin (HSA), is investigated. The prototropic transformation of the drug (NOF) is found to be remarkably modified following interaction with the protein as manifested through significant modulations of the photophysics of the drug. The predominant zwitterionic form of NOF in aqueous buffer phase undergoes transformation to the cationic form within the protein-encapsulated state. This implies the possible role of electrostatic interaction force in NOF-HSA binding. This postulate is further substantiated from the effect of ionic strength on the interaction process. To this end, the detailed study of the thermodynamics of the drug-protein interaction process from isothermal titration calorimetric (ITC) experiments is found to unfold the signature of electrostatic as well as hydrophobic interaction forces underlying the binding process. Thus, interplay of more than one interaction forces is argued to be responsible for the overall drug-protein binding. The ITC results reveal an important finding in terms of enthalpy-entropy compensation (EEC) characterizing the NOF-HSA binding. The effect of drug-binding on the native protein conformation has also been evaluated from circular dichroism (CD) spectroscopy which unveils partial rupture of the protein secondary structure. In conjunction to this, the functionality of the native protein (in terms of esterase-like activity) is found to be lowered as a result of binding with NOF. The AutoDock-based docking simulation unravels the probable binding location of NOF within the hydrophilic subdomain IA of HSA. The present program also focuses on exploring the dynamical aspects of the drug-protein interaction scenario. The rotational-relaxation dynamics of the protein-bound drug reveals the not-so-common "dip-and-rise" pattern.

  17. [The spectroscopic study on the interaction between edible pigment and human serum albumin in two-phase aqueous systems].

    PubMed

    Deng, Fan-zheng; Guo, Dong-fang; Wang, Hai-rong

    2007-02-01

    In polyethylene glycol 2000 (PEG)-(NH4)2SO4-edible pigment two-phase aqueous systems, the spectroscopic behaviour of the complexes of edible pigment and human serum albumin in PEG phase was investigated. Effects of different acidity, quantities of PEG and salt, reaction time, and coexistent matter on the determination of systems were discussed. Experimental results show that compared to BS spectra, the maximum wavelength of the complex of human serum albumin shifted to the red by 13 nm in buffer solution at pH 8, the maximum binding number of 40 was measured by molar ratio method, and the apparent molar absorptivity was 9.4 x 10(4) L x mol(-1) x cm(-1). The linear range was 0-21.07 mg x L(-1). With different surfactant, the interaction mechanism of protein and edible pigment was discussed.

  18. Spectroscopic characterization of the binding mechanism of fluorescein and carboxyfluorescein in human serum albumin

    NASA Astrophysics Data System (ADS)

    Sulaiman, Saba A. J.; Kulathunga, H. Udani; Abou-Zied, Osama K.

    2015-03-01

    Fluorescein (FL) and some of its precursors have proven to be effective fluorescent tracers in pharmaceutical and medical applications owing to their high quantum yield of fluorescence in physiological conditions and their high membrane permeability. In order to protect FL from metabolic effects during the process of its delivery, human serum albumin (HSA) has been used as a carrier because of its compatibility with the human body. In the present work, we used spectroscopic methods to characterize the binding mechanisms of FL and one of its derivatives, 5(6)- carboxyfluorescein (CFL), in the HSA protein. The absorbance change of the two ligands (FL and CFL) was quantified as a function of the HSA concentration and the results indicate a moderate binding strength for the two ligands inside HSA (1.00 +/- 0.12 x 104 M-1). The quenching effect of FL(CFL) on the fluorescence intensity of W214 (the sole tryptophan in HSA) indicates that FL and CFL occupy Site I in the protein which is known to bind several hydrophobic drugs. By performing site-competitive experiments, the location of the ligands is determined to be similar to that of the anticoagulant drug warfarin. At higher ratios of [ligand]/[HSA], we observed an upward curvature in the Stern-Volmer plots which indicates that the ligands occupy more pockets in Site I, close to W214. Our results indicate that both ligands bind in HSA with a moderate strength that should not affect their release when used as fluorescent reporters. The chemical and physical identities of the two ligands are also preserved inside the HSA binding sites.

  19. Binding of an anticancer drug, axitinib to human serum albumin: Fluorescence quenching and molecular docking study.

    PubMed

    Tayyab, Saad; Izzudin, Mohamad Mirza; Kabir, Md Zahirul; Feroz, Shevin R; Tee, Wei-Ven; Mohamad, Saharuddin B; Alias, Zazali

    2016-09-01

    Binding characteristics of a promising anticancer drug, axitinib (AXT) to human serum albumin (HSA), the major transport protein in human blood circulation, were studied using fluorescence, UV-vis absorption and circular dichroism (CD) spectroscopy as well as molecular docking analysis. A gradual decrease in the Stern-Volmer quenching constant with increasing temperature revealed the static mode of the protein fluorescence quenching upon AXT addition, thus confirmed AXT-HSA complex formation. This was also confirmed from alteration in the UV-vis spectrum of HSA upon AXT addition. Fluorescence quenching titration results demonstrated moderately strong binding affinity between AXT and HSA based on the binding constant value (1.08±0.06×10(5)M(-1)), obtained in 10mM sodium phosphate buffer, pH7.4 at 25°C. The sign and magnitude of the enthalpy change (∆H=-8.38kJmol(-1)) as well as the entropy change (∆S=+68.21Jmol(-1)K(-1)) clearly suggested involvement of both hydrophobic interactions and hydrogen bonding in AXT-HSA complex formation. These results were well supported by molecular docking results. Three-dimensional fluorescence spectral results indicated significant microenvironmental changes around Trp and Tyr residues of HSA upon complexation with AXT. AXT binding to the protein produced significant alterations in both secondary and tertiary structures of HSA, as revealed from the far-UV and the near-UV CD spectral results. Competitive drug displacement results obtained with phenylbutazone (site I marker), ketoprofen (site II marker) and hemin (site III marker) along with molecular docking results suggested Sudlow's site I, located in subdomain IIA of HSA, as the preferred binding site of AXT.

  20. Physiologically relevant plasma d,l-homocysteine concentrations mobilize Cd from human serum albumin.

    PubMed

    Sagmeister, Peter; Gibson, Matthew A; McDade, Kyle H; Gailer, Jürgen

    2016-08-01

    Although low-level chronic exposure of humans to cadmium (Cd(2+)) can result in a variety of adverse health effects, little is known about the role that its interactions with plasma proteins and small molecular weight (SMW) ligands in the bloodstream may play in delivering this metal to its target organs. To gain insight, a Cd-human serum albumin (HSA) 1:1 (molar ratio) complex was analyzed by size exclusion chromatography (SEC) coupled on-line to a flame atomic absorption spectrometer (FAAS). Using a phosphate buffered saline (PBS)-buffer mobile phase, the stability of the Cd-HSA complex was investigated in the presence of 2.0mM of SMW ligands, including taurine, acetaminophen, l-methionine, l-cysteine (Cys), d,l-homocysteine (hCys) or l-cysteine methyl-ester (Cys-Me). While taurine, acetaminophen and l-methionine did not affect its integrity, Cys, hCys and Cys-Me completely abstracted Cd from HSA. Subsequent investigations into the effect of 1.5, 1.0 and 0.5mM Cys and hCys on the integrity of the Cd-HSA complex revealed clear differences with regard to the nature of the eluting SMW-Cd species between these structurally related endogenous thiols. Interestingly, the Cd-specific chromatograms that were obtained for 0.5mM hCys revealed the elution of an apparent mixture of the parent Cd-HSA complex with a significant contribution of a structurally uncharacterized CdxhCysy species. Since this hCys concentration is encountered in blood plasma of hyperhomocysteinemia patients and since previous studies by others have revealed that a SH-containing carrier mediates the uptake of Cd into hepatocytes, our results suggest that plasma hCys may play a role in the toxicologically relevant translocation of Cd from the bloodstream to mammalian target organs. PMID:27294530

  1. Physiologically relevant plasma d,l-homocysteine concentrations mobilize Cd from human serum albumin.

    PubMed

    Sagmeister, Peter; Gibson, Matthew A; McDade, Kyle H; Gailer, Jürgen

    2016-08-01

    Although low-level chronic exposure of humans to cadmium (Cd(2+)) can result in a variety of adverse health effects, little is known about the role that its interactions with plasma proteins and small molecular weight (SMW) ligands in the bloodstream may play in delivering this metal to its target organs. To gain insight, a Cd-human serum albumin (HSA) 1:1 (molar ratio) complex was analyzed by size exclusion chromatography (SEC) coupled on-line to a flame atomic absorption spectrometer (FAAS). Using a phosphate buffered saline (PBS)-buffer mobile phase, the stability of the Cd-HSA complex was investigated in the presence of 2.0mM of SMW ligands, including taurine, acetaminophen, l-methionine, l-cysteine (Cys), d,l-homocysteine (hCys) or l-cysteine methyl-ester (Cys-Me). While taurine, acetaminophen and l-methionine did not affect its integrity, Cys, hCys and Cys-Me completely abstracted Cd from HSA. Subsequent investigations into the effect of 1.5, 1.0 and 0.5mM Cys and hCys on the integrity of the Cd-HSA complex revealed clear differences with regard to the nature of the eluting SMW-Cd species between these structurally related endogenous thiols. Interestingly, the Cd-specific chromatograms that were obtained for 0.5mM hCys revealed the elution of an apparent mixture of the parent Cd-HSA complex with a significant contribution of a structurally uncharacterized CdxhCysy species. Since this hCys concentration is encountered in blood plasma of hyperhomocysteinemia patients and since previous studies by others have revealed that a SH-containing carrier mediates the uptake of Cd into hepatocytes, our results suggest that plasma hCys may play a role in the toxicologically relevant translocation of Cd from the bloodstream to mammalian target organs.

  2. Binding of an anticancer drug, axitinib to human serum albumin: Fluorescence quenching and molecular docking study.

    PubMed

    Tayyab, Saad; Izzudin, Mohamad Mirza; Kabir, Md Zahirul; Feroz, Shevin R; Tee, Wei-Ven; Mohamad, Saharuddin B; Alias, Zazali

    2016-09-01

    Binding characteristics of a promising anticancer drug, axitinib (AXT) to human serum albumin (HSA), the major transport protein in human blood circulation, were studied using fluorescence, UV-vis absorption and circular dichroism (CD) spectroscopy as well as molecular docking analysis. A gradual decrease in the Stern-Volmer quenching constant with increasing temperature revealed the static mode of the protein fluorescence quenching upon AXT addition, thus confirmed AXT-HSA complex formation. This was also confirmed from alteration in the UV-vis spectrum of HSA upon AXT addition. Fluorescence quenching titration results demonstrated moderately strong binding affinity between AXT and HSA based on the binding constant value (1.08±0.06×10(5)M(-1)), obtained in 10mM sodium phosphate buffer, pH7.4 at 25°C. The sign and magnitude of the enthalpy change (∆H=-8.38kJmol(-1)) as well as the entropy change (∆S=+68.21Jmol(-1)K(-1)) clearly suggested involvement of both hydrophobic interactions and hydrogen bonding in AXT-HSA complex formation. These results were well supported by molecular docking results. Three-dimensional fluorescence spectral results indicated significant microenvironmental changes around Trp and Tyr residues of HSA upon complexation with AXT. AXT binding to the protein produced significant alterations in both secondary and tertiary structures of HSA, as revealed from the far-UV and the near-UV CD spectral results. Competitive drug displacement results obtained with phenylbutazone (site I marker), ketoprofen (site II marker) and hemin (site III marker) along with molecular docking results suggested Sudlow's site I, located in subdomain IIA of HSA, as the preferred binding site of AXT. PMID:27424099

  3. Appearance of annular ring-like intermediates during amyloid fibril formation from human serum albumin.

    PubMed

    Arya, Shruti; Kumari, Arpana; Dalal, Vijit; Bhattacharya, Mily; Mukhopadhyay, Samrat

    2015-09-21

    The self-assembly of proteins triggered by a conformational switch into highly ordered β-sheet rich amyloid fibrils has captivated burgeoning interest in recent years due to the involvement of amyloids in a variety of human diseases and a diverse range of biological functions. Here, we have investigated the mechanism of fibrillogenesis of human serum albumin (HSA), an all-α-helical protein, using an array of biophysical tools that include steady-state as well as time-resolved fluorescence, circular dichroism and Raman spectroscopy in conjunction with atomic force microscopy (AFM). Investigations into the temporal evolution of nanoscale morphology using AFM revealed the presence of ring-like intermediates that subsequently transformed into worm-like fibrils presumably by a ring-opening mechanism. Additionally, a multitude of morphologically-diverse oligomers were observed on the pathway to amyloid formation. Kinetic analysis using multiple structural probes in-tandem indicated that HSA amyloid assembly is a concerted process encompassing a major structural change that is primarily mediated by hydrophobic interactions between thermally-induced disordered segments originating in various domains. A slower growth kinetics of aggregates suggested that the protein structural reorganization is a prerequisite for fibril formation. Moreover, time-dependent Raman spectroscopic studies of HSA aggregation provided key molecular insights into the conformational transitions occurring within the protein amide backbone and at the residue-specific level. Our data revealed the emergence of conformationally-diverse disulfides as a consequence of structural reorganization and sequestration of tyrosines into the hydrophobic amyloid core comprising antiparallel cross β-sheets.

  4. Quantitation of 4,4′-methylene diphenyl diisocyanate human serum albumin adducts

    PubMed Central

    Luna, Leah G.; Green, Brett J.; Zhang, Fagen; Arnold, Scott M.; Siegel, Paul D.; Bartels, Michael J.

    2016-01-01

    4,4′-Methylene diphenyl diisocyanate (herein 4,4′-MDI) is used in the production of polyurethane foams, elastomers, coatings, adhesives and the like for a wide range of commercial products. Occupational exposure to MDI levels above current airborne exposure limits can elicit immune mediated hypersensitivity reactions such as occupational asthma in sensitive individuals. To accurately determine exposure, there has been increasing interest in developing analytical methods to measure internal biomarkers of exposure to MDI. Previous investigators have reported methodologies for measuring MDI diamine metabolites and MDI-Lysine (4,4′-MDI-Lys) adducts. The purpose of this study was to develop and validate an ultra performance liquid chromatography isotope dilution tandem mass spectrometry (UPLC-ID/MS/MS) quantitation method via a signature peptide approach to enable biomonitoring of 4,4′-MDI adducted to human serum albumin (HSA) in plasma. A murine, anti-4,4′-MDI monoclonal IgM antibody was bound to magnetic beads and utilized for enrichment of the MDI adducted HSA. Following enrichment, trypsin digestion was performed to generate the expected 414 site (primary site of adduction) 4,4′-MDI-adducted HSA signature peptide that was quantified by UPLC-ID/MS/MS. An Agilent 6530 UPLC/quadrupole time of flight MS (QTOF) system was utilized for intact adducted protein analysis and an Agilent 6490 UPLC/MS/MS system operated in multiple reaction monitoring (MRM) mode was utilized for quantification of the adducted signature peptide biomarker both for in chemico and worker serum samples. Worker serum samples were initially screened utilizing the previously developed 4,4′-MDI-Lys amino acid method and results showed that 12 samples were identified as quantifiable for 4,4′-MDI-Lys adducts. The signature peptide adduct approach was applied to the 12 worker samples identified as quantifiable for 4,4′-MDI-Lys adducts. Results indicated no positive results were obtained

  5. Novel curcumin-loaded human serum albumin nanoparticles surface functionalized with folate: characterization and in vitro/vivo evaluation.

    PubMed

    Song, Zhiwang; Lu, Yonglin; Zhang, Xia; Wang, Haiping; Han, Junyi; Dong, Chunyan

    2016-01-01

    Folate-conjugated, curcumin-loaded human serum albumin nanoparticles (F-CM-HSANPs) were obtained by the chemical conjugation of folate to the surface of the curcumin (CM)-loaded human serum albumin nanoparticles (NPs). The NPs were characterized by various parameters, including size, polydispersity, zeta potential, morphology, encapsulation efficiency, and drug release profile. The mean particle size of F-CM-HSANPs was 165.6±15.7 nm (polydispersity index <0.28), and the average encapsulation efficiency percentage and drug loading percentage of the F-CM-HSANPs were 88.7%±4.8% and 7.9%±0.4%, respectively. Applied in vitro, the CM NPs, after conjugation with folate, maintained sustained release, and a faster release of CM was more visibly observed than the unconjugated NPs. F-CM-HSANPs can prolong the retention time of CM significantly in vivo. However, after intravenous injection of F-CM-HSANPs, the pharmacokinetic parameters of CM were not significantly different from those of CM-loaded human serum albumin NPs. The improved antitumor activity of F-CM-HSANPs may be attributable to the protection of drug from enzymatic deactivation followed by the selective localization at the desired site. These results suggest that the intravenous injection of F-CM-HSANPs is likely to have an advantage in the current clinical CM formulation, because it does not require the use of a solubilization agent and it is better able to target the tumor tissue. PMID:27574403

  6. Novel curcumin-loaded human serum albumin nanoparticles surface functionalized with folate: characterization and in vitro/vivo evaluation

    PubMed Central

    Song, Zhiwang; Lu, Yonglin; Zhang, Xia; Wang, Haiping; Han, Junyi; Dong, Chunyan

    2016-01-01

    Folate-conjugated, curcumin-loaded human serum albumin nanoparticles (F-CM-HSANPs) were obtained by the chemical conjugation of folate to the surface of the curcumin (CM)-loaded human serum albumin nanoparticles (NPs). The NPs were characterized by various parameters, including size, polydispersity, zeta potential, morphology, encapsulation efficiency, and drug release profile. The mean particle size of F-CM-HSANPs was 165.6±15.7 nm (polydispersity index <0.28), and the average encapsulation efficiency percentage and drug loading percentage of the F-CM-HSANPs were 88.7%±4.8% and 7.9%±0.4%, respectively. Applied in vitro, the CM NPs, after conjugation with folate, maintained sustained release, and a faster release of CM was more visibly observed than the unconjugated NPs. F-CM-HSANPs can prolong the retention time of CM significantly in vivo. However, after intravenous injection of F-CM-HSANPs, the pharmacokinetic parameters of CM were not significantly different from those of CM-loaded human serum albumin NPs. The improved antitumor activity of F-CM-HSANPs may be attributable to the protection of drug from enzymatic deactivation followed by the selective localization at the desired site. These results suggest that the intravenous injection of F-CM-HSANPs is likely to have an advantage in the current clinical CM formulation, because it does not require the use of a solubilization agent and it is better able to target the tumor tissue. PMID:27574403

  7. Profiling Cys34 Adducts of Human Serum Albumin by Fixed-Step Selected Reaction Monitoring*

    PubMed Central

    Li, He; Grigoryan, Hasmik; Funk, William E.; Lu, Sixin Samantha; Rose, Sherri; Williams, Evan R.; Rappaport, Stephen M.

    2011-01-01

    A method is described for profiling putative adducts (or other unknown covalent modifications) at the Cys34 locus of human serum albumin (HSA), which represents the preferred reaction site for small electrophilic species in human serum. By comparing profiles of putative HSA-Cys34 adducts across populations of interest it is theoretically possible to explore environmental causes of degenerative diseases and cancer caused by both exogenous and endogenous chemicals. We report a novel application of selected-reaction-monitoring (SRM) mass spectrometry, termed fixed-step SRM (FS-SRM), that allows detection of essentially all HSA-Cys34 modifications over a specified range of mass increases (added masses). After tryptic digestion, HSA-Cys34 adducts are contained in the third largest peptide (T3), which contains 21 amino acids and an average mass of 2433.87 Da. The FS-SRM method does not require that exact masses of T3 adducts be known in advance but rather uses a theoretical list of T3-adduct m/z values separated by a fixed increment of 1.5. In terms of added masses, each triply charged parent ion represents a bin of ±2.3 Da between 9.1 Da and 351.1 Da. Synthetic T3 adducts were used to optimize FS-SRM and to establish screening rules based upon selected b- and y-series fragment ions. An isotopically labeled T3 adduct is added to protein digests to facilitate quantification of putative adducts. We used FS-SRM to generate putative adduct profiles from six archived specimens of HSA that had been pooled by gender, race, and smoking status. An average of 66 putative adduct hits (out of a possible 77) were detected in these samples. Putative adducts covered a wide range of concentrations, were most abundant in the mass range below 100 Da, and were more abundant in smokers than in nonsmokers. With minor modifications, the FS-SRM methodology can be applied to other nucleophilic sites and proteins. PMID:21193536

  8. Binding between Saikosaponin C and Human Serum Albumin by Fluorescence Spectroscopy and Molecular Docking.

    PubMed

    Chen, Yi-Cun; Wang, Hong-Mei; Niu, Qing-Xia; Ye, Dan-Yan; Liang, Guo-Wu

    2016-01-28

    Saikosaponin C (SSC) is one of the major active constituents of dried Radix bupleuri root (Chaihu in Chinese) that has been widely used in China to treat a variety of conditions, such as liver disease, for many centuries. The binding of SSC to human serum albumin (HSA) was explored by fluorescence, circular dichroism (CD), UV-vis spectrophotometry, and molecular docking to understand both the pharmacology and the basis of the clinical use of SSC/Chaihu. SSC produced a concentration-dependent quenching effect on the intrinsic fluorescence of HSA, accompanied by a blue shift in the fluorescence spectra. The Stern-Volmer equation showed that this quenching was dominated by static quenching. The binding constant of SSC with HSA was 3.72 × 10³ and 2.99 × 10³ L·mol(-1) at 26 °C and 36 °C, respectively, with a single binding site on each SSC and HSA molecule. Site competitive experiments demonstrated that SSC bound to site I (subdomain IIA) and site II (subdomain IIIA) in HSA. Analysis of thermodynamic parameters indicated that hydrogen bonding and van der Waals forces were mostly responsible for SSC-HSA association. The energy transfer efficiency and binding distance between SSC and HSA was calculated to be 0.23 J and 2.61 nm at 26 °C, respectively. Synchronous fluorescence and CD measurements indicated that SSC affected HSA conformation in the SSC-HSA complex. Molecular docking supported the experimental findings in conformational changes, binding sites and binding forces, and revealed binding of SSC at the interface between subdomains IIA-IIB.

  9. Expression and purification of recombinant human serum albumin from selectively terminable transgenic rice*

    PubMed Central

    Zhang, Qing; Yu, Hui; Zhang, Feng-zhen; Shen, Zhi-cheng

    2013-01-01

    Human serum albumin (HSA) is widely utilized for medical purposes and biochemical research. Transgenic rice has proved to be an attractive bioreactor for mass production of recombinant HSA (rHSA). However, transgene spread is a major environmental and food safety concern for transgenic rice expressing proteins of medical value. This study aimed to develop a selectively terminable transgenic rice line expressing HSA in rice seeds, and a simple process for recovery and purification of rHSA for economical manufacture. An HSA expression cassette was inserted into a T-DNA vector encoding an RNA interference (RNAi) cassette suppressing the CYP81A6 gene. This gene detoxifies the herbicide bentazon and is linked to the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) cassette which confers glyphosate tolerance. ANX Sepharose Fast Flow (ANX FF) anion exchange chromatography coupled with Butyl Sepharose High Performance (Butyl HP) hydrophobic interaction chromatography was used to purify rHSA. A transgenic rice line, HSA-84, was obtained with stable expression of rHSA of up to 0.72% of the total dry weight of the dehusked rice seeds. This line also demonstrated high sensitivity to bentazon, and thus could be killed selectively by a spray of bentazon. A two-step chromatography purification scheme was established to purify the rHSA from rice seeds to a purity of 99% with a recovery of 62.4%. Results from mass spectrometry and N-terminus sequencing suggested that the purified rHSA was identical to natural plasma-derived HSA. This study provides an alternative strategy for large-scale production of HSA with a built-in transgene safety control mechanism. PMID:24101203

  10. Study on the interaction of catechins with human serum albumin using spectroscopic and electrophoretic techniques

    NASA Astrophysics Data System (ADS)

    Trnková, Lucie; Boušová, Iva; Staňková, Veronika; Dršata, Jaroslav

    2011-01-01

    The interaction between eight naturally occurring flavanols (catechin, epicatechin, gallocatechin, epigallocatechin, catechin gallate, epicatechin gallate, gallocatechin gallate, and epigallocatechin gallate) and human serum albumin (HSA) has been investigated by spectroscopic (fluorescence quenching and UV-Vis absorption) and electrophoretic (native and SDS PAGE) techniques under simulated physiological conditions (pH 7.40, 37 °C). The spectroscopic results confirmed the complex formation for the tested systems. The binding constants and the number of binding sites were obtained by analysis of fluorescence data. The strongest binding affinity to HSA was found for epicatechin gallate and decreased in the order epicatechin gallate ⩾ catechin gallate > epigallocatechin gallate > gallocatechin gallate ≫ epicatechin ⩾ catechin > gallocatechin ⩾ epigallocatechin. All free energy changes possessed negative sign indicating the spontaneity of catechin-HSA systems formation. The binding distances between the donor (HSA) and the acceptors (catechins) estimated by the Förster theory revealed that non-radiation energy transfer from HSA to catechins occurred with high possibility. According to results obtained by native PAGE, the galloylated catechins increased the electrophoretic mobility of HSA, which indicated the change in the molecular charge of HSA, whilst the non-galloylated catechins caused no changes. The ability of aggregation and cross-linking of tested catechins with HSA was not proved by SDS-PAGE. The relationship between the structure characteristics of all tested catechins (e.g. presence of the galloyl moiety on the C-ring, the number of hydroxyl groups on the B-ring, and the spatial arrangement of the substituents on the C-ring) and their binding properties to HSA is discussed. The presented study contributes to the current knowledge in the area of protein-ligand binding, particularly catechin-HSA interactions.

  11. Expression and purification of recombinant human serum albumin from selectively terminable transgenic rice.

    PubMed

    Zhang, Qing; Yu, Hui; Zhang, Feng-zhen; Shen, Zhi-cheng

    2013-10-01

    Human serum albumin (HSA) is widely utilized for medical purposes and biochemical research. Transgenic rice has proved to be an attractive bioreactor for mass production of recombinant HSA (rHSA). However, transgene spread is a major environmental and food safety concern for transgenic rice expressing proteins of medical value. This study aimed to develop a selectively terminable transgenic rice line expressing HSA in rice seeds, and a simple process for recovery and purification of rHSA for economical manufacture. An HSA expression cassette was inserted into a T-DNA vector encoding an RNA interference (RNAi) cassette suppressing the CYP81A6 gene. This gene detoxifies the herbicide bentazon and is linked to the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) cassette which confers glyphosate tolerance. ANX Sepharose Fast Flow (ANX FF) anion exchange chromatography coupled with Butyl Sepharose High Performance (Butyl HP) hydrophobic interaction chromatography was used to purify rHSA. A transgenic rice line, HSA-84, was obtained with stable expression of rHSA of up to 0.72% of the total dry weight of the dehusked rice seeds. This line also demonstrated high sensitivity to bentazon, and thus could be killed selectively by a spray of bentazon. A two-step chromatography purification scheme was established to purify the rHSA from rice seeds to a purity of 99% with a recovery of 62.4%. Results from mass spectrometry and N-terminus sequencing suggested that the purified rHSA was identical to natural plasma-derived HSA. This study provides an alternative strategy for large-scale production of HSA with a built-in transgene safety control mechanism.

  12. Binding of methacycline to human serum albumin at subdomain IIA using multispectroscopic and molecular modeling methods.

    PubMed

    Dong, Chengyu; Lu, Ningning; Liu, Ying

    2013-01-01

    This study was designed to examine the interaction of methacyline (METC) with human serum albumin (HSA) by multispectroscopy and a molecular modeling method under simulative physiological conditions. The quenching mechanism was suggested to be static quenching based on fluorescence and ultraviolet-visible (UV-Vis) spectroscopy. According to the Vant' Hoff equation, the values of enthalpy (∆H) and entropy change (∆S) were calculated to be -95.29 kJ/mol and -218.13 J/mol/K, indicating that the main driving force of the interaction between HSA and METC were hydrogen bonds and van der Waals's forces. By performing displacement measurements, the specific binding of METC in the vicinity of Sudlow's site I of HSA was clarified. An apparent distance of 3.05 nm between Trp214 and METC was obtained via the fluorescence resonance energy transfer (FRET) method. Furthermore, the binding details between METC and HSA were further confirmed by molecular docking studies, which revealed that METC was bound at subdomain IIA through multiple interactions, such as hydrophobic effect, polar forces, hydrogen bonding, etc. The results of three-dimensional fluorescence and Fourier transform infrared (FTIR) spectroscopy showed that METC caused conformational and some microenvironmental changes in HSA and reduced the α-helix significantly in the range of 52.3-40.4% in HSA secondary structure. Moreover, the coexistence of metal ions such as Ca(2+), Al(3+), Fe(3+), Zn(2+), Cu(2+), Cr(3+) and Cd(2+) can decrease the binding constants of METC-HSA. PMID:23233363

  13. Spectroscopic analysis and molecular modeling on the interaction of jatrorrhizine with human serum albumin (HSA)

    NASA Astrophysics Data System (ADS)

    Li, Junfen; Li, Jinzeng; Jiao, Yong; Dong, Chuan

    2014-01-01

    In this work, the interaction of jatrorrhizine with human serum albumin (HSA) was studied by means of UV-vis and fluorescence spectra. The intrinsic fluorescence of HSA was quenched by jatrorrhizine, which was rationalized in terms of the static quenching mechanism. The results show that jatrorrhizine can obviously bind to HSA molecules. According to fluorescence quenching calculations, the bimolecular quenching constant (kq), apparent quenching constant (KSV) at different temperatures were obtained. The binding constants K are 4059 L mol-1 and 1438 L mol-1 at 299 K and 304 K respectively, and the number of binding sites n is almost 1. The thermodynamic parameters determined by the Van't Hoff analysis of the binding constants (ΔH -12.25 kJ mol-1 and ΔS 28.17 J mol-1 K-1) clearly indicate that the electrostatic force plays a major role in the process. The efficiency of energy transfer and the distance between the donor (HSA) and the acceptor (jatrorrhizine) were calculated as 22.2% and 3.19 nm according to Föster's non-radiative energy transfer theory. In addition, synchronous fluorescence spectroscopy reveals that jatrorrhizine can influence HSA's microstructure. That is, jatrorrhizine is more vicinal to tryptophane (Trp) residue than to tyrosine (Tyr) residue and the damage site is also mainly at Trp residue. Molecular modeling result shows that jatrorrhizine-HSA complex formed not only on the basis of electrostatic forces, but also on the basis of π-π staking and hydrogen bond. The research results will offer a reference for the studies on the biological effects and action mechanism of small molecule with protein.

  14. Exploring the interaction between picoplatin and human serum albumin: The effects on protein structure and activity.

    PubMed

    Wang, Yanqing; Wu, Peirong; Zhou, Xinchun; Zhang, Hongmei; Qiu, Ligan; Cao, Jian

    2016-09-01

    For the first time, the effects of picoplatin on the structure and esterase-like catalytic activity of human serum albumin (HSA) have been investigated by spectroscopic approaches and molecular modeling. The circular dichroism (CD) spectral examinations indicated that the binding of picoplatin with HSA induced a slight decrease of a-helix content of protein and unfolded the constituent polypeptides of the protein. The synchronous fluorescence and three-dimensional fluorescence spectral methods were used to estimate the effect of picoplatin on the micro-environmental changes of the Trp and Tyr residues of HSA, indicating that the micro-environment around the Tyr and Trp residue is partly disturbed by picoplatin. UV-vis absorption spectral result indicated the formation of the ground state complex between picoplatin with HSA. The ANS binding assay indicated the existence of competitive combination of picoplatin and ANS with HSA. The studies on the effects of picoplatin on the binding of HSA with bilirubin and heme showed that picoplatin binding caused a change of angle between two chromophores of bound bilirubin and the binding site of picoplatin does not locate in subdomain IB in HSA that bound with heme. The molecular modeling results showed that picoplatin binds to the connection between domain I and domain II by hydrophobic, hydrogen bonds, and van der Waals forces. In addition, HSA maintains most of its esterase activity in the presence of picoplatin. The investigations on how picoplatin interacts with HSA are important for the understanding of the anticancer mechanism and toxicity of platinum-based anticancer drug. PMID:27484966

  15. Hypohalous acid-modified human serum albumin induces neutrophil NADPH oxidase activation, degranulation, and shape change.

    PubMed

    Gorudko, Irina V; Grigorieva, Daria V; Shamova, Ekaterina V; Kostevich, Valeria A; Sokolov, Alexey V; Mikhalchik, Elena V; Cherenkevich, Sergey N; Arnhold, Jürgen; Panasenko, Oleg M

    2014-03-01

    Halogenated lipids, proteins, and lipoproteins formed in reactions with myeloperoxidase (MPO)-derived hypochlorous acid (HOCl) and hypobromous acid (HOBr) can contribute to the regulation of functional activity of cells and serve as mediators of inflammation. Human serum albumin (HSA) is the major plasma protein target of hypohalous acids. This study was performed to assess the potency of HSA modified by HOCl (HSA-Cl) and HOBr (HSA-Br) to elicit selected neutrophil responses. HSA-Cl/Br were found to induce neutrophil degranulation, generation of reactive oxygen intermediates, shape change, and actin cytoskeleton reorganization. Thus HSA-Cl/Br can initially act as a switch and then as a feeder of the "inflammatory loop" under oxidative stress. In HSA-Cl/Br-treated neutrophils, monoclonal antibodies against CD18, the β subunit of β2 integrins, reduced the production of superoxide anion radicals and hydrogen peroxide as well as MPO exocytosis, suggesting that CD18 contributed to neutrophil activation. HSA-Cl/Br-induced neutrophil responses were also inhibited by genistein, a broad-specificity tyrosine kinase inhibitor, and wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor, supporting the notion that activation of both tyrosine kinase and PI3K may play a role in neutrophil activation by HSA modified in MPO-dependent reactions. These results confirm the hypothesis that halogenated molecules formed in vivo via MPO-dependent reactions can be considered as a new class of biologically active substances potentially able to contribute to activation of myeloid cells in sites of inflammation and serve as inflammatory response modulators. PMID:24384524

  16. Optimization of human serum albumin monoliths for chiral separations and high-performance affinity chromatography

    PubMed Central

    Pfaunmiller, Erika L.; Hartmann, Mahli; Dupper, Courtney M.; Soman, Sony; Hage, David S.

    2012-01-01

    Various organic-based monoliths were prepared and optimized for immobilization of the protein human serum albumin (HSA) as a binding agent for chiral separations and high-performance affinity chromatography. These monoliths contained co-polymers based on glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) or GMA and trimethylolpropane trimethacrylate (TRIM). A mixture of cyclohexanol and 1-dodecanol was used as the porogen, with the ratio of these solvents being varied along with the polymerization temperature to generate a library of monoliths. These monoliths were used with both the Schiff base and epoxy immobilization methods and measured for their final content of HSA. Monoliths showing the highest protein content were further evaluated in chromatographic studies using R/S-warfarin and d/l-tryptophan as model chiral solutes. A 2.6–2.7-fold increase in HSA content was obtained in the final monoliths when compared to similar HSA monoliths prepared according to the literature. The increased protein content made it possible for the new monoliths to provide higher retention and/or two-fold faster separations for the tested solutes when using 4.6 mm i.d. × 50 mm columns. These monoliths were also used to create 4.6 mm i.d. × 10 mm HSA microcolumns that could separate the same chiral solutes in only 1.5–6.0 min. The approaches used in this study could be extended to the separation of other chiral solutes and to the optimization of organic monoliths for use with additional proteins as binding agents. PMID:23010249

  17. Study of the interaction of C60 fullerene with human serum albumin in aqueous solution

    SciTech Connect

    Li, Song; Zhao, Xiongce; Mo, Yiming; Cummings, Peter T; Heller, William T

    2013-01-01

    Concern about the toxicity of engineered nanoparticles, such as the prototypical nanomaterial C60 fullerene, continues to grow. While evidence continues to mount that C60 and its derivatives may pose health hazards, the specific molecular interactions of these particles with biological macromolecules require further investigation. To better understand the interaction of C60 with proteins, the protein human serum albumin (HSA) was studied in solution with C60 at C60:HSA molar ratios ranging from 1:2 to 4:1. HSA is the major protein component of blood plasma and plays a role in a variety of functions, such as the maintenance of blood pH and pressure. The C60-HSA interaction was probed by a combination of circular dichroism (CD) spectroscopy, small-angle neutron scattering (SANS) and atomistic molecular dynamics (MD) simulations to understand C60-driven changes in the structure of HSA in solution. The CD spectroscopy demonstrates that the secondary structure of the protein decreases in -helical content in response to the presence of C60. Similarly, C60 produces subtle changes in the solution conformation of HSA, as evidenced by the SANS data and MD. The data do not indicate that C60 is causing a change in the oligomerization state of the protein. Taken together results demonstrate that C60 interacts with HSA, but it does not strongly perturb the structure of the protein by unfolding it or inducing aggregation, suggesting a mechanism for transporting C60 throughout the body to accumulate in various tissues.

  18. Novel 7-(dimethylamino)fluorene-based fluorescent probes and their binding to human serum albumin.

    PubMed

    Park, Kwanghee Koh; Park, Joon Woo; Hamilton, Andrew D

    2009-10-21

    A novel solvatochromic fluorescent molecule, 9,9-dibutyl-7-(dimethylamino)-2-fluorenesulfonate 2 was synthesized from 2-nitrofluorene in moderate yield. The fluorescence spectra of 2 and 7-(dimethylamino)-2-fluorenesulfonate 1 shift to shorter wavelengths as the polarity of the medium decreases. Both 1 and 2 bind to hydrophobic sites of human serum albumin (HSA). The apparent binding constants were determined by fluorescence titration to be 0.37 x 10(6) M(-1) for 1 and 2.2 x 10(6) M(-1) for 2. The energy of the Trp-214 fluorescence of HSA is transferred to the HSA-bound fluorophores with near 100% efficiency. The covalent bonding of acrylodan (AC) to Cys-34 has little effect on the binding affinity of 2 to HSA or fluorescent behavior of HSA-bound 2. Bound 2 also has little effect on the fluorescence of AC, but 2-->AC and Trp-214-->2-->AC resonance energy transfers were observed. Competitive binding between the fluorene compounds and other ligands such as 1-anilino-8-naphthalenesulfonate, aspirin, S-(+)-ibuprofen and phenylbutazone were also studied fluorometrically. The results indicated that the primary binding site of 2 to HSA is site II in domain IIIA, whereas 1 binds to site I in domain IIA, but a different region from the phenylbutazone binding site. Because of its large molar absorptivity, strong fluorescence, sensitivity to its environment, and high binding constant to HSA, 2 can be used successfully in the study of proteins and their binding properties. PMID:19795061

  19. Dynamics of loop 1 of domain I in human serum albumin when dissolved in ionic liquids.

    PubMed

    Page, Taylor A; Kraut, Nadine D; Page, Phillip M; Baker, Gary A; Bright, Frank V

    2009-09-24

    We report on the rotational reorientation dynamics associated with loop 1 of domain I within a large multidomain protein (human serum albumin, HSA) when it is dissolved in binary mixtures of ionic liquid (1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([C4mim][Tf2N]), 1-butyl-3-methylimidazolium tetrafluoroborate ([C4mim][BF4]), or 1-butyl-3-methylimidazolium hexafluorophosphate ([C4mim][PF6])) and distilled deionized water (ddH2O) as a function of temperature and water loading. In IL/2% ddH2O (v/v) mixtures, loop 1 of domain I is more significantly denatured in comparison to the protein dissolved in aqueous solutions containing strong chemical denaturants (e.g., 8 M guanidine HCl (Gu.HCl) or urea). As water loading increases, there is evidence for progressive refolding of loop 1 of domain I followed by recoupling with domains I, II, and III in the [C4mim][BF4]/ddH2O mixtures at 20 degrees C. Above 30% (v/v) water, where domain I appears refolded, the Ac reporter molecule's semiangle steadily decreases from 35 degrees to 20 degrees with increasing water loading. From the perspective of domain I in HSA, this behavior is similar to the effects of dilution from 4 to 0 M Gu.HCl in aqueous solution. Overall, these results lend insight into the tangle of biocatalytic and structural/dynamical mechanisms that enzymes may undergo in ionic liquid-based systems. It will be particularly motivating to extend this work to include enzyme-attuned ionic liquids shown to improve biocatalytic performance beyond that possible in the native (predominantly aqueous) setting.

  20. Tandem mass spectral libraries of peptides in digests of individual proteins: Human Serum Albumin (HSA).

    PubMed

    Dong, Qian; Yan, Xinjian; Kilpatrick, Lisa E; Liang, Yuxue; Mirokhin, Yuri A; Roth, Jeri S; Rudnick, Paul A; Stein, Stephen E

    2014-09-01

    This work presents a method for creating a mass spectral library containing tandem spectra of identifiable peptide ions in the tryptic digestion of a single protein. Human serum albumin (HSA(1)) was selected for this purpose owing to its ubiquity, high level of characterization and availability of digest data. The underlying experimental data consisted of ∼3000 one-dimensional LC-ESI-MS/MS runs with ion-trap fragmentation. In order to generate a wide range of peptides, studies covered a broad set of instrument and digestion conditions using multiple sources of HSA and trypsin. Computer methods were developed to enable the reliable identification and reference spectrum extraction of all peptide ions identifiable by current sequence search methods. This process made use of both MS2 (tandem) spectra and MS1 (electrospray) data. Identified spectra were generated for 2918 different peptide ions, using a variety of manually-validated filters to ensure spectrum quality and identification reliability. The resulting library was composed of 10% conventional tryptic and 29% semitryptic peptide ions, along with 42% tryptic peptide ions with known or unknown modifications, which included both analytical artifacts and post-translational modifications (PTMs) present in the original HSA. The remaining 19% contained unexpected missed-cleavages or were under/over alkylated. The methods described can be extended to create equivalent spectral libraries for any target protein. Such libraries have a number of applications in addition to their known advantages of speed and sensitivity, including the ready re-identification of known PTMs, rejection of artifact spectra and a means of assessing sample and digestion quality.

  1. Binding of naproxen enantiomers to human serum albumin studied by fluorescence and room-temperature phosphorescence

    NASA Astrophysics Data System (ADS)

    Lammers, Ivonne; Lhiaubet-Vallet, Virginie; Ariese, Freek; Miranda, Miguel A.; Gooijer, Cees

    2013-03-01

    The interaction of the enantiomers of the non-steroidal anti-inflammatory drug naproxen (NPX) with human serum albumin (HSA) has been investigated using fluorescence and phosphorescence spectroscopy in the steady-state and time-resolved mode. The absorption, fluorescence excitation, and fluorescence emission spectra of (S)-NPX and (R)-NPX differ in shape in the presence of HSA, indicating that these enantiomers experience a different environment when bound. In solutions containing 0.2 M KI, complexation with HSA results in a strongly increased NPX fluorescence intensity and a decreased NPX phosphorescence intensity due to the inhibition of the collisional interaction with the heavy atom iodide. Fluorescence intensity curves obtained upon selective excitation of NPX show 8-fold different slopes for bound and free NPX. No significant difference in the binding constants of (3.8 ± 0.6) × 105 M-1 for (S)-NPX and (3.9 ± 0.6) × 105 M-1 for (R)-NPX was found. Furthermore, the addition of NPX quenches the phosphorescence of the single tryptophan in HSA (Trp-214) based on Dexter energy transfer. The short-range nature of this mechanism explains the upward curvature of the Stern-Volmer plot observed for HSA: At low concentrations NPX binds to HSA at a distance from Trp-214 and no quenching occurs, whereas at high NPX concentrations the phosphorescence intensity decreases due to dynamic quenching by NPX diffusing into site I from the bulk solution. The dynamic quenching observed in the Stern-Volmer plots based on the longest phosphorescence lifetime indicates an overall binding constant to HSA of about 3 × 105 M-1 for both enantiomers.

  2. Synthesis of the native copper(II)-transport site of human serum albumin and its copper(II)-binding properties.

    PubMed Central

    Iyer, K S; Lau, S J; Laurie, S H; Sarkar, B

    1978-01-01

    A derivative of the native-sequence tripeptide of the specific Cu(II)-transport site of human serum albumin, L-aspartyl-L-alanyl-L-histidine N-methylamide, was synthesized, and its binding to Cu(II) was examined to determine the influence of the side-chain groups on the Cu(II) binding. The equilibria involved in the Cu(II)-L-aspartyl-L-alanyl-L-histidine N-methylamide system were investigated by analytical potentiometry. Three complex species were found in the pH range 4-10. The same species were identified in both the visible and circular-dichroism spectra. The main species present in the physiological pH range is shown to have the same ligands around the square-planar Cu(II) ion as those reported for albumin and tripeptides diglycyl-L-histidine and its N-methylamide derivative. The results obtained from competition experiments showed that this tripeptide has a higher affinity towards Cu(II) than has albumin itself. The overall findings are compared with those from albumin. At neutral pH the side chains do not play any important role in the Cu(II) binding, but at low pH the beta-carboxyl group of the N-terminal aspartic residue becomes important. A possible competition site on albumin for Cu(II) at low pH is discussed. PMID:24440

  3. Subnanosecond fluorescence spectroscopy of human serum albumin as a method to estimate the efficiency of the depression therapy

    NASA Astrophysics Data System (ADS)

    Syrejshchikova, T. I.; Gryzunov, Yu. A.; Smolina, N. V.; Komar, A. A.; Uzbekov, M. G.; Misionzhnik, E. J.; Maksimova, N. M.

    2010-05-01

    The efficiency of the therapy of psychiatric diseases is estimated using the fluorescence measurements of the conformational changes of human serum albumin in the course of medical treatment. The fluorescence decay curves of the CAPIDAN probe (N-carboxyphenylimide of the dimethylaminonaphthalic acid) in the blood serum are measured. The probe is specifically bound to the albumin drug binding sites and exhibits fluorescence as a reporter ligand. A variation in the conformation of the albumin molecule substantially affects the CAPIDAN fluorescence decay curve on the subnanosecond time scale. A subnanosecond pulsed laser or a Pico-Quant LED excitation source and a fast photon detector with a time resolution of about 50 ps are used for the kinetic measurements. The blood sera of ten patients suffering from depression and treated at the Institute of Psychiatry were preliminary clinically tested. Blood for analysis was taken from each patient prior to the treatment and on the third week of treatment. For ten patients, the analysis of the fluorescence decay curves of the probe in the blood serum using the three-exponential fitting shows that the difference between the amplitudes of the decay function corresponding to the long-lived (9 ns) fluorescence of the probe prior to and after the therapeutic procedure reliably differs from zero at a significance level of 1% ( p < 0.01).

  4. Comparative studies on drug binding to the purified and pharmaceutical-grade human serum albumins: Bridging between basic research and clinical applications of albumin.

    PubMed

    Ashrafi-Kooshk, Mohammad Reza; Ebrahimi, Farangis; Ranjbar, Samira; Ghobadi, Sirous; Moradi, Nastaran; Khodarahmi, Reza

    2015-09-01

    Human serum albumin (HSA), the most abundant protein in blood plasma, is a monomeric multidomain protein that possesses an extraordinary capacity for binding, so that serves as a circulating depot for endogenous and exogenous compounds. During the heat sterilization process, the structure of pharmaceutical-grade HSA may change and some of its activities may be lost. In this study, to provide deeper insight on this issue, we investigated drug-binding and some physicochemical properties of purified albumin (PA) and pharmaceutical-grade albumin (PGA) using two known drugs (indomethacin and ibuprofen). PGA displayed significantly lower drug binding capacity compared to PA. Analysis of the quenching and thermodynamic parameters indicated that intermolecular interactions between the drugs and the proteins are different from each other. Surface hydrophobicity as well as the stability of PGA decreased compared to PA, also surface hydrophobicity of PA and PGA increased upon drugs binding. Also, kinetic analysis of pseudo-esterase activities indicated that Km and Vmax parameters for PGA enzymatic activity are more and less than those of PA, respectively. This in vitro study demonstrates that the specific drug binding of PGA is significantly reduced. Such studies can act as connecting bridge between basic research discoveries and clinical applications.

  5. Unraveling the binding mechanism of asiatic acid with human serum albumin and its biological implications.

    PubMed

    Gokara, Mahesh; Malavath, Tirupathi; Kalangi, Suresh Kumar; Reddana, Pallu; Subramanyam, Rajagopal

    2014-01-01

    Asiatic acid (AsA), a naturally occurring pentacyclictriterpenoid found in Centella asiatica, plays a major role in neuroprotection, anticancer, antioxidant, and hepatoprotective activities. Human serum albumin (HSA), a blood plasma protein, participates in the regulation of plasma osmotic pressure and transports endogenous and exogenous substances. The study undertaken to analyze the drug-binding mechanisms of HSA is crucial in understanding the bioavailability of drugs. In this study, we analyzed the cytotoxic activity of AsA on HepG2 (human hepatocellular carcinoma) cell lines and its binding, conformational, docking, molecular simulation studies with HSA under physiological pH 7.2. These studies revealed a clear decrease in the viability of HepG2 cells upon exposure to AsA in a dose-dependent manner with an IC50 of 45 μM. Further studies showed the quenching of intrinsic fluorescence of HSA by AsA with a binding constant of KAsA = 3.86 ± 0.01 × 10(4) M(-1), which corresponds to the free energy of (ΔG) -6.3 kcal M(-1) at 25 °C. Circular dichroism (CD) studies revealed that there is a clear decrease in the α-helical content from 57.50 ± 2.4 to 50% ± 2.3 and an increase in the β-turns from 25 ± 0.65 to 29% ± 0.91 and random coils from 17.5% ± 0.95 to 21% ± 1.2, suggesting partial unfolding of HSA. Autodock studies revealed that the AsA is bound to the subdomain IIA with hydrophobic and hydrophilic interactions. From molecular dynamics, simulation data (RMSD, Rg and RMSF) emphasized the local conformational changes and rigidity of the residues of both HSA and HSA-AsA complexes. PMID:23844909

  6. Existence of Different Structural Intermediates on the Fibrillation Pathway of Human Serum Albumin

    PubMed Central

    Juárez, Josué; Taboada, Pablo; Mosquera, Víctor

    2009-01-01

    The fibrillation propensity of the multidomain protein human serum albumin (HSA) was analyzed under different solution conditions. The aggregation kinetics, protein conformational changes upon self-assembly, and structure of the different intermediates on the fibrillation pathway were determined by means of thioflavin T (ThT) fluorescence and Congo Red absorbance; far- and near-ultraviolet circular dichroism; tryptophan fluorescence; Fourier transform infrared spectroscopy; x-ray diffraction; and transmission electron, scanning electron, atomic force, and microscopies. HSA fibrillation extends over several days of incubation without the presence of a lag phase, except for HSA samples incubated at acidic pH and room temperature in the absence of electrolyte. The absence of a lag phase occurs if the initial aggregation is a downhill process that does not require a highly organized and unstable nucleus. The fibrillation process is accompanied by a progressive increase in the β-sheet (up to 26%) and unordered conformation at the expense of α-helical conformation, as revealed by ThT fluorescence and circular dichroism and Fourier transform infrared spectroscopies, but changes in the secondary structure contents depend on solution conditions. These changes also involve the presence of different structural intermediates in the aggregation pathway, such as oligomeric clusters (globules), bead-like structures, and ring-shaped aggregates. We suggest that fibril formation may take place through the role of association-competent oligomeric intermediates, resulting in a kinetic pathway via clustering of these oligomeric species to yield protofibrils and then fibrils. The resultant fibrils are elongated but curly, and differ in length depending on solution conditions. Under acidic conditions, circular fibrils are commonly observed if the fibrils are sufficiently flexible and long enough for the ends to find themselves regularly in close proximity to each other. These fibrils

  7. Preparation of protein imprinted materials by hierarchical imprinting techniques and application in selective depletion of albumin from human serum

    NASA Astrophysics Data System (ADS)

    Liu, Jinxiang; Deng, Qiliang; Tao, Dingyin; Yang, Kaiguang; Zhang, Lihua; Liang, Zhen; Zhang, Yukui

    2014-06-01

    Hierarchical imprinting was developed to prepare the protein imprinted materials, as the artificial antibody, for the selective depletion of HSA from the human serum proteome. Porcine serum albumin (PSA) was employed as the dummy template for the fabrication of the recognition sites. To demonstrate the advantages of the hierarchical imprinting, molecularly imprinted polymers prepared by hierarchical imprinting technique (h-MIPs) were compared with those obtained by bulk imprinting (b-MIPs), in terms of the binding capacity, adsorption kinetics, selectivity and synthesis reproducibility. The binding capacity of h-MIPs could reach 12 mg g-1. And saturation binding could be reached in less than 20 min for the h-MIPs. In the protein mixture, h-MIPs exhibit excellent selectivity for PSA, with imprinting factors as about 3.6, much higher than those for non-template proteins. For the proteomic application, the identified protein group number in serum treated by h-MIPs was increased to 422, which is 21% higher than that obtained from the original serum, meanwhile the identified protein group number for the Albumin Removal kit was only 376. The results demonstrate that protein imprinted polymers prepared by hierarchical imprinting technique, might become the artificial antibodies for the selective depletion of high abundance proteins in proteome study.

  8. Sandwich enzyme-linked immunosorbent assay for the quantification of human serum albumin fragment 408-423 in bodily fluids.

    PubMed

    Mohr, Katharina B; Zirafi, Onofrio; Hennies, Mark; Wiese, Sebastian; Kirchhoff, Frank; Münch, Jan

    2015-05-01

    Urinary levels of human serum albumin (hSA) fragment 408-423 have been proposed to represent an early marker for graft-versus-host disease (GvHD) and chronic kidney diseases. Here, we developed an enzyme-linked immunosorbent assay (ELISA) for the quantification of hSA(408-423). The sandwich ELISA has a detection limit of 0.5ng/ml and is highly specific for hSA(408-423) because it does not cross-react with other albumin fragments or the full-length precursor. This ELISA allows rapid and convenient quantification of hSA(408-423) in bodily fluids, further clarifying the prognostic and diagnostic value of this peptide in GvHD, kidney disease, and other disorders.

  9. Reevaluation of ANS binding to human and bovine serum albumins: key role of equilibrium microdialysis in ligand - receptor binding characterization.

    PubMed

    Kuznetsova, Irina M; Sulatskaya, Anna I; Povarova, Olga I; Turoverov, Konstantin K

    2012-01-01

    In this work we return to the problem of the determination of ligand-receptor binding stoichiometry and binding constants. In many cases the ligand is a fluorescent dye which has low fluorescence quantum yield in free state but forms highly fluorescent complex with target receptor. That is why many researchers use dye fluorescence for determination of its binding parameters with receptor, but they leave out of account that fluorescence intensity is proportional to the part of the light absorbed by the solution rather than to the concentration of bound dye. We showed how ligand-receptor binding parameters can be determined by spectrophotometry of the solutions prepared by equilibrium microdialysis. We determined the binding parameters of ANS - human serum albumin (HSA) and ANS - bovine serum albumin (BSA) interaction, absorption spectra, concentration and molar extinction coefficient, as well as fluorescence quantum yield of the bound dye. It was found that HSA and BSA have two binding modes with significantly different affinity to ANS. Correct determination of the binding parameters of ligand-receptor interaction is important for fundamental investigations and practical aspects of molecule medicine and pharmaceutics. The data obtained for albumins are important in connection with their role as drugs transporters.

  10. Correlated and Anticorrelated Domain Movement of Human Serum Albumin: A Peek into the Complexity of the Crowded Milieu.

    PubMed

    Biswas, Saikat; Chowdhury, Pramit Kumar

    2016-06-01

    Protein dynamics in cells have been shown to be markedly different from that in dilute solutions because of the highly crowded cellular interior. The volume exclusion arising from the high concentration of macromolecules present can affect both equilibrium and kinetic processes involving protein conformational changes. While global changes in structure leading to modulations in the stability of the protein have been well-documented, local changes that can have a large bearing on the functional aspects of these biomolecules are rare to come across. Using the multidomain serum protein human serum albumin and a fluorescence resonance energy transfer (FRET)-based approach, with fluorescent reporters in each of its three domains, we, in this article, have provided a detailed mapping of variations in the interdomain distances (as a function of pH) in the presence of five macromolecular crowding agents, differing based on their constituent monomers and average molecular weight(s). From the observation of correlated domain movements for dextran based crowding agents to anticorrelated motion induced by Ficoll 70, and both correlated and anticorrelated action for PEG8000 (PEG8), our results reveal the inherent complexity of a crowded milieu with the serum protein serving as an able sensor for decoding such variations. Differences in the manner in which the macromolecular crowders of similar average molecular weights influence the protein conformational ensemble also provide insights into the possible variations at the molecular level that these polymeric molecules possess. Evidence is presented in support of the fact that for the large molecular weight crowding agents and PEG8, soft interactions predominate over hard sphere potentials. Finally, the nature of domain movements encountered for the serum protein are of immense significance with respect to the function of human serum albumin (HSA) as a prolific binder and transporter of small molecules. PMID:27163260

  11. Spectroscopic investigations of the interactions of tramadol hydrochloride and 5-azacytidine drugs with human serum albumin and human hemoglobin proteins.

    PubMed

    Tunç, Sibel; Cetinkaya, Ahmet; Duman, Osman

    2013-03-01

    The interactions of tramadol hydrochloride (THC) and 5-azacytidine (AZA) drugs with human serum albumin (HSA) and human hemoglobin (HMG) proteins were investigated by fluorescence, UV absorption and circular dichroism (CD) spectroscopy at pH 7.4 and different temperatures. The UV absorption spectra and the fluorescence quenching of HSA and HMG proteins indicated the formation of HSA-THC and HMG-THC complexes via static quenching mechanism. AZA did not interact with HSA and HMG proteins. It was found that the formation of HMG-THC complex was stronger than that of HSA-THC complex. The stability of HSA-THC and HMG-THC complexes decreased with increasing temperature. The number of binding site was found as one for HSA-THC and HMG-THC systems. Negative enthalpy change (ΔH) and Gibbs free energy change (ΔG) and positive entropy change (ΔS) values were obtained for these systems. The binding of THC-HSA and HMG proteins was spontaneous and exothermic. In addition, electrostatic interactions between protein and drug molecules played an important role in the binding processes. The results of CD analysis revealed that the addition of THC led to a significant conformational change in the secondary structure of HSA protein, on the contrary to HMG protein. PMID:23428887

  12. Serum Albumin and Ca2+ Are Natural Competence Inducers in the Human Pathogen Acinetobacter baumannii.

    PubMed

    Traglia, German Matias; Quinn, Brettni; Schramm, Sareda T J; Soler-Bistue, Alfonso; Ramirez, Maria Soledad

    2016-08-01

    The increasing frequency of bacteria showing antimicrobial resistance (AMR) raises the menace of entering into a postantibiotic era. Horizontal gene transfer (HGT) is one of the prime reasons for AMR acquisition. Acinetobacter baumannii is a nosocomial pathogen with outstanding abilities to survive in the hospital environment and to acquire resistance determinants. Its capacity to incorporate exogenous DNA is a major source of AMR genes; however, few studies have addressed this subject. The transformation machinery as well as the factors that induce natural competence in A. baumannii are unknown. In this study, we demonstrate that naturally competent strain A118 increases its natural transformation frequency upon the addition of Ca(2+)or albumin. We show that comEA and pilQ are involved in this process since their expression levels are increased upon the addition of these compounds. An unspecific protein, like casein, does not reproduce this effect, showing that albumin's effect is specific. Our work describes the first specific inducers of natural competence in A. baumannii Overall, our results suggest that the main protein in blood enhances HGT in A. baumannii, contributing to the increase of AMR in this threatening human pathogen.

  13. Identification and mapping of linear antibody epitopes in human serum albumin using high-density Peptide arrays.

    PubMed

    Hansen, Lajla Bruntse; Buus, Soren; Schafer-Nielsen, Claus

    2013-01-01

    We have recently developed a high-density photolithographic, peptide array technology with a theoretical upper limit of 2 million different peptides per array of 2 cm(2). Here, we have used this to perform complete and exhaustive analyses of linear B cell epitopes of a medium sized protein target using human serum albumin (HSA) as an example. All possible overlapping 15-mers from HSA were synthesized and probed with a commercially available polyclonal rabbit anti-HSA antibody preparation. To allow for identification of even the weakest epitopes and at the same time perform a detailed characterization of key residues involved in antibody binding, the array also included complete single substitution scans (i.e. including each of the 20 common amino acids) at each position of each 15-mer peptide. As specificity controls, all possible 15-mer peptides from bovine serum albumin (BSA) and from rabbit serum albumin (RSA) were included as well. The resulting layout contained more than 200.000 peptide fields and could be synthesized in a single array on a microscope slide. More than 20 linear epitope candidates were identified and characterized at high resolution i.e. identifying which amino acids in which positions were needed, or not needed, for antibody interaction. As expected, moderate cross-reaction with some peptides in BSA was identified whereas no cross-reaction was observed with peptides from RSA. We conclude that high-density peptide microarrays are a very powerful methodology to identify and characterize linear antibody epitopes, and should advance detailed description of individual specificities at the single antibody level as well as serologic analysis at the proteome-wide level.

  14. Differential Effects of Methoxy Group on the Interaction of Curcuminoids with Two Major Ligand Binding Sites of Human Serum Albumin

    PubMed Central

    Sato, Hiroki; Chuang, Victor Tuan Giam; Yamasaki, Keishi; Yamaotsu, Noriyuki; Watanabe, Hiroshi; Nagumo, Kohei; Anraku, Makoto; Kadowaki, Daisuke; Ishima, Yu; Hirono, Shuichi; Otagiri, Masaki; Maruyama, Toru

    2014-01-01

    Curcuminoids are a group of compounds with a similar chemical backbone structure but containing different numbers of methoxy groups that have therapeutic potential due to their anti-inflammatory and anti-oxidant properties. They mainly bind to albumin in plasma. These findings influence their body disposition and biological activities. Spectroscopic analysis using site specific probes on human serum albumin (HSA) clearly indicated that curcumin (Cur), demethylcurcumin (Dmc) and bisdemethoxycurcumin (Bdmc) bind to both Site I (sub-site Ia and Ib) and Site II on HSA. At pH 7.4, the binding constants for Site I were relatively comparable between curcuminoids, while the binding constants for Site II at pH 7.4 were increased in order Cur < Dmc < Bdmc. Binding experiments using HSA mutants showed that Trp214 and Arg218 at Site I, and Tyr411 and Arg410 at Site II are involved in the binding of curcuminoids. The molecular docking of all curcuminoids to the Site I pocket showed that curcuminoids stacked with Phe211 and Trp214, and interacted with hydrophobic and aromatic amino acid residues. In contrast, each curcuminoid interacted with Site II in a different manner depending whether a methoxy group was present or absent. A detailed analysis of curcuminoids-albumin interactions would provide valuable information in terms of understanding the pharmacokinetics and the biological activities of this class of compounds. PMID:24498401

  15. Evidence that L-Arginine inhibits glycation of human serum albumin (HSA) in vitro

    SciTech Connect

    Servetnick, D.A.; Wiesenfeld, P.L.; Szepesi, B. )

    1990-02-26

    Previous work by Brownlee has shown that glycation of bovine serum albumin can be reduced in the presence of aminoguanidine (AG). Presumably, the guanidinium group on AG interferes with further rearrangement of amadori products to advanced glycosylated end products (AGE). Since L-arginine (ARG) also contains a guanidinium group, its ability to inhibit the formation of AGE products was investigated. HSA was incubated at 37{degrees}C in the presence or absence of glucose; with glucose and fructose; or with sugars in the presence or absence of ARG or AG. A tracer amount of U-{sup 14}C-glucose was added to each tube containing sugars. Protein bound glucose was separated from unreacted glucose by gel filtration. Radioactivity, total protein, fluorescence, and glucose concentration were measured. Preliminary data show enhanced binding of {sup 14}C-glucose to HSA with fructose at all time points. A 30-40% decrease in {sup 14}C-glucose incorporation was observed when ARG or AG as present. ARG and AG were equally effective in inhibiting incorporation of {sup 14}C-glucose. FPLC analysis is in progress to determine the type and degree of HSA crosslinking during the 2 week incubation period.

  16. Water-Soluble alpha,beta-unsaturated aldehydes of cigarette smoke induce carbonylation of human serum albumin.

    PubMed

    Colombo, Graziano; Aldini, Giancarlo; Orioli, Marica; Giustarini, Daniela; Gornati, Rosalba; Rossi, Ranieri; Colombo, Roberto; Carini, Marina; Milzani, Aldo; Dalle-Donne, Isabella

    2010-03-01

    Cigarette smoking is a major risk factor for developing pulmonary and cardiovascular diseases as well as some forms of cancer. Understanding the mechanisms by which smoking contributes to disease remains a major research focus. Increased levels of carbonylated serum proteins are present in smokers; albumin is the major carbonylated protein in the bronchoalveolar lavage fluid of older smokers. We have investigated the susceptibility of human serum albumin (HSA) to alpha,beta-unsaturated aldehyde-induced carbonylation when exposed to whole-phase cigarette smoke extract (CSE). Fluorescence studies with fluorescent probes showed depletion of HSA Cys34 free thiol and marked decrease of free Lys residues. Spectrophotometric and immunochemical carbonyl assays after carbonyl derivatization with 2,4-dinitrophenylhydrazine revealed the formation of covalent carbonyl adducts. Nanoscale capillary liquid chromatography and electrospray tandem mass spectrometry analysis detected acrolein and crotonaldehyde Michael adducts at Cys34, Lys525, Lys351, and His39 at all the CSE concentrations tested. Lys541 and Lys545 were also found to form a Schiff base with acrolein. The carbonyl scavenger drugs, hydralazine and pyridoxamine, partially prevented CSE-induced HSA carbonylation. Carbonylation of HSA associated with cigarette smoking might result in modifications of its antioxidant properties and transport functions of both endogenous and exogenous compounds.

  17. Influence of the binding of reduced NAMI-A to human serum albumin on the pharmacokinetics and biological activity.

    PubMed

    Novohradský, V; Bergamo, A; Cocchietto, M; Zajac, J; Brabec, V; Mestroni, G; Sava, G

    2015-01-28

    NAMI-A is a ruthenium-based drug endowed with the unique property of selectively targeting solid tumour metastases. Although two clinical studies had already been completed, limited information exists on the behavior of NAMI-A after injection into the bloodstream. PK data in humans informs us of a rather low free drug concentration, of a relatively high half-life time of elimination and of a linear relationship between the administered dose and the corresponding AUC for up to toxic doses. In the present study, we examined the chemical kinetics of albumin binding with or without the presence of reducing agents, and we evaluated how these chemical aspects might influence the in vivo PK and the in vitro ability of NAMI-A to inhibit cell migration, which is a bona fide, rapid and easy way to suggest anti-metastatic properties. The experimental data support the binding of NAMI-A to serum albumin. The reaction is facilitated when the drug is in its reduced form and, in agreement with already reported data, the adduct formed with albumin maintains the biological activity of the ruthenium drug. The formation of the adduct is favored by low ratios of NAMI-A : HSA and by the reduction of the drug with ascorbic acid. The difference in in vivo PK and the faster binding to albumin of the reduced NAMI-A seem to suggest that the drug is not rapidly reduced immediately upon injection, even at low doses. Most probably, cell and protein binding prevail over the reduction of the drug. This observation supports the thesis that the reduction of the drug before injection must be considered relevant for the pharmacological activity of NAMI-A against tumour metastases. PMID:25489765

  18. Comparative analysis of the interaction of capecitabine and gefitinib with human serum albumin using (19)F nuclear magnetic resonance-based approach.

    PubMed

    Wu, Di; Yan, Jin; Sun, Pingchuan; Xu, Kailin; Li, Shanshan; Yang, Hongqi; Li, Hui

    2016-09-10

    Monitoring the interaction between drugs and proteins is critical to understanding drug transport and metabolism underlying pharmacodynamics. The binding capacities to human serum albumin of two anticancer drugs, capecitabine and gefitinib, were compared via an approach combining (19)F NMR, (1)H saturation transfer difference (STD) NMR, circular dichroism and docking simulations. Results showed that the two drugs interaction with human serum albumin caused (19)F NMR signal shifted to different directions. Capecitabine had accurate binding site and higher binding affinity than gefitinib. This study provided fresh insights into ligand-protein interaction and the strength of (19)F NMR approach in biomedical research was well illustrated in this case.

  19. Comparative analysis of the interaction of capecitabine and gefitinib with human serum albumin using (19)F nuclear magnetic resonance-based approach.

    PubMed

    Wu, Di; Yan, Jin; Sun, Pingchuan; Xu, Kailin; Li, Shanshan; Yang, Hongqi; Li, Hui

    2016-09-10

    Monitoring the interaction between drugs and proteins is critical to understanding drug transport and metabolism underlying pharmacodynamics. The binding capacities to human serum albumin of two anticancer drugs, capecitabine and gefitinib, were compared via an approach combining (19)F NMR, (1)H saturation transfer difference (STD) NMR, circular dichroism and docking simulations. Results showed that the two drugs interaction with human serum albumin caused (19)F NMR signal shifted to different directions. Capecitabine had accurate binding site and higher binding affinity than gefitinib. This study provided fresh insights into ligand-protein interaction and the strength of (19)F NMR approach in biomedical research was well illustrated in this case. PMID:27392172

  20. O2-binding albumin thin films: solid membranes of poly(ethylene glycol)-conjugated human serum albumin incorporating iron porphyrin.

    PubMed

    Nakagawa, Akito; Komatsu, Teruyuki; Huang, Yubin; Lu, Gang; Tsuchida, Eishun

    2007-01-01

    Poly(ethylene glycol) (PEG)-conjugated human serum albumin (HSA) incorporating the tetrakis(alpha,alpha,alpha,alpha-o-amidophenyl)porphinatoiron(II) derivative (FeP) [PEG(HSA-FeP)] is a unique plasma protein-based O2 carrier as a red blood cell substitute. The aqueous solution of PEG(HSA-FeP) [mw of PEG: 2-kDa (PEG2) or 5-kDa (PEG5)] was evaporated on a glass surface to produce a red-colored solid membrane. Scanning electron microscopy observations revealed that the PEG2(HSA-FeP) membrane consisted of two parts: (i) a surface layer made of a fibrous component (10 microm thickness), and (ii) a bottom layer of an amorphous phase (5 microm thickness). The condensed solution provided a thick membrane (70 microm), which also has the amorphous bottom layer. On the other hand, the PEG5(HSA-FeP) produced homogeneous membrane made of the fibrous component. The FeP active sites in the solid membrane formed very stable O2-adduct complexes at 37 degrees C with a half-lifetime of 40 h. The O2-binding affinity of the PEG2(HSA-FeP) membrane (P1/2 = 40 Torr, 25 degrees C) was 4-fold lower than that in aqueous solution, which is kinetically due to the low association rate constant. The membrane was soluble again in water and organic solvents (ethanol and chloroform) without deformation of the secondary structure of the protein. The addition of hyaluronic acid gave a free-standing flexible thin film, and it can also bind and release O2 as well. These O2-carrying albumin membranes with a micrometer-thickness would be of significant medical importance for a variety of clinical treatments.

  1. Experimental investigation of the serum albumin fascia microstructure

    NASA Astrophysics Data System (ADS)

    Buzoverya, M. E.; Shcherbak, Yu. P.; Shishpor, I. V.

    2012-09-01

    The results of theoretical and experimental investigation of biological liquids are reported. Structural effects observed in fascias are considered with account of the molecular features of albumin and the concept of supramolecular organization of polymers. It is revealed that the morphology of human serum albumin fascias depends on the concentration and quality of the solvent. It is shown that the water-salt fascias of albumin are more structured than water solutions with the same concentration.

  2. Exploring the binding of 4-thiothymidine with human serum albumin by spectroscopy, atomic force microscopy, and molecular modeling methods.

    PubMed

    Zhang, Juling; Gu, Huaimin; Zhang, Xiaohui

    2014-01-30

    The interaction of 4-thiothymidine (S(4)TdR) with human serum albumin (HSA) was studied by equilibrium dialysis under normal physiological conditions. In this work, the mechanism of the interaction between S(4)TdR and human serum albumin (HSA) was exploited by fluorescence, UV, CD circular, and SERS spectroscopic. Fluorescence and UV spectroscopy suggest that HSA intensities are significantly decreased when adding S(4)TdR to HAS, and the quenching mechanism of the fluorescence is static. Also, the ΔG, ΔH, and ΔS values across temperature indicated that hydrophobic interaction was the predominant binding force. The CD circular results show that there is little change in the secondary structure of HSA except the environment of amino acid changes when adding S(4)TdR to HSA. The surface-enhanced Raman scattering (SERS) shows that the interaction between S(4)TdR and HSA can be achieved through different binding sites which are probably located in the II A and III A hydrophobic pockets of HSA which correspond to Sudlow's I and II binding sites. In addition, the molecular modeling displays that S(4)TdR-HSA complex is stabilized by hydrophobic forces, which result from amino acid residues. The atomic force microscopy results revealed that the single HSA molecular dimensions were larger after interaction of 4-thiothymidine. This work would be useful to understand the state of the transportation, distribution, and metabolism of the anticancer drugs in the human body, and it could provide a useful biochemistry parameter for the development of new anti-cancer drugs and research of pharmacology mechanisms. PMID:24370945

  3. Mechanistic and conformational studies on the interaction of food dye amaranth with human serum albumin by multispectroscopic methods.

    PubMed

    Zhang, Guowen; Ma, Yadi

    2013-01-15

    The mechanism of interaction between food dye amaranth and human serum albumin (HSA) in physiological buffer (pH 7.4) was investigated by fluorescence, UV-vis absorption, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy. Results obtained from analysis of fluorescence spectra indicated that amaranth had a strong ability to quench the intrinsic fluorescence of HSA through a static quenching procedure. The negative value of enthalpy change and positive value of entropy change elucidated that the binding of amaranth to HSA was driven mainly by hydrophobic and hydrogen bonding interactions. The surface hydrophobicity of HSA increased after binding with amaranth. The binding distance between HSA and amaranth was estimated to be 3.03 nm and subdomain IIA (Sudlow site I) was the primary binding site for amaranth on HSA. The results of CD and FT-IR spectra showed that binding of amaranth to HSA induced conformational changes of HSA.

  4. Mechanistic and conformational studies on the interaction of food dye amaranth with human serum albumin by multispectroscopic methods.

    PubMed

    Zhang, Guowen; Ma, Yadi

    2013-01-15

    The mechanism of interaction between food dye amaranth and human serum albumin (HSA) in physiological buffer (pH 7.4) was investigated by fluorescence, UV-vis absorption, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy. Results obtained from analysis of fluorescence spectra indicated that amaranth had a strong ability to quench the intrinsic fluorescence of HSA through a static quenching procedure. The negative value of enthalpy change and positive value of entropy change elucidated that the binding of amaranth to HSA was driven mainly by hydrophobic and hydrogen bonding interactions. The surface hydrophobicity of HSA increased after binding with amaranth. The binding distance between HSA and amaranth was estimated to be 3.03 nm and subdomain IIA (Sudlow site I) was the primary binding site for amaranth on HSA. The results of CD and FT-IR spectra showed that binding of amaranth to HSA induced conformational changes of HSA. PMID:23122082

  5. The orientation of protoberberine alkaloids and their binding activities to human serum albumin by surface-enhanced Raman scattering

    NASA Astrophysics Data System (ADS)

    Zhang, Wei; Zhao, Yu; Bai, Xueyuan; Wang, Yingping; Zhao, Daqing

    2011-03-01

    Raman and surface-enhanced Raman scattering (SERS) technique are reliably used to compare relative intensity shifts and to investigate the adsorption geometry of protoberberine alkaloids on Ag nanoparticles. We report joint application of fluorescence and SERS spectroscopy to study the interaction between protoberberine alkaloids and human serum albumin (HSA). We propose SERS technique to improve the quenching interaction caused by protoberberine alkaloids which are used to be applied in recognition process of fluorescent drugs with large biomolecules. The fluorescence results show that the fluorescence intensity of HSA is significantly decreased in presence of protoberberine alkaloids. The SERS technique demonstrates obvious advantages over direct measurements in discriminating and identifying pharmaceutical molecules. By means of this method, we are able to detect important information concerning the orientation of protoberberine alkaloids when interacting with HSA. We also show that the nitrogen atom is free, but a benzene ring and two adjacent methoxy groups are involved in the spontaneously electrostatic inducement and subsequently binding with HSA.

  6. Study of interaction between human serum albumin and three antioxidants: ascorbic acid, α-tocopherol, and proanthocyanidins.

    PubMed

    Li, Xiangrong; Chen, Dejun; Wang, Gongke; Lu, Yan

    2013-01-01

    Ascorbic acid, α-tocopherol and proanthocyanidins are three classic dietary antioxidants. In this study, the interaction between the three antioxidants and human serum albumin (HSA) was investigated by several spectroscopic techniques. Experimental results proved that the three antioxidants quench the fluorescence of HSA through a static (proanthocyanidins) or static-dynamic combined quenching mechanism (ascorbic acid and α-tocopherol). Thermodynamic investigations revealed that the combination between ascorbic acid or proanthocyanidins and HSA was driven mainly by electrostatic interaction, and the hydrophobic interactions play a major role for α-tocopherol-HSA association. Binding site I was found to be the primary binding site for ascorbic acid and proanthocyanidins, and site II for α-tocopherol. Additionally, the three antioxidants may induce conformational and microenvironmental changes of HSA. PMID:24140914

  7. Induction of antibodies to nuclear antigens in rabbits by immunization with hydralazine-human serum albumin conjugates.

    PubMed Central

    Yamauchi, Y; Litwin, A; Adams, L; Zimmer, H; Hess, E V

    1975-01-01

    The antihypertensive drug hydralazine can induce in man a syndrome similar to spontaneous systemic lupus erythematosus (SLE). The pathogenesis of this drug-induced syndrome is not understood. In this investigation, five groups of rabbits were studied: group I, 10 rabbits hyperimmunized with hydralazine conjugated to human serum albumin (HSA) in complete Freund's adjuvant (CFA); group II, four rabbits with HSA in CFA; group III, four rabbits with CFA alone; group IV, five rabbits with hydralazine conjugated to rabbit serum albumin (RSA); and group V, four rabbits with a major metabolite of hydralazine conjugated to HSA. The rabbits immunized with hydralazine-HSA developed rising titers of antibodies to hydralazine and progressively increasing amounts of antibodies to both single-stranded and native DNA. The antibodies to DNA were cross-reactive with hydralazine as determined by inhibition of DNA binding and DNA hemagglutination tests. Similar results were obtained in rabbits immunized with the metabolite-HSA compound except the major hapten antibody response was to the metabolite. The DNA antibodies in this group were also capable of being absorbed by metabolite-HSA as well as hydralazine-HSA, indicative of the cross-reactivity between hydralazine and its metabolite. Immunization with hydralazine-RSA caused rabbits to produce antibodies to hydralazine but not to DNA, indicating the requirement for an immune response to the carrier protein in order for antibodies reactive with DNA to be produced. Thus, hyperimmunization of rabbits with hydralazine-protein conjugates may provide a useful animal model of SLE. The data suggests that an immune response to hydralazine may be important in human hydralazine-induced SLE. Images PMID:808562

  8. Probing the interaction of human serum albumin with DPPH in the absence and presence of the eight antioxidants.

    PubMed

    Li, Xiangrong; Chen, Dejun; Wang, Gongke; Lu, Yan

    2015-02-25

    Albumin represents a very abundant and important circulating antioxidant in plasma. DPPH radical is also called 2,2-diphenyl-1-picrylhydrazyl. It has been widely used for measuring the efficiency of antioxidants. In this paper, the ability of human serum albumin (HSA) to scavenge DPPH radical was investigated using UV-vis absorption spectra. The interaction between HSA and DPPH was investigated in the absence and presence of eight popular antioxidants using fluorescence spectroscopy. These results indicate the antioxidant activity of HSA against DPPH radical is similar to glutathione and the value of IC50 is 5.200×10(-5) mol L(-1). In addition, the fluorescence experiments indicate the quenching mechanism of HSA, by DPPH, is a static process. The quenching process of DPPH with HSA is easily affected by the eight antioxidants, however, they cannot change the quenching mechanism of DPPH with HSA. The binding of DPPH to HSA primarily takes place in subdomain IIA and exists two classes of binding sites with two different interaction behaviors. The decreased binding constants and the number of binding sites of DPPH with HSA by the introduction of the eight antioxidants may result from the competition of the eight antioxidants and DPPH binding to HSA. The binding of DPPH to HSA may induce the micro-environment of the lone Trp-214 from polar to slightly nonpolar.

  9. Probing the interaction of human serum albumin with DPPH in the absence and presence of the eight antioxidants

    NASA Astrophysics Data System (ADS)

    Li, Xiangrong; Chen, Dejun; Wang, Gongke; Lu, Yan

    2015-02-01

    Albumin represents a very abundant and important circulating antioxidant in plasma. DPPH radical is also called 2,2-diphenyl-1-picrylhydrazyl. It has been widely used for measuring the efficiency of antioxidants. In this paper, the ability of human serum albumin (HSA) to scavenge DPPH radical was investigated using UV-vis absorption spectra. The interaction between HSA and DPPH was investigated in the absence and presence of eight popular antioxidants using fluorescence spectroscopy. These results indicate the antioxidant activity of HSA against DPPH radical is similar to glutathione and the value of IC50 is 5.200 × 10-5 mol L-1. In addition, the fluorescence experiments indicate the quenching mechanism of HSA, by DPPH, is a static process. The quenching process of DPPH with HSA is easily affected by the eight antioxidants, however, they cannot change the quenching mechanism of DPPH with HSA. The binding of DPPH to HSA primarily takes place in subdomain IIA and exists two classes of binding sites with two different interaction behaviors. The decreased binding constants and the number of binding sites of DPPH with HSA by the introduction of the eight antioxidants may result from the competition of the eight antioxidants and DPPH binding to HSA. The binding of DPPH to HSA may induce the micro-environment of the lone Trp-214 from polar to slightly nonpolar.

  10. Evaluation of an immunoaffinity extraction column for enrichment of adducts between human serum albumin and hexahydrophthalic anhydride in plasma.

    PubMed

    Johannesson, Gunvor A; Kristiansson, Monica H; Jönsson, Bo A G; Lindh, Christian H

    2008-03-01

    An immunoaffinity extraction (IAE) column was prepared for extraction of adducts between human serum albumin (HSA) and hexahydrophthalic anhydride (HHPA). HHPA is a strong sensitizer inducing immunoglobulin E antibodies in vivo. Polyclonal antibodies from a rabbit immunized with keyhole limpet hemocyananin-HHPA conjugate were purified using a Protein A Sepharose gel. To obtain antibodies with optimal affinity towards HHPA-protein adducts, HHPA-specific antibodies were selected using an N-hydroxysuccinimide-Sepharose column coupled with albumin-HHPA conjugate. Antibodies eluted from this column at pH 2.2 were selected to prepare the IAE column. The column was evaluated using 2 mL plasma spiked with HSA-HHPA conjugate. The column was eluted with glycine buffer at pH 2.0. The conjugates in the eluate were hydrolyzed to the corresponding HHP acid and quantified by mass spectrometry. The average recovery of HHPA adducts in 11 experiments was 68% with a coefficient of variation (CV) of 7%. The column's capacity to bind protein-HHPA adducts was found to be linear in the range of 0.15-1.2 nmol conjugate. The evaluation showed that the IAE column had adequate affinity towards the HHPA adducts and that the adducts could be extracted with good recovery and precision from a large volume of plasma.

  11. Radiochemical analysis of Tc-99m human serum albumin with high-pressure liquid chromatography: concise communication.

    PubMed

    Vallabhajosula, S; Goldsmith, S J; Pollina, R; Lipszyc, H

    1982-04-01

    High-pressure liquid chromatography (HPLC) can be performed with an aqueous size-exclusion column to separate proteins or other macromolecules on the basis of molecular size. An HPLC system with a Spherogel-TSK SW column was modified to detect simultaneously uv absorption and radioactivity. Characteristic retention times (RT) were determined for pure human serum albumin (HSA) (RT = 17 min) and pertechnetate (RT = 28.5 min). When analysis was performed on Tc-99m HSA preparations, Tc-99m radioactivity was resolved into five different peaks, with RT ranging from 10.2 to 28.5 min. Less than 2% radioactivity was associated with the pertechnetate peak, whereas the remaining Tc-99m was protein bound. Most of the activity (90%) corresponded to the albumin peak, and 7% was bound to contaminants of high molecular weight with RTs of 10.2 and 14 min. Rapid separation of various radiochemical components differing in molecular size provides an improved basis for understanding the biodistribution of a Tc-99m HSA preparation. This technique would be useful for the preparation and analysis of various radiolabeled macromolecules such as enzymes, immunoglobulins, and other proteins.

  12. Radiochemical analysis of Tc-99m human serum albumin with high-pressure liquid chromatography: concise communication

    SciTech Connect

    Vallabhajosula, S.; Goldsmith, S.J.; Pollina, R.; Lipszyc, H.

    1982-04-01

    High-pressure liquid chromatography (HPLC) can be performed with an aqueous size-exclusion column to separate proteins or other macromolecules on the basis of molecular size. An HPLC system with a Spherogel-TSK SW column was modified to detect simultaneously uv absorption and radioactivity. Characteristic retention times (RT) were determined for pure human serum albumin (HSA) (RT = 17 min) and pertechnetate (RT = 28.5 min). When analysis was performed on Tc-99m HSA preparations, Tc-99m radioactivity was resolved into five different peaks, with RT ranging from 10.2 to 28.5 min. Less than 2% radioactivity was associated with the pertechnetate peak, whereas the remaining Tc-99m was protein bound. Most of the activity (90%) corresponded to the albumin peak, and 7% was bound to contaminants of high molecular weight with RTs of 10.2 and 14 min. Rapid separation of various radiochemical components differing in molecular size provides an improved basis for understanding the biodistribution of a Tc-99m HSA preparation. This technique would be useful for the preparation and analysis of various radiolabeled macromolecules such as enzymes, immunoglobulins, and other proteins.

  13. Radiochemical analysis of /sup 99m/Tc human serum albumin with high-pressure liquid chromatography: concise communication

    SciTech Connect

    Vallabhajosula, S.; Goldsmith, S.J.; Pollina, R.; Lipszyc, H.

    1982-04-01

    High-pressure liquid chromatography (HPLC) can be performed with an aqueous size-exclusion column to separate proteins or other macromolecules on the basis of molecular size. An HPLC system with a Spherogel-TSK SW column was modified to detect simultaneously uv absorption and radioactivity. Characteristic retention times (RT) were determined for pure human serum albumin (HSA) (RT . 17 min) and pertechnetate (RT . 28.5 min). When analysis was performed on /sup 99m/Tc HSA preparations, /sup 99m/Tc radioactivity was resolved into five different peaks, with RT ranging from 10.2 to 28.5 min. Less than 2% radioactivity was associated with the pertechnetate peak, whereas the remaining /sup 99m/Tc was protein bound. Most of the activity (90%) corresponded to the albumin peak, and 7% was bound to contaminants of high molecular weight with RTs of 10.2 and 14 min. Rapid separation of various radiochemical components differing in molecular size provides an improved basis for understanding the biodistribution of a /sup 99m/Tc HSA preparation. This technique would be useful for the preparation and analysis of various radiolabeled macromolecules such as enzymes, immunoglobulins, and other proteins.

  14. A molecular dynamics approach to ligand-receptor interaction in the aspirin-human serum albumin complex.

    PubMed

    Alvarez, H Ariel; McCarthy, Andrés N; Grigera, J Raúl

    2012-01-01

    In this work, we present a study of the interaction between human serum albumin (HSA) and acetylsalicylic acid (ASA, C(9)H(8)O(4)) by molecular dynamics simulations (MD). Starting from an experimentally resolved structure of the complex, we performed the extraction of the ligand by means of the application of an external force. After stabilization of the system, we quantified the force used to remove the ASA from its specific site of binding to HSA and calculated the mechanical nonequilibrium external work done during this process. We obtain a reasonable value for the upper boundary of the Gibbs free energy difference (an equilibrium thermodynamic potential) between the complexed and noncomplexed states. To achieve this goal, we used the finite sampling estimator of the average work, calculated from the Jarzynski Equality. To evaluate the effect of the solvent, we calculated the so-called "viscous work," that is, the work done to move the aspirin in the same trajectory through the solvent in absence of the protein, so as to assess the relevance of its contribution to the total work. The results are in good agreement with the available experimental data for the albumin affinity constant for aspirin, obtained through quenching fluorescence methods.

  15. A Molecular Dynamics Approach to Ligand-Receptor Interaction in the Aspirin-Human Serum Albumin Complex

    PubMed Central

    Alvarez, H. Ariel; McCarthy, Andrés N.; Grigera, J. Raúl

    2012-01-01

    In this work, we present a study of the interaction between human serum albumin (HSA) and acetylsalicylic acid (ASA, C9H8O4) by molecular dynamics simulations (MD). Starting from an experimentally resolved structure of the complex, we performed the extraction of the ligand by means of the application of an external force. After stabilization of the system, we quantified the force used to remove the ASA from its specific site of binding to HSA and calculated the mechanical nonequilibrium external work done during this process. We obtain a reasonable value for the upper boundary of the Gibbs free energy difference (an equilibrium thermodynamic potential) between the complexed and noncomplexed states. To achieve this goal, we used the finite sampling estimator of the average work, calculated from the Jarzynski Equality. To evaluate the effect of the solvent, we calculated the so-called “viscous work,” that is, the work done to move the aspirin in the same trajectory through the solvent in absence of the protein, so as to assess the relevance of its contribution to the total work. The results are in good agreement with the available experimental data for the albumin affinity constant for aspirin, obtained through quenching fluorescence methods. PMID:23251150

  16. Interaction of coffee compounds with serum albumins. Part II: Diterpenes.

    PubMed

    Guercia, Elena; Forzato, Cristina; Navarini, Luciano; Berti, Federico

    2016-05-15

    Cafestol and 16-O-methylcafestol are diterpenes present in coffee, but whilst cafestol is found in both Coffea canephora and Coffea arabica, 16-O-methylcafestol (16-OMC) was reported to be specific of only C. canephora. The interactions of such compounds, with serum albumins, have been studied. Three albumins have been considered, namely human serum albumin (HSA), fatty acid free HSA (ffHSA) and bovine serum albumin (BSA). The proteins interact with the diterpenes at the interface between Sudlow site I and the fatty acid binding site 6 in a very peculiar way, leading to a significant change in the secondary structure. The diterpenes do not displace reference binding drugs of site 2, but rather they enhance the affinity of the site for the drugs. They, therefore, may alter the pharmacokinetic profile of albumin - bound drugs.

  17. Antioxidant capacity and structural changes of human serum albumin from patients in advanced stages of diabetic nephropathy and the effect of the dialysis.

    PubMed

    Rosas-Díaz, Marisol; Camarillo-Cadena, Menandro; Hernández-Arana, Andrés; Ramón-Gallegos, Eva; Medina-Navarro, Rafael

    2015-06-01

    Changes in the antioxidant capacity of albumin and alterations of the albumin structural conformation were examined in patients in advanced stages of diabetes nephropathy. Human serum albumin was purified from diabetic patients in pre-dialysis (glomerular filtration rate [GFR] between 15 and 29 ml min(-1) 1.73 m(-2)) and those in dialysis (GFR ≤ 15 ml min(-1) 1.73 m(-2)) and then compared with albumin from patients with a normal GFR (>90 ml min(-1) m(-2)). We evaluated the antioxidant capacity of albumin using an enhanced chemiluminescence-based assay and thiol group content, and the structural changes were evaluated by circular dichroism and fluorescence spectroscopy. The antioxidant capacity and thiol content of albumin from patients in advanced stages of diabetic nephropathy were markedly reduced. The circular dichroism spectra showed a mean albumin α-helix content reduction from 44 to 37 % and from 44 to 30 % between the control group and pre-dialysis and dialysis patients, respectively. Additionally, the fluorescence intensity was reduced by 4.2 and 13 % for the groups 4 and 5, respectively, in relation with the control. These data provide evidence for the partial denaturation of albumin and exacerbated oxidative stress among patients in advanced stages of diabetes nephropathy before and even after dialysis.

  18. Catabolism of (64)Cu and Cy5.5-labeled human serum albumin in a tumor xenograft model.

    PubMed

    Kang, Choong Mo; Kim, Hyunjung; Koo, Hyun-Jung; Park, Jin Won; An, Gwang Il; Choi, Joon Young; Lee, Kyung-Han; Kim, Byung-Tae; Choe, Yearn Seong

    2016-07-01

    Human serum albumin (HSA), the most abundant protein in blood plasma, has been used as a drug carrier for the last few decades. Residualizingly radiolabeled serum albumin has been reported to be avidly taken up by tumors of sarcoma-bearing mice and to most likely undergo lysosomal degradation. In this study, we prepared (64)Cu-1,4,7,10-tetraazacyclododecane-N,N',N″,N'″-tetraacetic acid (DOTA) and Cy5.5-conjugated HSA (dual probe), and evaluated its tumor uptake and catabolism. Two dual probes were prepared using different DOTA conjugation sites of HSA (one via Lys residues and the other via the Cys residue). (64)Cu-DOTA-Lys-HSA-Cy5.5 (dual probe-Lys) exhibited higher uptake by RR1022 sarcoma cells in vitro than (64)Cu-DOTA-Cys-HSA-Cy5.5 (dual probe-Cys). In RR1022 tumor-bearing mice, the two dual probes showed a similar level of tumor uptake, but uptake of dual probe-Lys was reduced in the liver and spleen compared to dual probe-Cys, probably because of the presence of a higher number of DOTA molecules in the former. At 24 and 48 h after injection, dual probe-Lys was intact or partially degraded in blood, liver, kidney, and tumor samples, but (64)Cu-DOTA-Lys was observed in the urine using radioactivity detection. Similarly, Cy5.5-Lys was observed in the urine using fluorescence detection. These results indicate that dual probe-Lys may be useful for predicting the catabolic fate of drug-HSA conjugates. PMID:27098932

  19. Serum albumin and risk of venous thromboembolism

    PubMed Central

    Folsom, Aaron. R.; Lutsey, Pamela L.; Heckbert, Susan R.; Cushman, Mary

    2010-01-01

    Summary The incidence of venous thromboembolism (VTE) is increased in patients with albuminuria. However, whether a low serum albumin concentration is associated with increased risk of VTE has been a matter of controversy. We determined the association of serum albumin with VTE incidence in two large, prospective, population-based cohorts: the Atherosclerosis Risk in Communities (ARIC) Study (n = 15,300) and the Cardiovascular Health Study (CHS) (n = 5,400). Validated VTE occurrence (n=462 in ARIC and n=174 in CHS) was ascertained during follow-up. In both studies, after adjustment for age, sex, race, use of hormone replacement therapy, estimated GFR, history of cancer, and diabetes, serum albumin tended to be associated inversely with VTE. The adjusted hazard ratio per standard deviation lower albumin was 1.18 (95% CI = 1.08, 1.31) in ARIC and 1.10 (95% CI = 0.94, 1.29) in CHS. The hazard ratio for albumin below (versus above) the fifth percentile was 1.28 (95% CI = 0.90, 1.84) in ARIC and 1.80 (95% CI = 1.11, 2.93) in CHS. In conclusion, low serum albumin was a modest marker of increased VTE risk. The observed association likely does not reflect cause and effect, but rather that low serum albumin reflects a hyperinflammatory or hypercoagulable state. Whether this association has clinical relevance warrants further study. PMID:20390234

  20. Serum albumin complexation of acetylsalicylic acid metabolites.

    PubMed

    Jurkowski, Wiktor; Porebski, Grzegorz; Obtułowicz, Krystyna; Roterman, Irena

    2009-06-01

    One possible origin of the type I hypersensitivity reaction is reaction of drugs such as acetylsalicylic acid and its metabolites being complexed with human serum albumin. Albumin, being transporting molecule abundant in blood plasma is able to bind large array of ligands varying from small single carbon particles to long hydrophobic tailed lipidic acids (e.g. myristic acid). This non specificity is possible because of multi domain scaffold and large flexibility of inter-domain loops, which results in serious reorientation of domains. Hypothesis that acetylsalicylic acid metabolites may play indirect role in activation of allergic reaction has been tested. Binding of acetylsalicylic acid metabolites in intra-domain space causes significant increase of liability of domains IIIA and IIIB. One of metabolites, salicyluric acid, once is bound causes distortion and partial unfolding of helices in domains IA, IIB and IIIB. Changed are both directions and amplitude of relative motions as well as intra-domain distances. In result albumin is able to cross-link of adjacent IgE receptors which subsequently starts allergic reaction.

  1. Serum albumin complexation of acetylsalicylic acid metabolites.

    PubMed

    Jurkowski, Wiktor; Porebski, Grzegorz; Obtułowicz, Krystyna; Roterman, Irena

    2009-06-01

    One possible origin of the type I hypersensitivity reaction is reaction of drugs such as acetylsalicylic acid and its metabolites being complexed with human serum albumin. Albumin, being transporting molecule abundant in blood plasma is able to bind large array of ligands varying from small single carbon particles to long hydrophobic tailed lipidic acids (e.g. myristic acid). This non specificity is possible because of multi domain scaffold and large flexibility of inter-domain loops, which results in serious reorientation of domains. Hypothesis that acetylsalicylic acid metabolites may play indirect role in activation of allergic reaction has been tested. Binding of acetylsalicylic acid metabolites in intra-domain space causes significant increase of liability of domains IIIA and IIIB. One of metabolites, salicyluric acid, once is bound causes distortion and partial unfolding of helices in domains IA, IIB and IIIB. Changed are both directions and amplitude of relative motions as well as intra-domain distances. In result albumin is able to cross-link of adjacent IgE receptors which subsequently starts allergic reaction. PMID:19689242

  2. Biodegradable human serum albumin nanoparticles as contrast agents for the detection of hepatocellular carcinoma by magnetic resonance imaging.

    PubMed

    Watcharin, Waralee; Schmithals, Christian; Pleli, Thomas; Köberle, Verena; Korkusuz, Hüdayi; Huebner, Frank; Zeuzem, Stefan; Korf, Hans W; Vogl, Thomas J; Rittmeyer, Claudia; Terfort, Andreas; Piiper, Albrecht; Gelperina, Svetlana; Kreuter, Jörg

    2014-05-01

    Tumor visualization by magnetic resonance imaging (MRI) and nanoparticle-based contrast agents may improve the imaging of solid tumors such as hepatocellular carcinoma (HCC). In particular, human serum albumin (HSA) nanoparticles appear to be a suitable carrier due to their safety and feasibility of functionalization. In the present study HSA nanoparticles were conjugated with gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) using carbodiimide chemistry. The nanoparticles had a uniform spherical shape and a diameter of 235±19nm. For better optical visualization in vitro and in vivo, the HSA-Gd nanoparticles were additionally labeled with rhodamine 123. As shown by confocal microscopy and flow cytometry analysis, the fluorescent nanoparticles were readily taken up by Huh-7 hepatocellular carcinoma cells. After 24h incubation in blood serum, less than 5% of the Gd(III) was released from the particles, which suggests that this nanoparticulate system may be stable in vivo and, therefore, may serve as potentially safe T1 MRI contrast agent for MRI of hepatocellular carcinoma.

  3. Extending the half-life of a fab fragment through generation of a humanized anti-human serum albumin Fv domain: An investigation into the correlation between affinity and serum half-life

    PubMed Central

    Adams, Ralph; Griffin, Laura; Compson, Joanne E.; Jairaj, Mark; Baker, Terry; Ceska, Tom; West, Shauna; Zaccheo, Oliver; Davé, Emma; Lawson, Alastair DG.; Humphreys, David P.; Heywood, Sam

    2016-01-01

    ABSTRACT We generated an anti-albumin antibody, CA645, to link its Fv domain to an antigen-binding fragment (Fab), thereby extending the serum half-life of the Fab. CA645 was demonstrated to bind human, cynomolgus, and mouse serum albumin with similar affinity (1–7 nM), and to bind human serum albumin (HSA) when it is in complex with common known ligands. Importantly for half-life extension, CA645 binds HSA with similar affinity within the physiologically relevant range of pH 5.0 – pH 7.4, and does not have a deleterious effect on the binding of HSA to neonatal Fc receptor (FcRn). A crystal structure of humanized CA645 Fab in complex with HSA was solved and showed that CA645 Fab binds to domain II of HSA. Superimposition with the crystal structure of FcRn bound to HSA confirmed that CA645 does not block HSA binding to FcRn. In mice, the serum half-life of humanized CA645 Fab is 84.2 h. This is a significant extension in comparison with < 1 h for a non-HSA binding CA645 Fab variant. The Fab-HSA structure was used to design a series of mutants with reduced affinity to investigate the correlation between the affinity for albumin and serum half-life. Reduction in the affinity for MSA by 144-fold from 2.2 nM to 316 nM had no effect on serum half-life. Strikingly, despite a reduction in affinity to 62 µM, an extension in serum half-life of 26.4 h was still obtained. CA645 Fab and the CA645 Fab-HSA complex have been deposited in the Protein Data Bank (PDB) with accession codes, 5FUZ and 5FUO, respectively. PMID:27315033

  4. Proteolytic processing of human serum albumin generates EPI-X4, an endogenous antagonist of CXCR4.

    PubMed

    Zirafi, Onofrio; Hermann, Patrick C; Münch, Jan

    2016-06-01

    The chemokine receptor CXCR4 is an important G protein-coupled receptor. Signaling via CXCL12 regulates a number of important biologic processes, including immune responses, organogenesis, or hematopoiesis. Dysregulation of CXCR4 signaling is associated with a variety of diseases, such as cancer development and metastasis, immunodeficiencies, or chronic inflammation. Here, we review our findings on endogenous peptide inhibitor of CXCR4 as a novel antagonist of CXCR4. This peptide is a 16-residue fragment of human serum albumin and was isolated as an inhibitor of CXCR4-tropic human immunodeficiency virus type 1 from a blood-derived peptide library. Endogenous peptide inhibitor of CXCR4 binds the second extracellular loop of CXCR4, thereby preventing engagement of CXCL12 and antagonizing the receptor. Consequently, endogenous peptide inhibitor of CXCR4 inhibits CXCL12-mediated migration of CXCR4-expressing cells in vitro, mobilizes hematopoietic stem cells, and suppresses inflammatory responses in vivo. We discuss the generation of endogenous peptide inhibitor of CXCR4, its relevance as biomarker for disease, and its role in human immunodeficiency virus/acquired immunodeficiency syndrome pathogenesis and cancer. Furthermore, we discuss why optimized endogenous peptide inhibitor of CXCR4 derivatives might have advantages over other CXCR4 antagonists. PMID:26965637

  5. Isolation and characterization of serum albumin from Camelus dromedarius

    PubMed Central

    MALIK, AJAMALUDDIN; AL-SENAIDY, ABDULRAHMAN; SKRZYPCZAK-JANKUN, EWA; JANKUN, JERZY

    2013-01-01

    Serum albumin constitutes 35–50 mg/ml of plasma proteins and performs various physiological activities including the regulation of osmotic pressure on blood, maintaining buffering of the blood pH, carrying different fatty acids and other small molecules, such as bilirubin, hormones, drugs and metal ions, as well as participating in immunological responses. Serum albumin is an extensively used protein in biotechnological and pharmaceutical industries. The camel (Camelus dromedarius) is well tailored to successfully survive in extremely hot and dry climates. Plasma osmolality in the camel increases during water-deprived conditions. In such circumstances serum albumin is crucial in the regulation of blood pressure. The study of biochemical, biophysical and immunological aspects of camel serum albumin (CSA) are likely to provide molecular insights into camel physiology and may render it an alternative to human serum albumin (HSA) and bovine serum albumin (BSA) in all cases. However, these proteins are currently not available or cannot be utilized due to a variety of considerations. In this study, 12 mg of highly pure CSA was obtained from 1 ml plasma. Coomassie Brilliant Blue staining of SDS-PAGE yielded one band and RP-HPLC results revealed a single sharp peak, indicating homogenous preparation of the CSA. The charge/mass ratio and surface hydrophobicity of the CSA was similar to that of BSA. Mass spectrometry analysis of the purified protein confirmed the identity of CSA. PMID:24137219

  6. Spectroscopic analysis of the impact of oxidative stress on the structure of human serum albumin (HSA) in terms of its binding properties

    NASA Astrophysics Data System (ADS)

    Maciążek-Jurczyk, M.; Sułkowska, A.

    2015-02-01

    Oxygen metabolism has an important role in the pathogenesis of rheumatoid arthritis (RA). Reactive oxygen species (ROS) are produced in the course of cellular oxidative phosphorylation and by activated phagocytic cells during oxidative bursts, exceed the physiological buffering capacity and result in oxidative stress. ROS result in oxidation of serum albumin, which causes a number of structural changes in the spatial structure, may influence the binding and cause significant drug interactions, particularly in polytherapy. During the oxidation modification of amino acid residues, particularly cysteine and methionine may occur. The aim of the study was to investigate the influence of oxidative stress on human serum albumin (HSA) structure and evaluate of possible alterations in the binding of the drug to oxidized human serum albumin (oHSA). HSA was oxidized by a chloramine-T (CT). CT reacts rapidly with sulfhydryl groups and at pH 7.4 the reaction was monitored by spectroscopic techniques. Modification of free thiol group in the Cys residue in HSA was quantitatively determined by the use of Ellman's reagent. Changes of albumin conformation were examined by comparison of modified (oHSA) and nonmodified human serum albumin (HSA) absorption spectra, emission spectra, red-edge shift (REES) and synchronous spectroscopy. Studies of absorption spectra indicated that changes in the value of absorbance associated with spectral changes in the region of 200-250 nm involve structural alterations in peptide backbone conformation. Synchronous fluorescence spectroscopy technique confirmed changes of position of tryptophanyl and tyrosyl residues fluorescent band caused by CT. Moreover analysis of REES effect allowed to observe structural changes caused by CT in the region of the hydrophobic pocket containing the tryptophanyl residue. Effect of oxidative stress on binding of anti-rheumatic drugs, sulfasalazine (SSZ) and sulindac (SLD) in the high and low affinity binding sites was

  7. Study on the interaction of artificial and natural food colorants with human serum albumin: A computational point of view.

    PubMed

    Masone, Diego; Chanforan, Céline

    2015-06-01

    Due to the high amount of artificial food colorants present in infants' diets, their adverse effects have been of major concern among the literature. Artificial food colorants have been suggested to affect children's behavior, being hyperactivity the most common disorder. In this study we compare binding affinities of a group of artificial colorants (sunset yellow, quinoline yellow, carmoisine, allura red and tartrazine) and their natural industrial equivalents (carminic acid, curcumin, peonidin-3-glucoside, cyanidin-3-glucoside) to human serum albumin (HSA) by a docking approach and further refinement through atomistic molecular dynamics simulations. Due to the protein-ligand conformational interface complexity, we used collective variable driven molecular dynamics to refine docking predictions and to score them according to a hydrogen-bond criterion. With this protocol, we were able to rank ligand affinities to HSA and to compare between the studied natural and artificial food additives. Our results show that the five artificial colorants studied bind better to HSA than their equivalent natural options, in terms of their H-bonding network, supporting the hypothesis of their potential risk to human health. PMID:25935119

  8. Significance of technetium-99m human serum albumin diethylenetriamine pentaacetic acid scintigraphy in patients with nephrotic syndrome.

    PubMed

    Takashima, Tsuyoshi; Kishi, Tomoya; Onozawa, Koji; Rikitake, Shuichi; Miyazono, Motoaki; Otsuka, Takateru; Irie, Hiroyuki; Iwakiri, Ryuichi; Fujimoto, Kazuma; Ikeda, Yuji

    2015-01-01

    It is thought that a large amount of albumin leaking from the glomerulus in nephrotic syndrome (NS) is reabsorbed at the proximal tubule and catabolized. Therefore, it is possible the final quantity of urinary protein does not always reflect the amount of leakage of protein from the glomerulus. We experienced two cases without nephrotic range proteinuria thought to involve hypoproteinemia due to the same pathophysiology as NS. On these patients, we performed protein leakage scintigraphy with technetium-99m human serum albumin diethylenetriamine pentaacetic acid (99mTc-HSAD) to exclude a diagnosis of protein-losing gastroenteropathy and observed diffuse positive accumulation in the kidneys with more intense uptake in the kidney than the liver on the anterior view 24 hours after 99mTc-HSAD administration. In healthy adults intravenously given 99mTc-HSAD, the same dynamics are observed as in albumin metabolism, and the organ radioactivity of the liver and kidneys after 24 hours is equal. Therefore, we thought it was possible that the renal uptake 24 hours after 99mTc-HSAD administration was a characteristic finding of NS. In order to confirm it, the subjects were divided into two groups: the NS group (n = 10) and the non-NS group (n = 7). We defined more intense uptake in the kidney than the liver on the anterior view 24 hours after 99mTc-HSAD administration as Dense Kidney (+). Furthermore, we designed regions of interest in the right and left kidneys and liver on anterior and posterior images, then calculated the kidney-liver ratio. Nine of the ten patients had Dense Kidney (+) in the NS group, compared to none in the non-NS group. And the kidney-liver ratio was significantly higher in the NS group than in the non-NS group on each view in the bilateral kidneys. In conclusion, our results suggest that the renal uptake 24 hours after 99mTc-HSAD administration is a characteristic finding of NS.

  9. Significance of Technetium-99m Human Serum Albumin Diethylenetriamine Pentaacetic Acid Scintigraphy in Patients with Nephrotic Syndrome

    PubMed Central

    Takashima, Tsuyoshi; Kishi, Tomoya; Onozawa, Koji; Rikitake, Shuichi; Miyazono, Motoaki; Otsuka, Takateru; Irie, Hiroyuki; Iwakiri, Ryuichi; Fujimoto, Kazuma; Ikeda, Yuji

    2015-01-01

    It is thought that a large amount of albumin leaking from the glomerulus in nephrotic syndrome (NS) is reabsorbed at the proximal tubule and catabolized. Therefore, it is possible the final quantity of urinary protein does not always reflect the amount of leakage of protein from the glomerulus. We experienced two cases without nephrotic range proteinuria thought to involve hypoproteinemia due to the same pathophysiology as NS. On these patients, we performed protein leakage scintigraphy with technetium-99m human serum albumin diethylenetriamine pentaacetic acid (99mTc-HSAD) to exclude a diagnosis of protein-losing gastroenteropathy and observed diffuse positive accumulation in the kidneys with more intense uptake in the kidney than the liver on the anterior view 24 hours after 99mTc-HSAD administration. In healthy adults intravenously given 99mTc-HSAD, the same dynamics are observed as in albumin metabolism, and the organ radioactivity of the liver and kidneys after 24 hours is equal. Therefore, we thought it was possible that the renal uptake 24 hours after 99mTc-HSAD administration was a characteristic finding of NS. In order to confirm it, the subjects were divided into two groups: the NS group (n = 10) and the non-NS group (n = 7). We defined more intense uptake in the kidney than the liver on the anterior view 24 hours after 99mTc-HSAD administration as Dense Kidney (+). Furthermore, we designed regions of interest in the right and left kidneys and liver on anterior and posterior images, then calculated the kidney-liver ratio. Nine of the ten patients had Dense Kidney (+) in the NS group, compared to none in the non-NS group. And the kidney-liver ratio was significantly higher in the NS group than in the non-NS group on each view in the bilateral kidneys. In conclusion, our results suggest that the renal uptake 24 hours after 99mTc-HSAD administration is a characteristic finding of NS. PMID:25859658

  10. [Modified albumin in harp seal blood serum].

    PubMed

    Erokhina, I A

    1999-01-01

    The content of modified albumin (Am) in harp seal (Pagophilus groenlandica Erxleben, 1777) blood serum was studied. Am was determined by paper electrophoresis by means of re-precipitation in the trichloroacetic acid-ethanol system. Modified albumin content in normal seal pups' blood serum increased from 1990 to 1994. The Am level in undernourished pups was stable from year to year and higher than in normal pups. In oceanarium investigations it was revealed a low albumin resistance to denaturation and the dependence of Am content on the animals' physiological state. Thus there is a possibility to regard modified albumin content as one of the significant parameters in biomonitoring of harp seal population and, moreover, as a supplementary criterion for estimation of seals' health state in captivity.

  11. Albumin-deficient mouse models for studying metabolism of human albumin and pharmacokinetics of albumin-based drugs

    PubMed Central

    Roopenian, Derry C; Low, Benjamin E; Christianson, Gregory J; Proetzel, Gabriele; Sproule, Thomas J; Wiles, Michael V

    2015-01-01

    Serum albumin is the major determinant of blood colloidal osmotic pressure acting as a depot and distributor of compounds including drugs. In humans, serum albumin exhibits an unusually long half-life mainly due to protection from catabolism by neonatal Fc receptor (FcRn)-mediated recycling. These properties make albumin an attractive courier of therapeutically-active compounds. However, pharmaceutical research and development of albumin-based therapeutics has been hampered by the lack of appropriate preclinical animal models. To overcome this, we developed and describe the first mouse with a genetic deficiency in albumin and its incorporation into an existing humanized FcRn mouse model, B6.Cg-Fcgrttm1Dcr Tg(FCGRT)32Dcr/DcrJ (Tg32). Albumin-deficient strains (Alb-/-) were created by TALEN-mediated disruption of the albumin (Alb) gene directly in fertilized oocytes derived from Tg32 mice and its non-transgenic background control, C57BL/6J (B6). The resulting Alb-/- strains are analbuminemic but healthy. Intravenous administration of human albumin to Tg32-Alb-/- mFcRn-/- hFcRnTg/Tg) mice results in a remarkably extended human albumin serum half-life of ∼24 days, comparable to that found in humans, and in contrast to half-lives of 2.6–5.8 d observed in B6, B6-Alb-/- and Tg32 strains. This striking increase can be explained by the absence of competing endogenous mouse albumin and the presence of an active human FcRn. These novel albumin-deficient models provide unique tools for investigating the biology and pathobiology of serum albumin and are a more appropriate rodent surrogates for evaluating human serum albumin pharmacokinetics and albumin-based compounds. PMID:25654695

  12. Caffeic acid phenethyl ester exhibiting distinctive binding interaction with human serum albumin implies the pharmacokinetic basis of propolis bioactive components.

    PubMed

    Li, Hongliang; Wu, Fan; Tan, Jing; Wang, Kai; Zhang, Cuiping; Zheng, Huoqing; Hu, Fuliang

    2016-04-15

    Caffeic acid phenethyl ester (CAPE), as one of the major bioactive components present in propolis, exhibits versatile bioactivities, especially for its potent cytotoxic effects on several cancer cell models. To understand the pharmacokinetic characteristics of CAPE, the binding interaction between CAPE and human serum albumin (HSA) was investigated in vitro using multiple spectroscopic methods and molecular docking. The results reveal that CAPE exhibits a distinctive binding interaction with HSA comparing with other propolis components. The association constant K(A) (L mol(-1)) of the binding reaches 10(6) order of magnitude, which is significantly stronger than the other components of propolis. Based on the theory of fluorescence resonance energy transfer, the binding distance was calculated as 5.7 nm, which is longer than that of the other components of propolis. The thermodynamic results indicate that the binding is mainly driven by hydrogen bonds and van der Waals force. The docking and drugs (warfarin and ibuprofen) competitive results show that CAPE is located in the subdomain IIA (Sudlow's site I, FA7) of HSA, and Gln196 and Lys199 contribute to the hydrogen bonds. Circular dichroism spectra suggest an alteration of the secondary structure of HSA due to its partial unfolding in the presence of CAPE.

  13. A study of the adsorption of the amphiphilic penicillins cloxacillin and dicloxacillin onto human serum albumin using surface tension isotherms

    NASA Astrophysics Data System (ADS)

    Barbosa, Silvia; Leis, David; Taboada, Pablo; Attwood, David; Mosquera, Victor

    The interaction of human serum albumin (HSA) with two structurally similar anionic amphiphilic penicillins, cloxacillin and dicloxacillin, at 25°C has been examined by surface tension measurements under conditions at which the HSA molecule was positively (pH 4.5) or negatively charged (pH 7.4). Measurements were at fixed HSA concentrations (0.0125 and 0.125% w/v) and at drug concentrations over a range including, where possible, the critical micelle concentration (cmc). Interaction between anionic drugs and positively charged HSA at pH 7.4 resulted in an increase of the cmc of each drug as a consequence of its removal from solution by adsorption. Limited data for cloxacillin at pH 4.5 indicated an apparent decrease of the cmc in the presence of HSA suggesting a facilitation of the aggregation by association with the protein. Changes in the surface tension-log (drug concentration) plots in the presence of HSA have been discussed in terms of the adsorption of drug at the air-solution and protein-solution interfaces. Standard free energy changes associated with the micellization of both drugs and their adsorption at the air-solution interface have been calculated and compared.

  14. Caffeic acid phenethyl ester exhibiting distinctive binding interaction with human serum albumin implies the pharmacokinetic basis of propolis bioactive components.

    PubMed

    Li, Hongliang; Wu, Fan; Tan, Jing; Wang, Kai; Zhang, Cuiping; Zheng, Huoqing; Hu, Fuliang

    2016-04-15

    Caffeic acid phenethyl ester (CAPE), as one of the major bioactive components present in propolis, exhibits versatile bioactivities, especially for its potent cytotoxic effects on several cancer cell models. To understand the pharmacokinetic characteristics of CAPE, the binding interaction between CAPE and human serum albumin (HSA) was investigated in vitro using multiple spectroscopic methods and molecular docking. The results reveal that CAPE exhibits a distinctive binding interaction with HSA comparing with other propolis components. The association constant K(A) (L mol(-1)) of the binding reaches 10(6) order of magnitude, which is significantly stronger than the other components of propolis. Based on the theory of fluorescence resonance energy transfer, the binding distance was calculated as 5.7 nm, which is longer than that of the other components of propolis. The thermodynamic results indicate that the binding is mainly driven by hydrogen bonds and van der Waals force. The docking and drugs (warfarin and ibuprofen) competitive results show that CAPE is located in the subdomain IIA (Sudlow's site I, FA7) of HSA, and Gln196 and Lys199 contribute to the hydrogen bonds. Circular dichroism spectra suggest an alteration of the secondary structure of HSA due to its partial unfolding in the presence of CAPE. PMID:26829518

  15. Synthesis, characterization and the interaction of some new water-soluble metal Schiff base complexes with human serum albumin.

    PubMed

    Asadi, Mozaffar; Asadi, Zahra; Sadi, Somaye Barzegar; Zarei, Leila; Baigi, Fatemeh Moosavi; Amirghofran, Zahra

    2014-03-25

    Some new water-soluble Schiff base complexes of Na2[M(L)(H2O)n]; (M=Zn, Cu, Ni, Mn) with a new water-soluble Schiff base ligand where L denotes an asymmetric N2O2 Schiff base ligands; N,N'-bis(5-sulfosalicyliden)-3,4-diaminobenzophenone (5-SO3-3,4-salbenz) were synthesized and characterized. The formation constants of the water soluble Schiff base complexes were calculated by Ketelaar's equation. The theoretical molecular structure for the complexes was computed by using the HF method and the 6-311G basis set. The mechanism of binding of Na2[M(L)(H2O)n] with human serum albumin (HSA) was studied by fluorescence spectroscopic technique. The results of fluorescence titration showed that the intrinsic fluorescence of HSA was quenched by the complexes; which was rationalized in terms of the dynamic quenching mechanism. The values of Stern-Volmer constants, quenching rate constants, binding constants, binding sites and average aggregation number of HSA have been determined. The thermodynamic parameters, were calculated by van't Hoff equation, indicate that the binding is entropy driven and enthalpically disfavored. Based on the Förster theory of non-radiation energy transfer, the efficiency of energy transfer and the distance between the donor (Trp residues) and the acceptor (complex) were obtained. Finally, the growth inhibitory effects of the complexes toward the K562 cancer cell line were measured.

  16. Study on the interaction of the epilepsy drug, zonisamide with human serum albumin (HSA) by spectroscopic and molecular docking techniques

    NASA Astrophysics Data System (ADS)

    Shahabadi, Nahid; Khorshidi, Aref; Moghadam, Neda Hossinpour

    2013-10-01

    In the present investigation, an attempt has been made to study the interaction of zonisamide (ZNS) with the transport protein, human serum albumin (HSA) employing UV-Vis, fluorometric, circular dichroism (CD) and molecular docking techniques. The results indicated that binding of ZNS to HSA caused strong fluorescence quenching of HSA through static quenching mechanism, hydrogen bonds and van der Waals contacts are the major forces in the stability of protein ZNS complex and the process of the binding of ZNS with HSA was driven by enthalpy (ΔH = -193.442 kJ mol-1). The results of CD and UV-Vis spectroscopy showed that the binding of this drug to HSA induced conformational changes in HSA. Furthermore, the study of molecular docking also indicated that zonisamide could strongly bind to the site I (subdomain IIA) of HSA mainly by hydrophobic interaction and there were hydrogen bond interactions between this drug and HSA, also known as the warfarin binding site.

  17. Study on the interaction of a copper(II) complex containing the artificial sweetener aspartame with human serum albumin.

    PubMed

    Shahabadi, Nahid; Khodaei, Mohammad Mehdi; Kashanian, Soheila; Kheirdoosh, Fahimeh; Filli, Soraya Moradi

    2014-05-01

    A copper(II) complex containing aspartame (APM) as ligand, Cu(APM)2Cl2·2H2O, was synthesized and characterized. In vitro binding interaction of this complex with human serum albumin (HSA) was studied at physiological pH. Binding studies of this complex with HSA are useful for understanding the Cu(APM)2Cl2·2H2O-HSA interaction mechanism and providing guidance for the application and design of new and more efficient artificial sweeteners drive. The interaction was investigated by spectrophotometric, spectrofluorometric, competition experiment and circular dichroism. Hyperchromicity observed in UV absorption band of Cu(APM)2Cl2·2H2O. A strong fluorescence quenching reaction of HSA to Cu(APM)2Cl2·2H2O was observed and the binding constant (Kf) and corresponding numbers of binding sites (n) were calculated at different temperatures. Thermodynamic parameters, enthalpy change (∆H) and entropy change (∆S) were calculated to be -458.67 kJ mol(-1) and -1,339 J mol(-1 )K(-1) respectively. According to the van't Hoff equation, the reaction is predominantly enthalpically driven. In conformity with experimental results, we suggest that Cu(APM)2Cl2·2H2O interacts with HSA. In comparison with previous study, it is found that the Cu(II) complex binds stronger than aspartame. PMID:24481880

  18. Generation of TALE nickase-mediated gene-targeted cows expressing human serum albumin in mammary glands.

    PubMed

    Luo, Yan; Wang, Yongsheng; Liu, Jun; Cui, Chenchen; Wu, Yongyan; Lan, Hui; Chen, Qi; Liu, Xu; Quan, Fusheng; Guo, Zekun; Zhang, Yong

    2016-02-08

    Targeting exogenous genes at milk protein loci via gene-targeting technology is an ideal strategy for producing large quantities of pharmaceutical proteins. Transcription-activator-like effector (TALE) nucleases (TALENs) are an efficient genome-editing tool. However, the off-target effects may lead to unintended gene mutations. In this study, we constructed TALENs and TALE nickases directed against exon 2 of the bovine β-lactoglobulin (BLG) locus. The nickases can induce a site-specific DNA single-strand break, without inducing double-strand break and nonhomologous end joining mediated gene mutation, and lower cell apoptosis rate than TALENs. After co-transfecting the bovine fetal fibroblasts with human serum albumin (HSA) gene-targeting vector and TALE nickase expression vectors, approximately 4.8% (40/835) of the cell clones contained HSA at BLG locus. Unexpectedly, one homozygous gene-targeted cell clone (1/835, 0.1%) was obtained by targeting both alleles of BLG in a single round of transfection. The recombinant protein mimicking the endogenous BLG was highly expressed and correctly folded in the mammary glands of the targeted cows, and the expression level of HSA was significantly increased in the homozygous targeted cows. Results suggested that the combination of TALE nickase-mediated gene targeting and somatic cell nuclear transfer is a feasible and safe approach in producing gene-targeted livestock.

  19. Surface characterization of human serum albumin and sodium perfluorooctanoate mixed solutions by pendant drop tensiometry and circular dichroism.

    PubMed

    Messina, Paula; Prieto, Gerardo; Dodero, Verónica; Cabrerizo-Vílchez, M A; Maldonado-Valderrama, J; Ruso, Juan M; Sarmiento, Félix

    2006-06-15

    The interfacial behavior of mixed human serum albumin (HSA)/sodium perfluorooctanoate (C8FONa) solutions is examined by using two experimental techniques, pendant drop tensiometry and circular dichroism spectroscopy. Through the analysis of the surface tension of the mixed solutions, surface competitive adsorption at the air-water interface between C8FONa and HSA is detected. The dynamic adsorption curves exhibit the distinct regimes in their time-dependent surface tension. The nature of these regimes is further analyzed in terms of the variation of the molecules surface areas. As a consequence, a compact and dense structure was formed where protein molecules were interconnected and overlapped. Thus, a reduction of the area occupied per molecule from 100 to 0.2 nm(2) is interpreted as a gel-like structure at the surface. The presence of the surfactant seems to favor the formation of this interfacial structure. Finally, measurements of circular dichroism suggests a compaction of the protein due to the association with the surfactant given by an increase of alpha-helix structure in the complexes as compared to that of pure protein.

  20. Ultraviolet-circular dichroism spectroscopy and potentiometric study of the interaction between human serum albumin and sodium perfluorooctanoate.

    PubMed

    Messina, Paula; Prieto, Gerardo; Dodero, Verónica; Ruso, Juan M; Schulz, Pablo; Sarmiento, Félix

    2005-12-15

    The interaction of a fluorinated surfactant, sodium perfluorooctanoate, with human serum albumin (HSA) has been investigated by a combination of ultraviolet-circular dichroism (UV-CD) spectroscopy and potentiometry (by a home-built ion-selective electrode) techniques to detect and characterize the conformational transitions of HSA. By using difference spectroscopy, the transition was followed as a function of temperature, and the data were analyzed to obtain the parameters characterizing the thermodynamics of unfolding. The results indicate that the presence of surfactant drastically changes the melting unfolding, acting as a structure stabilizer and delaying the unfolding process. Potentiometric measurements were used to determine the binding isotherms and binding capacity for this system. The isotherm shows a high affinity of surfactant molecules for HSA. The average number of surfactant molecules absorbed per protein molecule (at 28 mM of surfactant concentration) was found to be approximately 900, about 6 g of surfactant per gram of protein. The shape of the binding capacity curve and the relation between binding capacity and extend of cooperativity were examined. From these analysis, the values of g (number of ligand-binding sites), KH (Hill binding constant), and nH (Hill coefficient) were determined.

  1. Interactions of benzotriazole UV stabilizers with human serum albumin: Atomic insights revealed by biosensors, spectroscopies and molecular dynamics simulations.

    PubMed

    Zhuang, Shulin; Wang, Haifei; Ding, Keke; Wang, Jiaying; Pan, Liumeng; Lu, Yanli; Liu, Qingjun; Zhang, Chunlong

    2016-02-01

    Benzotriazole UV stabilizers (BZTs) belong to one prominent group of ultraviolet (UV) stabilizers and are widely used in various plastics materials. Their large production volumes, frequent detections in the environment and potential toxicities have raised increasing public concern. BZTs can be transported in vivo by transport proteins in plasma and the binding association to transport proteins may serve as a significant parameter to evaluate the bioaccumulative potential. We utilized a novel HSA biosensor, circular dichroism spectroscopy, fluorescence spectroscopy to detect the dynamic interactions of six BZTs (UV-326, UV-327, UV-328, UV-329, UV-P, and BZT) with human serum albumin (HSA), and characterized the corresponding structure-activity relationships (SAR) by molecular dynamics simulations. All test BZTs potently bind at Sudlow site I of HSA with a binding constant of 10(4) L/mol at 298 K. Minor changes in the moieties of BZTs affect their interactions with HSA and differently induce conformations of HSA. Their binding reduced electrochemical impedance spectra and α-helix content of HSA, caused slight red-shifted emission, and changed fluorescence lifetime components of HSA in a concentration-dependent mode. UV-327 and UV-329 form hydrogen bonds with HSA, while UV-329, UV-P and BZT bind HSA with more favorable electrostatic interactions. Our in vitro and in silico study offered a significant framework toward the understanding of risk assessment of BZTs and provides guide for future design of environmental benign BZTs-related materials.

  2. Interaction of meropenem with 'N' and 'B' isoforms of human serum albumin: a spectroscopic and molecular docking study.

    PubMed

    Rehman, Md Tabish; Ahmed, Sarfraz; Khan, Asad U

    2016-09-01

    Carbapenems are used to control the outbreak of β-lactamases expressing bacteria. The effectiveness of drugs is influenced by its interaction with human serum albumin (HSA). Strong binding of carbapenems to HSA may lead to decreased bioavailability of the drug. The non-optimal drug dosage will provide a positive selection pressure on bacteria to develop resistance. Here, we investigated the interaction between meropenem and HSA at physiological pH 7.5 (N-isoform HSA) and non-physiological pH 9.2 (B-isoform HSA). Results showed that meropenem quenches the fluorescence of both 'N' and 'B' isoforms of HSA (ΔG < 0 and binding constant ~10(4) M(-1)). Electrostatic interactions and van der Waal interactions along with H-bonds stabilized the complex of meropenem with 'N' and 'B' isoforms of HSA, respectively. Molecular docking results revealed that meropenem binds to HSA near Sudlow's site II (subdomain IIIA) close to Trp-214 with a contribution of a few residues of subdomain IIA. CD spectroscopy showed a change in the conformation of both the isoforms of HSA upon meropenem binding. The catalytic efficiency of HSA (only N-isoform) on p-nitrophenyl acetate was increased primarily due to a decrease in Km and an increase in kcat values. This study provides an insight into the molecular basis of interaction between meropenem and HSA.

  3. Safety assessment of genetically modified rice expressing human serum albumin from urine metabonomics and fecal bacterial profile.

    PubMed

    Qi, Xiaozhe; Chen, Siyuan; Sheng, Yao; Guo, Mingzhang; Liu, Yifei; He, Xiaoyun; Huang, Kunlun; Xu, Wentao

    2015-02-01

    The genetically modified (GM) rice expressing human serum albumin (HSA) is used for non-food purposes; however, its food safety assessment should be conducted due to the probability of accidental mixture with conventional food. In this research, Sprague Dawley rats were fed diets containing 50% (wt/wt) GM rice expressing HSA or non-GM rice for 90 days. Urine metabolites were detected by (1)H NMR to examine the changes of the metabolites in the dynamic process of metabolism. Fecal bacterial profiles were detected by denaturing gradient gel electrophoresis to reflect intestinal health. Additionally, short chain fatty acids and fecal enzymes were investigated. The results showed that compared with rats fed the non-GM rice, some significant differences were observed in rats fed with the GM rice; however, these changes were not significantly different from the control diet group. Additionally, the gut microbiota was associated with blood indexes and urine metabolites. In conclusion, the GM rice diet is as safe as the traditional daily diet. Furthermore, urine metabonomics and fecal bacterial profiles provide a non-invasive food safety assessment rat model for genetically modified crops that are used for non-food/feed purposes. Fecal bacterial profiles have the potential for predicting the change of blood indexes in future.

  4. Investigation of siRNA-loaded polyethylenimine-coated human serum albumin nanoparticle complexes for the treatment of breast cancer.

    PubMed

    Abbasi, Sana; Paul, Arghya; Prakash, Satya

    2011-11-01

    Small interfering RNA (siRNA) molecules have great potential for developing into a future therapy for breast cancer. To overcome the issues related to rapid degradation and low transfection of naked siRNA, polyethylenimine (PEI)-coated human serum albumin (HSA) nanoparticles have been characterized and studied here for efficient siRNA delivery to the MCF-7 breast cancer cell line. The optimized nanoparticles were ~90 nm in size, carrying a surface charge of +26 mV and a polydispersity index (PDI) less than 0.25. The shape and morphology of the particles was studied using electron microscopy. A cytotoxicity assessment of the nanoparticles showed no correlation of cytotoxicity with HSA concentration, while using high molecular weight PEI (MW of 70 against 25 kDa) showed higher cytotoxicity. The optimal transfection achieved of fluorescin-tagged siRNA loaded into PEI-coated HSA nanoparticles was 61.66 ± 6.8%, prepared with 6.25 μg of PEI (25 kDa) added per mg of HSA and 20 mg/ml HSA, indicating that this nonviral vector may serve as a promising gene delivery system.

  5. Startling temperature effect on proteins when confined: single molecular level behaviour of human serum albumin in a reverse micelle.

    PubMed

    Sengupta, Bhaswati; Yadav, Rajeev; Sen, Pratik

    2016-06-01

    The present work reports the effect of confinement, and temperature therein, on the conformational fluctuation dynamics of domain-I of human serum albumin (HSA) by fluorescence correlation spectroscopy (FCS). The water-pool of a sodium bis(2-ethylhexyl)sulfosuccinate (AOT) reverse micelle has been used as the confined environment. It was observed that the conformational fluctuation time is about 6 times smaller compared to bulk medium when confined in a water-pool of 3.5 nm radius. On increasing the size of the water-pool the conformational fluctuation time was found to increase monotonically and approaches the bulk value. The effect of confinement is on par with the general belief about the restricted motion of a macromolecule upon confinement. However, the effect of temperature was found to be surprising. An increase in the temperature from 298 K to 313 K induces a larger change in the conformational fluctuation time in HSA, when confined. In the bulk medium, apparently there is no change in the conformational fluctuation time in the aforementioned temperature range, whereas, when HSA is present in an AOT water-pool of radius 3.5 nm, about an 88% increase in the fluctuation time was observed. The observed prominent thermal effect on the conformational dynamics of domain-I of HSA in the water-pool of an AOT reverse micelle as compared to in the bulk medium was concluded to arise from the confined solvent effect.

  6. Enhanced production of human serum albumin by fed-batch culture of Hansenula polymorpha with high-purity oxygen.

    PubMed

    Youn, Jong Kyu; Shang, Longan; Kim, Moon Il; Jeong, Chang Moon; Chang, Ho Nam; Hahm, Moon Sun; Rhee, Sang Ki; Kang, Hyun Ah

    2010-11-01

    Fed-batch cultures of Hansenula polymorpha were studied to develop an efficient biosystem to produce recombinant human serum albumin (HSA). To comply with this purpose, we used high purity oxygen supplying strategy to increase viable cell density in a bioreactor and enhance the production of target protein. A mutant strain, H. polymorpha GOT7 was utilized in this study as a host strain in both 5-L and 30-L scale fermentors. To supply high purity oxygen into a bioreactor, nearly 100 % high purity oxygen from commercial bomb or higher than 93 % oxygen available in-situ from a pressure swing adsorption oxygen generator (PSA) was employed. Under the optimal fermentation of H. polymorpha with high purity oxygen, the final cell densities and produced HSA concentrations were 24.6 g/L and 5.1 g/L in the 5-L fermentor, and 24.8 g/L and 4.5 g/L in the 30-L fermentor, respectively. These were about 2-10 times higher than those obtained in air-based fed-batch fermentations. The discrepancies between the 5-L and 30-L fermentors with air supply were presumably due to the higher contribution of surface aeration over submerged aeration in the 5-L fermentor. This study, therefore, proved the positive effect of high purity oxygen to enhance viable cell density as well as target recombinant protein production in microbial fermentations. PMID:21124059

  7. Characterisation of interaction between food colourant allura red AC and human serum albumin: multispectroscopic analyses and docking simulations.

    PubMed

    Wu, Di; Yan, Jin; Wang, Jing; Wang, Qing; Li, Hui

    2015-03-01

    Binding interaction of human serum albumin (HSA) with allura red AC, a food colourant, was investigated at the molecular level through fluorescence, ultraviolet-visible, circular dichroism (CD) and Raman spectroscopies, as well as protein-ligand docking studies to better understand the chemical absorption, distribution and transportation of colourants. Results show that allura red AC has the ability to quench the intrinsic fluorescence of HSA through static quenching. The negative values of the thermodynamic parameters ΔG, ΔH, and ΔS indicated that hydrogen bond and van der Waals forces are dominant in the binding between the food colourant and HSA. The CD and Raman spectra showed that the binding of allura red AC to HSA induces the rearrangement of the carbonyl hydrogen-bonding network of polypeptides, which changes the HSA secondary structure. This colourant is bound to HSA in site I, and the binding mode was further analysed with the use of the CDOCKER algorithm in Discovery Studio.

  8. Chloramphenicol binding to human serum albumin: Determination of binding constants and binding sites by steady-state fluorescence

    NASA Astrophysics Data System (ADS)

    Ding, Fei; Zhao, Guangyu; Chen, Shoucong; Liu, Feng; Sun, Ying; Zhang, Li

    2009-07-01

    The interaction between chloramphenicol and human serum albumin (HSA) was studied by fluorescence, UV/vis, circular dichroism (CD) and three-dimensional fluorescence spectroscopy. Fluorescence data revealed that the fluorescence quenching of HSA by chloramphenicol was the result of the formation of drug-HSA complex, and the effective quenching constants ( Ka) were 2.852 × 10 4, 2.765 × 10 4, 2.638 × 10 4 and 2.542 × 10 4 M -1 at 287, 295, 303 and 311 K, respectively. The thermodynamic parameters, enthalpy change (Δ H) and entropy change (Δ S) for the reaction were calculated to be -3.634 kJ mol -1 and 72.66 J mol -1 K -1 according to van't Hoff equation. The results indicated that the hydrophobic and electrostatic interactions played a major role in the binding of drug to HSA. The distance r between donor and acceptor was obtained to be 3.63 nm according to Förster's theory. Site marker competitive experiments indicated that the binding of drug to HSA primarily took place in subdomain IIA. The alterations of HSA secondary structure in the presence of chloramphenicol were confirmed by the evidences from synchronous fluorescence, CD and three-dimensional fluorescence spectra. In addition, the effect of common ions on the binding constants of drug-HSA complex was also discussed.

  9. Energetic domains and conformational analysis of human serum albumin upon co-incubation with sodium benzoate and glucose.

    PubMed

    Taghavi, F; Moosavi-Movahedi, A A; Bohlooli, M; Habibi-Rezaei, M; Hadi Alijanvand, H; Amanlou, M; Sheibani, N; Saboury, A A; Ahmad, F

    2014-01-01

    Sodium benzoate (SB), a powerful inhibitor of microbial growth, is one of the most commonly used food preservative. Here, we determined the effects of SB on human serum albumin (HSA) structure in the presence or absence of glucose after 35 days of incubation under physiological conditions. The biochemical, biophysical, and molecular approaches including free amine content assay (TNBSA assay), fluorescence, and circular dichroism spectroscopy (CD), differential scanning calorimetry (DSC), and molecular docking and LIGPLOT studies were utilized for structural studies. The TNBSA results indicated that SB has the ability to bind Lys residues in HSA through covalent bonds. The docking and LIGPLOT studies also determined another specific site via hydrophobic interactions. The CD results showed more structural helicity for HSA incubated with SB, while HSA incubated with glucose had the least, and HSA incubated with glucose + SB had medium helicity. Fluorescence spectrophotometry results demonstrated partial unfolding of HSA incubated with SB in the presence or absence of glucose, while maximum partial unfolding was observed in HSA incubated with glucose. These results were confirmed by DSC and its deconvoluted thermograms. The DSC results also showed significant changes in HSA energetic structural domains due to HSA incubation with SB in the presence or absence of glucose. Together, our studies showed the formation of three different intermediates and indicate that biomolecular investigation are effective in providing new insight into safety determinations especially in health-related conditions including diabetes.

  10. Study on the interaction of a copper(II) complex containing the artificial sweetener aspartame with human serum albumin.

    PubMed

    Shahabadi, Nahid; Khodaei, Mohammad Mehdi; Kashanian, Soheila; Kheirdoosh, Fahimeh; Filli, Soraya Moradi

    2014-05-01

    A copper(II) complex containing aspartame (APM) as ligand, Cu(APM)2Cl2·2H2O, was synthesized and characterized. In vitro binding interaction of this complex with human serum albumin (HSA) was studied at physiological pH. Binding studies of this complex with HSA are useful for understanding the Cu(APM)2Cl2·2H2O-HSA interaction mechanism and providing guidance for the application and design of new and more efficient artificial sweeteners drive. The interaction was investigated by spectrophotometric, spectrofluorometric, competition experiment and circular dichroism. Hyperchromicity observed in UV absorption band of Cu(APM)2Cl2·2H2O. A strong fluorescence quenching reaction of HSA to Cu(APM)2Cl2·2H2O was observed and the binding constant (Kf) and corresponding numbers of binding sites (n) were calculated at different temperatures. Thermodynamic parameters, enthalpy change (∆H) and entropy change (∆S) were calculated to be -458.67 kJ mol(-1) and -1,339 J mol(-1 )K(-1) respectively. According to the van't Hoff equation, the reaction is predominantly enthalpically driven. In conformity with experimental results, we suggest that Cu(APM)2Cl2·2H2O interacts with HSA. In comparison with previous study, it is found that the Cu(II) complex binds stronger than aspartame.

  11. Abacavir and warfarin modulate allosterically kinetics of NO dissociation from ferrous nitrosylated human serum heme-albumin

    SciTech Connect

    Ascenzi, Paolo Imperi, Francesco; Coletta, Massimo; Fasano, Mauro

    2008-05-02

    Human serum albumin (HSA) participates to heme scavenging, in turn HSA-heme binds gaseous diatomic ligands at the heme-Fe-atom. Here, the effect of abacavir and warfarin on denitrosylation kinetics of HSA-heme-Fe(II)-NO (i.e., k{sub off}) is reported. In the absence of drugs, the value of k{sub off} is (1.3 {+-} 0.2) x 10{sup -4} s{sup -1}. Abacavir and warfarin facilitate NO dissociation from HSA-heme-Fe(II)-NO, the k{sub off} value increases to (8.6 {+-} 0.9) x 10{sup -4} s{sup -1}. From the dependence of k{sub off} on the drug concentration, values of the dissociation equilibrium constant for the abacavir and warfarin binding to HSA-heme-Fe(II)-NO (i.e., K = (1.2 {+-} 0.2) x 10{sup -3} M and (6.2 {+-} 0.7) x 10{sup -5} M, respectively) were determined. The increase of k{sub off} values reflects the stabilization of the basic form of HSA-heme-Fe by ligands (e.g., abacavir and warfarin) that bind to Sudlow's site I. This event parallels the stabilization of the six-coordinate derivative of the HSA-heme-Fe(II)-NO atom. Present data highlight the allosteric modulation of HSA-heme-Fe(II) reactivity by heterotropic effectors.

  12. Multifunctional Effect of Human Serum Albumin Reduces Alzheimer's Disease Related Pathologies in the 3xTg Mouse Model.

    PubMed

    Ezra, Assaf; Rabinovich-Nikitin, Inna; Rabinovich-Toidman, Polina; Solomon, Beka

    2015-01-01

    Alzheimer's disease (AD), the prevalent dementia in the elderly, involves many related and interdependent pathologies that manifests simultaneously, eventually leading to cognitive impairment and death. No treatment is currently available; however, an agent addressing several key pathologies simultaneously has a better therapeutic potential. Human serum albumin (HSA) is a highly versatile protein, harboring multifunctional properties that are relevant to key pathologies underlying AD. This study provides insight into the mechanism for HSA's therapeutic effect. In vivo, a myriad of beneficial effects were observed by pumps infusing HSA intracerebroventricularly, for the first time in an AD 3xTg mice model. A significant effect on amyloid-β (Aβ) pathology was observed. Aβ1-42, soluble oligomers, and total plaque area were reduced. Neuroblastoma SHSY5Y cell line confirmed that the reduction in Aβ1-42 toxicity was due to direct binding rather than other properties of HSA. Total and hyperphosphorylated tau were reduced along with an increase in tubulin, suggesting increased microtubule stability. HSA treatment also reduced brain inflammation, affecting both astrocytes and microglia markers. Finally, evidence for blood-brain barrier and myelin integrity repair was observed. These multidimensional beneficial effects of intracranial administrated HSA, together or individually, contributed to an improvement in cognitive tests, suggesting a non-immune or Aβ efflux dependent means for treating AD.

  13. Structure of the methyl orange-binding site on human serum albumin and its color-change mechanism.

    PubMed

    Ito, Shigenori; Yamamoto, Daisuke

    2015-01-01

    The goal in this study was to clarify the color-change mechanisms of methyl orange (MO) bound to human serum albumin (HSA) and the structure of the binding site. The absorbance of the MOHSA complex was measured at 560 nm in solutions of varying pH (pH 2.4-6.6). The obtained pH-dependent experimental data were consistent with the data calculated using the Henderson-Hasselbalch equation and pKa values (3.8, MO; 1.4, carboxyl group). The extent of the binding of MO to an HSA molecule was determined to be 1-4 by performing surface plasmon resonance analysis. Furthermore, the binding of MO to HSA was inhibited by warfarin. A fitting model of MO to HSA was created to evaluate these results based on PDB data (warfarin-HSA complex: 2BXD) and protein-structure analysis. The color-change mechanism of the MO-HSA complex appears to be as follows: the dissociated sulfo group of MO binds to Arg218/Lys444 sidechains through electrostatic interaction in the warfarin-binding site, and, subsequently, the color change occurs through a proton exchange between the diazenyl group and the γ-carboxyl group of Glu292. The color-changed MO is fixed in the warfarin-binding site. These results could support the development of a reliable dye-binding method and of a new method for staining diverse tissues that is based on a validated mechanism.

  14. Potassium sorbate as an AGE activator for human serum albumin in the presence and absence of glucose.

    PubMed

    Taghavi, F; Moosavi-Movahedi, A A; Bohlooli, M; Hadi Alijanvand, H; Salami, M; Maghami, P; Saboury, A A; Farhadi, M; Yousefi, R; Habibi-Rezaei, M; Sheibani, N

    2013-11-01

    Advanced glycation end products (AGEs) are the predominant intermediates of glycation process, and mediate oxidative stress and complications of diabetes. Potassium sorbate (PS) as a widespread preservative is an oxidative agent and used in different dairy and drug products, which can readily enter biological matrices. Here we studied the PS interference with glycation of human serum albumin (HSA) in the presence of glucose (Glc) using various techniques. These included TNBSA assay, circular dichroism, fluorescence spectroscopy, differential scanning calorimetry (DSC), Th T assay, and atomic force microscopy. Our results indicated that HSA glycation was accelerated in the presence of PS. Furthermore, PS produced AGEs in the absence of glucose. Secondary and tertiary structural changes were also observed in HSA incubated with glucose in the presence or absence of PS through beta-sheet inducing effects. Th T assay demonstrated the role of PS in HSA fibril formation in the presence or absence of glucose. Atomic force microscopy determined different amyloid fibril formation in HSA incubated with PS in the presence or absence of glucose. Together our results indicated that PS has a stimulatory effect on glycation and fibrillation of HSA in the presence or absence of glucose, and could exacerbate complication of diabetes. PMID:24001567

  15. Interactions of benzotriazole UV stabilizers with human serum albumin: Atomic insights revealed by biosensors, spectroscopies and molecular dynamics simulations.

    PubMed

    Zhuang, Shulin; Wang, Haifei; Ding, Keke; Wang, Jiaying; Pan, Liumeng; Lu, Yanli; Liu, Qingjun; Zhang, Chunlong

    2016-02-01

    Benzotriazole UV stabilizers (BZTs) belong to one prominent group of ultraviolet (UV) stabilizers and are widely used in various plastics materials. Their large production volumes, frequent detections in the environment and potential toxicities have raised increasing public concern. BZTs can be transported in vivo by transport proteins in plasma and the binding association to transport proteins may serve as a significant parameter to evaluate the bioaccumulative potential. We utilized a novel HSA biosensor, circular dichroism spectroscopy, fluorescence spectroscopy to detect the dynamic interactions of six BZTs (UV-326, UV-327, UV-328, UV-329, UV-P, and BZT) with human serum albumin (HSA), and characterized the corresponding structure-activity relationships (SAR) by molecular dynamics simulations. All test BZTs potently bind at Sudlow site I of HSA with a binding constant of 10(4) L/mol at 298 K. Minor changes in the moieties of BZTs affect their interactions with HSA and differently induce conformations of HSA. Their binding reduced electrochemical impedance spectra and α-helix content of HSA, caused slight red-shifted emission, and changed fluorescence lifetime components of HSA in a concentration-dependent mode. UV-327 and UV-329 form hydrogen bonds with HSA, while UV-329, UV-P and BZT bind HSA with more favorable electrostatic interactions. Our in vitro and in silico study offered a significant framework toward the understanding of risk assessment of BZTs and provides guide for future design of environmental benign BZTs-related materials. PMID:26454115

  16. Structural alterations of human serum albumin caused by glycative and oxidative stressors revealed by circular dichroism analysis.

    PubMed

    Monacelli, Fiammetta; Storace, Daniela; D'Arrigo, Cristina; Sanguineti, Roberta; Borghi, Roberta; Pacini, Davide; Furfaro, Anna L; Pronzato, Maria A; Odetti, Patrizio; Traverso, Nicola

    2013-01-01

    The aim of this work was to evaluate the ability of oxidative and glycative stressors to modify properties of human serum albumin (HSA) by analyzing markers of glycation (pentosidine) and oxidation (advanced oxidative protein products (AOPPs)) and assessing fluorescence and circular dichroism. HSA was incubated for up to 21 days with ribose, ascorbic acid (AA) and diethylenetriamine pentacetate (DTPA) in various combinations in order to evaluate influences of these substances on the structure of HSA. Ribose was included as a strong glycative molecule, AA as a modulator of oxidative stress, and DTPA as an inhibitor of metal-catalyzed oxidation. Ribose induced a significant increase in pentosidine levels. AA and DTPA prevented the accumulation of pentosidine, especially at later time points. Ribose induced a mild increase in AOPP formation, while AA was a strong inducer of AOPP formation. Ribose, in combination with AA, further increased the formation of AOPP. DTPA prevented the AA-induced generation of AOPP. Ribose was also a potent inducer of fluorescence at 335nm ex/385nm em, which is typical of pentosidine. AA and DTPA prevented this fluorescence. Circular dichroism showed complex results, in which AA and DTPA were strong modifiers of the percentages of the alpha-helical structure of HSA, while ribose affected the structure of HSA only at later time points. PMID:23702842

  17. Generation of TALE nickase-mediated gene-targeted cows expressing human serum albumin in mammary glands

    PubMed Central

    Luo, Yan; Wang, Yongsheng; Liu, Jun; Cui, Chenchen; Wu, Yongyan; Lan, Hui; Chen, Qi; Liu, Xu; Quan, Fusheng; Guo, Zekun; Zhang, Yong

    2016-01-01

    Targeting exogenous genes at milk protein loci via gene-targeting technology is an ideal strategy for producing large quantities of pharmaceutical proteins. Transcription- activator-like effector (TALE) nucleases (TALENs) are an efficient genome-editing tool. However, the off-target effects may lead to unintended gene mutations. In this study, we constructed TALENs and TALE nickases directed against exon 2 of the bovine β-lactoglobulin (BLG) locus. The nickases can induce a site-specific DNA single-strand break, without inducing double-strand break and nonhomologous end joining mediated gene mutation, and lower cell apoptosis rate than TALENs. After co-transfecting the bovine fetal fibroblasts with human serum albumin (HSA) gene-targeting vector and TALE nickase expression vectors, approximately 4.8% (40/835) of the cell clones contained HSA at BLG locus. Unexpectedly, one homozygous gene-targeted cell clone (1/835, 0.1%) was obtained by targeting both alleles of BLG in a single round of transfection. The recombinant protein mimicking the endogenous BLG was highly expressed and correctly folded in the mammary glands of the targeted cows, and the expression level of HSA was significantly increased in the homozygous targeted cows. Results suggested that the combination of TALE nickase-mediated gene targeting and somatic cell nuclear transfer is a feasible and safe approach in producing gene-targeted livestock. PMID:26853907

  18. Effect of tanshinone IIA on the noncovalent interaction between warfarin and human serum albumin studied by electrospray ionization mass spectrometry.

    PubMed

    Liu, Jie; Wang, Xiaoru; Cai, Zongwei; Lee, Frank S C

    2008-10-01

    Enhanced anticoagulation and/or even bleeding are often observed when patients on long-term warfarin (WAR) therapy consumed Danshen, a well-known medicinal herb in traditional Chinese medicine (TCM). This study demonstrates that altered WAR metabolism, arising from its interaction with the active components in Danshen, played a significant role in this curative effect. Mass spectrometric techniques including ESI-ITMS (electrospray ionization ion-trap mass spectrometry) and ESI-TOF (time-of-flight)-MS have been developed for the study of such drug-herb interactions. The experimental approach involved a detailed analysis and comparison of WAR metabolites in vivo from blood or urine of rats that had been orally administrated with WAR, either singly or together with the representative bioactive component of Danshen-lipid soluble TIIA (Tanshinon IIA), and a study of the interaction of human serum albumin (HSA), WAR, and water-soluble sodium tanshinone IIA sulfonate (STS) in vitro. Results demonstrate that TIIA accelerates the metabolic rate of WAR, whereas STS displaces WAR from the WAR-HSA complex, resulting in an increase of free WAR concentration in blood. It is suggested that the elevated level and enhanced metabolism of WAR is responsible for the over-anticoagulation effect observed. PMID:18657993

  19. Exploring the site-selective binding of jatrorrhizine to human serum albumin: Spectroscopic and molecular modeling approaches

    NASA Astrophysics Data System (ADS)

    Mi, Ran; Hu, Yan-Jun; Fan, Xiao-Yang; Ouyang, Yu; Bai, Ai-Min

    2014-01-01

    This paper exploring the site-selective binding of jatrorrhizine to human serum albumin (HSA) under physiological conditions (pH = 7.4). The investigation was carried out using fluorescence spectroscopy, UV-vis spectroscopy, and molecular modeling. The results of fluorescence quenching and UV-vis absorption spectra experiments indicated the formation of the complex of HSA-jatrorrhizine. Binding parameters calculating from Stern-Volmer method and Scatchard method were calculated at 298, 304 and 310 K, with the corresponding thermodynamic parameters ΔG, ΔH and ΔS as well. Binding parameters calculating from Stern-Volmer method and Scatchard method showed that jatrorrhizine bind to HSA with the binding affinities of the order 104 L mol-1. The thermodynamic parameters studies revealed that the binding was characterized by negative enthalpy and positive entropy changes and the electrostatic interactions play a major role for jatrorrhizine-HSA association. Site marker competitive displacement experiments and molecular modeling calculation demonstrating that jatrorrhizine is mainly located within the hydrophobic pocket of the subdomain IIIA of HSA. Furthermore, the synchronous fluorescence spectra suggested that the association between jatrorrhizine and HSA changed molecular conformation of HSA.

  20. Investigation of the Interaction between Patulin and Human Serum Albumin by a Spectroscopic Method, Atomic Force Microscopy, and Molecular Modeling

    PubMed Central

    Yuqin, Li; Guirong, You; Zhen, Yang; Caihong, Liu; Baoxiu, Jia; Jiao, Chen; Yurong, Guo

    2014-01-01

    The interaction of patulin with human serum albumin (HSA) was studied in vitro under normal physiological conditions. The study was performed using fluorescence, ultraviolet-visible spectroscopy (UV-Vis), circular dichroism (CD), atomic force microscopy (AFM), and molecular modeling techniques. The quenching mechanism was investigated using the association constants, the number of binding sites, and basic thermodynamic parameters. A dynamic quenching mechanism occurred between HSA and patulin, and the binding constants (K) were 2.60 × 104, 4.59 × 104, and 7.01 × 104 M−1 at 288, 300, and 310 K, respectively. Based on fluorescence resonance energy transfer, the distance between the HSA and patulin was determined to be 2.847 nm. The ΔG0, ΔH0, and ΔS0 values across various temperatures indicated that hydrophobic interaction was the predominant binding force. The UV-Vis and CD results confirmed that the secondary structure of HSA was altered in the presence of patulin. The AFM results revealed that the individual HSA molecule dimensions were larger after interaction with patulin. In addition, molecular modeling showed that the patulin-HSA complex was stabilized by hydrophobic and hydrogen bond forces. The study results suggested that a weak intermolecular interaction occurred between patulin and HSA. Overall, the results are potentially useful for elucidating the toxigenicity of patulin when it is combined with the biomolecular function effect, transmembrane transport, toxicological, testing and other experiments. PMID:25110690

  1. Interaction of ionic detergent cethyltrimethylammonium bromide with human serum albumin at various values of pH: Spectroscopic study

    NASA Astrophysics Data System (ADS)

    Vlasova, Irina M.; Vlasov, Alexander A.; Saletsky, Alexander M.

    2010-12-01

    The interaction of cationic detergent cethyltrimethylammonium bromide (CTAB) with human serum albumin (HSA) at various values of pH has been studied using steady-state tryptophan fluorescence of HSA, polarized tryptophan fluorescence of HSA, fluorescence of nanomarker eosin in solutions of HSA and CTAB and Raman spectroscopy. By methods of fluorescent analysis the qualitative rearrangements of HSA globules at denaturation under action of CTAB are registered, whereas by Raman spectroscopy the quantitative changes of secondary structure of HSA at CTAB-induced denaturation are determined. It is shown that denaturation of HSA, taking place at interaction of cationic detergent CTAB with HSA, has one-stage character. At interaction of CTAB with HSA the deepest denaturation of HSA is reached at concentration of 4 mM CTAB (in the range of pH 3.5-8.0). More intensive denaturation of HSA under action of CTAB takes place at values of pH, higher than the isoelectric point of HSA (pI 4.7).

  2. Safety assessment of genetically modified rice expressing human serum albumin from urine metabonomics and fecal bacterial profile.

    PubMed

    Qi, Xiaozhe; Chen, Siyuan; Sheng, Yao; Guo, Mingzhang; Liu, Yifei; He, Xiaoyun; Huang, Kunlun; Xu, Wentao

    2015-02-01

    The genetically modified (GM) rice expressing human serum albumin (HSA) is used for non-food purposes; however, its food safety assessment should be conducted due to the probability of accidental mixture with conventional food. In this research, Sprague Dawley rats were fed diets containing 50% (wt/wt) GM rice expressing HSA or non-GM rice for 90 days. Urine metabolites were detected by (1)H NMR to examine the changes of the metabolites in the dynamic process of metabolism. Fecal bacterial profiles were detected by denaturing gradient gel electrophoresis to reflect intestinal health. Additionally, short chain fatty acids and fecal enzymes were investigated. The results showed that compared with rats fed the non-GM rice, some significant differences were observed in rats fed with the GM rice; however, these changes were not significantly different from the control diet group. Additionally, the gut microbiota was associated with blood indexes and urine metabolites. In conclusion, the GM rice diet is as safe as the traditional daily diet. Furthermore, urine metabonomics and fecal bacterial profiles provide a non-invasive food safety assessment rat model for genetically modified crops that are used for non-food/feed purposes. Fecal bacterial profiles have the potential for predicting the change of blood indexes in future. PMID:25478734

  3. Determination of human serum alpha1-acid glycoprotein and albumin binding of various marketed and preclinical kinase inhibitors.

    PubMed

    Zsila, Ferenc; Fitos, Ilona; Bencze, Gyula; Kéri, György; Orfi, László

    2009-01-01

    There are about 380 protein kinase inhibitors in drug development as of today and 15 drugs have been marketed already for the treatment of cancer. This time 139 validated kinase targets are in the focus of drug research of pharmaceutical companies and big efforts are made for the development of new, druglike kinase inhibitors. Plasma protein binding is an important factor of the ADME profiling of a drug compound. Human serum albumin (HSA) and alpha(1)-acid glycoprotein (AAG) are the most relevant drug carriers in blood plasma. Since previous literature data indicated that AAG is the principal plasma binding component of some kinase inhibitors the present work focuses on the comprehensive evaluation of AAG binding of a series of marketed and experimental kinase inhibitors by using circular dichroism (CD) spectroscopy approach. HSA binding was also evaluated by affinity chromatography. Protein binding interactions of twenty-six kinase inhibitors are characterized. The contribution of AAG and HSA binding data to the pharmacokinetic profiles of the investigated therapeutic agents is discussed. Structural, biological and drug binding properties of AAG as well as the applicability of the CD method in studying drug-protein binding interactions are also briefly reviewed.

  4. Structural alterations of human serum albumin caused by glycative and oxidative stressors revealed by circular dichroism analysis.

    PubMed

    Monacelli, Fiammetta; Storace, Daniela; D'Arrigo, Cristina; Sanguineti, Roberta; Borghi, Roberta; Pacini, Davide; Furfaro, Anna L; Pronzato, Maria A; Odetti, Patrizio; Traverso, Nicola

    2013-01-01

    The aim of this work was to evaluate the ability of oxidative and glycative stressors to modify properties of human serum albumin (HSA) by analyzing markers of glycation (pentosidine) and oxidation (advanced oxidative protein products (AOPPs)) and assessing fluorescence and circular dichroism. HSA was incubated for up to 21 days with ribose, ascorbic acid (AA) and diethylenetriamine pentacetate (DTPA) in various combinations in order to evaluate influences of these substances on the structure of HSA. Ribose was included as a strong glycative molecule, AA as a modulator of oxidative stress, and DTPA as an inhibitor of metal-catalyzed oxidation. Ribose induced a significant increase in pentosidine levels. AA and DTPA prevented the accumulation of pentosidine, especially at later time points. Ribose induced a mild increase in AOPP formation, while AA was a strong inducer of AOPP formation. Ribose, in combination with AA, further increased the formation of AOPP. DTPA prevented the AA-induced generation of AOPP. Ribose was also a potent inducer of fluorescence at 335nm ex/385nm em, which is typical of pentosidine. AA and DTPA prevented this fluorescence. Circular dichroism showed complex results, in which AA and DTPA were strong modifiers of the percentages of the alpha-helical structure of HSA, while ribose affected the structure of HSA only at later time points.

  5. Structural Alterations of Human Serum Albumin Caused by Glycative and Oxidative Stressors Revealed by Circular Dichroism Analysis

    PubMed Central

    Monacelli, Fiammetta; Storace, Daniela; D’Arrigo, Cristina; Sanguineti, Roberta; Borghi, Roberta; Pacini, Davide; Furfaro, Anna L.; Pronzato, Maria A.; Odetti, Patrizio; Traverso, Nicola

    2013-01-01

    The aim of this work was to evaluate the ability of oxidative and glycative stressors to modify properties of human serum albumin (HSA) by analyzing markers of glycation (pentosidine) and oxidation (advanced oxidative protein products (AOPPs)) and assessing fluorescence and circular dichroism. HSA was incubated for up to 21 days with ribose, ascorbic acid (AA) and diethylenetriamine pentacetate (DTPA) in various combinations in order to evaluate influences of these substances on the structure of HSA. Ribose was included as a strong glycative molecule, AA as a modulator of oxidative stress, and DTPA as an inhibitor of metal-catalyzed oxidation. Ribose induced a significant increase in pentosidine levels. AA and DTPA prevented the accumulation of pentosidine, especially at later time points. Ribose induced a mild increase in AOPP formation, while AA was a strong inducer of AOPP formation. Ribose, in combination with AA, further increased the formation of AOPP. DTPA prevented the AA-induced generation of AOPP. Ribose was also a potent inducer of fluorescence at 335nm ex/385nm em, which is typical of pentosidine. AA and DTPA prevented this fluorescence. Circular dichroism showed complex results, in which AA and DTPA were strong modifiers of the percentages of the alpha-helical structure of HSA, while ribose affected the structure of HSA only at later time points. PMID:23702842

  6. Gold Nanoparticle-Based Facile Detection of Human Serum Albumin and Its Application as an INHIBIT Logic Gate.

    PubMed

    Huang, Zhenzhen; Wang, Haonan; Yang, Wensheng

    2015-05-01

    In this work, a facile colorimetric method is developed for quantitative detection of human serum albumin (HSA) based on the antiaggregation effect of gold nanoparticles (Au NPs) in the presence of HSA. The citrate-capped Au NPs undergo a color change from red to blue when melamine is added as a cross-linker to induce the aggregation of the NPs. Such an aggregation is efficiently suppressed upon the adsorption of HSA on the particle surface. This method provides the advantages of simplicity and cost-efficiency for quantitative detection of HSA with a detection limit of ∼1.4 nM by monitoring the colorimetric changes of the Au NPs with UV-vis spectroscopy. In addition, this approach shows good selectivity for HSA over various amino acids, peptides, and proteins and is qualified for detection of HSA in a biological sample. Such an antiaggregation effect can be further extended to fabricate an INHIBIT logic gate by using HSA and melamine as inputs and the color changes of Au NPs as outputs, which may have application potentials in point-of-care medical diagnosis. PMID:25850684

  7. Synthesis, characterization and the interaction of some new water-soluble metal Schiff base complexes with human serum albumin

    NASA Astrophysics Data System (ADS)

    Asadi, Mozaffar; Asadi, Zahra; Sadi, Somaye Barzegar; Zarei, Leila; Baigi, Fatemeh Moosavi; Amirghofran, Zahra

    2014-03-01

    Some new water-soluble Schiff base complexes of Na2[M(L)(H2O)n]; (M = Zn, Cu, Ni, Mn) with a new water-soluble Schiff base ligand where L denotes an asymmetric N2O2 Schiff base ligands; N,N";-bis(5-sulfosalicyliden)-3,4-diaminobenzophenone (5-SO3-3,4-salbenz) were synthesized and characterized. The formation constants of the water soluble Schiff base complexes were calculated by Ketelaar's equation. The theoretical molecular structure for the complexes was computed by using the HF method and the 6-311G basis set. The mechanism of binding of Na2[M(L)(H2O)n] with human serum albumin (HSA) was studied by fluorescence spectroscopic technique. The results of fluorescence titration showed that the intrinsic fluorescence of HSA was quenched by the complexes; which was rationalized in terms of the dynamic quenching mechanism. The values of Stern-Volmer constants, quenching rate constants, binding constants, binding sites and average aggregation number of HSA have been determined. The thermodynamic parameters, were calculated by van't Hoff equation, indicate that the binding is entropy driven and enthalpically disfavored. Based on the Förster theory of non-radiation energy transfer, the efficiency of energy transfer and the distance between the donor (Trp residues) and the acceptor (complex) were obtained. Finally, the growth inhibitory effects of the complexes toward the K562 cancer cell line were measured.

  8. Evidence that Chemical Chaperone 4-Phenylbutyric Acid Binds to Human Serum Albumin at Fatty Acid Binding Sites

    PubMed Central

    James, Joel; Shihabudeen, Mohamed Sham; Kulshrestha, Shweta; Goel, Varun; Thirumurugan, Kavitha

    2015-01-01

    Endoplasmic reticulum stress elicits unfolded protein response to counteract the accumulating unfolded protein load inside a cell. The chemical chaperone, 4-Phenylbutyric acid (4-PBA) is a FDA approved drug that alleviates endoplasmic reticulum stress by assisting protein folding. It is found efficacious to augment pathological conditions like type 2 diabetes, obesity and neurodegeneration. This study explores the binding nature of 4-PBA with human serum albumin (HSA) through spectroscopic and molecular dynamics approaches, and the results show that 4-PBA has high binding specificity to Sudlow Site II (Fatty acid binding site 3, subdomain IIIA). Ligand displacement studies, RMSD stabilization profiles and MM-PBSA binding free energy calculation confirm the same. The binding constant as calculated from fluorescence spectroscopic studies was found to be kPBA = 2.69 x 105 M-1. Like long chain fatty acids, 4-PBA induces conformational changes on HSA as shown by circular dichroism, and it elicits stable binding at Sudlow Site II (fatty acid binding site 3) by forming strong hydrogen bonding and a salt bridge between domain II and III of HSA. This minimizes the fluctuation of HSA backbone as shown by limited conformational space occupancy in the principal component analysis. The overall hydrophobicity of W214 pocket (located at subdomain IIA), increases upon occupancy of 4-PBA at any FA site. Descriptors of this pocket formed by residues from other subdomains largely play a role in compensating the dynamic movement of W214. PMID:26181488

  9. Study of the effect of Cal-Red on the secondary structure of human serum albumin by spectroscopic techniques

    NASA Astrophysics Data System (ADS)

    Dong, Lijun; Chen, Xingguo; Hu, Zhide

    2007-11-01

    The effect of Cal-Red on the structure of human serum albumin (HSA) was studied using Resonance light scattering (RLS), Fourier transformed Infrared (FT-IR) and Circular dichroism (CD) spectroscopic methods. The RLS spectroscopic results show that the RLS intensity of HSA was significantly increased in the presence of Cal-Red. The binding parameters of HSA with Cal-Red were studied at different temperatures of 289, 299, 309 and 319 K at pH 4.1. It is indicated by the Scatchard plots that the binding constant K decreased from 4.03 × 10 8 to 7.59 × 10 7 l/mol and the maximum binding number N decreased from 215 to 152 with increasing the temperature, respectively. The binding process was exothermic and spontaneous, as indicated by the thermodynamic analyses, and the major part of the binding energy is hydrophobic interaction. The enthalpy change Δ H0, the free energy change Δ G0 and the entropy change Δ S0 of 289 K were calculated to be -42.75 kJ/mol, -47.56 kJ/mol and 16.66 J/mol K, respectively. The alterations of protein secondary structure in the presence of Cal-Red in aqueous solution were quantitatively calculated from FT-IR and CD spectroscopy with reductions of α-helices content about 5%, β-turn from 10% to 2% and with increases of β-sheet from 38% to 51%.

  10. Studies on the interaction of total saponins of panax notoginseng and human serum albumin by Fourier transform infrared spectroscopy

    NASA Astrophysics Data System (ADS)

    Liu, Yuan; Xie, Meng-Xia; Kang, Juan; Zheng, Dong

    2003-10-01

    Total saponins of panax notoginseng (TPNS), isolated from the roots of panax notoginseng (Burk) F.H. Chen, have been considered as the main active components of San-Chi and have various therapeutical actions. Their interactions with human serum albumin have been investigated by Fourier transformed infrared spectrometry and fluorescence methods. The results showed that TPNS combined with HSA through C=O and CN groups of polypeptide chain. The drug-protein combination caused the significant loss of α-helix structure and the microenvironment changes of the tyrosine residues in protein at higher drug concentration. Combining the curve-fitting results of amide I and amide III bands, the alterations of protein secondary structure after drug complexation were quantitatively determined. The α-helix structure has a decrease of ≈6%, from 55 to 49% and the β-sheet increased ≈3%, from 23 to 26% at high drug concentration. However, no major alterations were observed for the β-turn and random coil structures up on drug-protein binding.

  11. Effect of functionalized magnetic MnFe2O4 nanoparticles on fibrillation of human serum albumin.

    PubMed

    Sen, Shubhatam; Konar, Suraj; Pathak, Amita; Dasgupta, Swagata; DasGupta, Sunando

    2014-10-01

    Pathogenesis of amyloid-related diseases is related to nonnative folding of proteins with the formation of insoluble deposits in the extracellular space of various tissues. Having the unique properties of small size, large surface area, biodegradability, and relative nontoxicity, magnetic nanoparticles have drawn a lot of attention in biomedical applications. Herein, we demonstrate the effect of bare and differently functionalized magnetic MnFe2O4 nanoparticles on fibrillation of human serum albumin in vitro. The process has been monitored using Thioflavin T fluorescence, Congo red binding assay, circular dichroism, fluorescence microscopy, and transmission electron microscopy. From our experimental results, amine functionalized MnFe2O4 nanoparticles are found to inhibit formation of fibrils more effectively than bare ones, while carboxylated nanoparticles do not have a significant effect on fibrillation. This study has explored the prospects of using specific magnetic nanoparticles with appropriate modification to control self-assembly of proteins and may act as a precursor in therapeutic applications. PMID:25247718

  12. Rapid Screening of Drug-Protein Binding Using High-Performance Affinity Chromatography with Columns Containing Immobilized Human Serum Albumin

    PubMed Central

    Li, Ying-Fei; Zhang, Xiao-Qiong; Hu, Wei-Yu; Li, Zheng; Liu, Ping-Xia; Zhang, Zhen-Qing

    2013-01-01

    For drug candidates, a plasma protein binding (PPB) more than 90% is more meaningful and deserves further investigation in development. In the study, a high-performance liquid chromatography method employing column containing immobilized human serum albumin (HSA) to screen in vitro PPB of leading compounds was established and successfully applied to tested compounds. Good correlation (a coefficient correlation of 0.96) was attained between the reciprocal values (X) of experimentally obtained retention time of reference compounds eluted through HSA column and the reported PPB values (Y) with a correlation equation of Y = 92.03 − 97.01X. The method was successfully applied to six test compounds, and the result was confirmed by the conventional ultrafiltration technique, and both yielded equal results. However, due to the particular protein immobilized to column, the method cannot be applied for all compounds and should be exploited judiciously based on the value of the logarithmic measure of the acid dissociation constant (pKa) as per the requirement. If α1-acid glycoprotein and other plasma proteins could be immobilized like HSA with their actual ratio in plasma to column simultaneously, the result attained using immobilized column may be more accurate, and the method could be applied to more compounds without pKa limitation. PMID:23607050

  13. The Effect of Human Serum Albumin and Hematocrit on the Cake Collapse Temperature of Lyophilized Red Blood Cells.

    PubMed

    Runyon, Daniel E; Higgins, Adam Z

    2015-10-01

    Freeze-drying, or lyophilization, has shown great promise in addressing many of the logistical challenges of storing and preserving red blood cells (RBCs). A crucial part of any RBC lyophilization protocol is the primary drying temperature, which affects the sample drying rate and the dried cake's ability to form a stable glassy solid. Primary drying is most efficient just below the temperature at which the porous structure of the cake begins to collapse, known as the cake collapse temperature. In this short report, we utilize freeze-drying microscopy to examine the effects of human serum albumin (HSA) and hematocrit on the cake collapse temperature. Increasing the hematocrit from 0% to 20% significantly raised the cake collapse temperature from - 37.8°C to -34.8°C. Addition of 5% HSA to a 20% hematocrit RBC suspension further increased the cake collapse temperature to -20.4°C. These data provide a basis for future study of the relationship between cake collapse and overall cell survival, with the object of building a clinically-viable RBC lyophilization protocol.

  14. Studies on the synthesis, characterization, human serum albumin binding and biological activity of single chain surfactant-cobalt(III) complexes.

    PubMed

    Vignesh, G; Sugumar, K; Arunachalam, S; Vignesh, S; Arthur James, R; Arun, R; Premkumar, K

    2016-03-01

    The interaction of surfactant-cobalt(III) complexes [Co(bpy)(dien)TA](ClO4)3 · 3H2O (1) and [Co(dien)(phen)TA](ClO4)3 · 4H2O (2), where bpy = 2,2'-bipyridine, dien = diethylenetriamine, phen = 1,10-phenanthroline and TA = tetradecylamine with human serum albumin (HSA) under physiological conditions was analyzed using steady state, synchronous, 3D fluorescence, UV/visabsorption and circular dichroism spectroscopic techniques. The results show that these complexes cause the fluorescence quenching of HSA through a static mechanism. The binding constant (Kb ) and number of binding-sites (n) were obtained at different temperatures. The corresponding thermodynamic parameters (∆G°, ∆H° and ∆S°) and Ea were also obtained. According to Förster's non-radiation energy transfer theory, the binding distance (r) between the complexes and HSA were calculated. The results of synchronous and 3D fluorescence spectroscopy indicate that the binding process has changed considerably the polarity around the fluorophores, along with changes in the conformation of the protein. The antimicrobial and anticancer activities of the complexes were tested and the results show that the complexes have good activities against pathogenic microorganisms and cancer cells. PMID:26250655

  15. Dynamics of acrylodan-labeled bovine and human serum albumin sequestered within aerosol-OT reverse micelles.

    PubMed

    Lundgren, J S; Heitz, M P; Bright, F V

    1995-10-15

    We investigate the effects of hydration on acrylodan-labeled bovine and human serum albumin (BSA-Ac and HSA-Ac) in aerosol-OT (AOT) reverse micelles solubilized in n-heptane. Time-resolved fluorescence intensity decay experiments reveal a dipolar relaxation process surrounding the acrylodan cybotactic region. This process is best described by a two-term rate law wherein the average relaxation increases with increased hydration. However, the actual rate constants describing the relaxation process either remain unchanged or actually decrease with increased hydration. The results illustrate that the fractional contribution associated with the individual relaxation pathways causes the observed changes in relaxation dynamics. The recovered rotational reorientation dynamics of the acrylodan residue are also affected by the extent of protein hydration. As hydration is increased, the semiangle through which the acrylodan residue precesses increases by 10 degrees for both protein systems. Interestingly, the recovered semiangles for the native proteins equal those recovered at lower hydration when the proteins are sequestered within the AOT reverse micelle. These results demonstrate the importance of hydration on protein behavior in environments where water is limited (e.g., biosensor interfaces and sol--gel-derived biocomposites).

  16. Based on SERS conformational studies of ginsenoside Rb1 and its metabolites before and after combined with human serum albumin

    NASA Astrophysics Data System (ADS)

    Zhang, Wei; Wang, Yingping; Bai, Xueyuan; Zhao, Bing

    2015-02-01

    Surface-enhanced Raman scattering (SERS) and fluorescence spectroscopy were employed to probe the interaction of the pharmaceutical and natural product molecules, ginsenoside Rb1, Rd, Rg3 and compound K (CK), with human serum albumin (HSA). Normal Raman spectra of these four ginsenosides were obtained from solid powder on glass slide. Based on the unsplit peak at 1445 cm-1, the stacking modes of ginsenoside Rb1, Rd, Rg3 and CK were quite similar, when the deconvolution of alkyl chain was not occurred. SERS spectra of ginsenoside Rb1, Rd, Rg3 and CK were obtained from a colloidal silver surface on a self-assembled SERS substrate, the most enhanced modes were those with certain motions perpendicular to the metal surface, such as C24dbnd C25 stretch and Csbnd H out-of-plane bending from alkyl chain. The SERS spectra were used to predict similar perpendicular orientation of flexible alkyl chain and parallel orientation of carbocyclic rings on Ag colloid particles. Therefore, when combined with HSA, the transformations of four ginsenosides still exhibit similar, although in different binding cavities in subdomain IIA and IIIA by making the methyls at C26 and C27 perpendicular plugging into the hydrophobic site of HSA, while the aglycone and glucose nearby are perpendicularly exposed outside to fit other suitable active targeting sites.

  17. Silicon-substituted hydroxyapatite composite coating by using vacuum-plasma spraying and its interaction with human serum albumin.

    PubMed

    Xiao, Feng-Juan; Peng, Lei; Zhang, Ying; Yun, Li-Jiang

    2009-08-01

    The incorporation of silicon can improve the bioactivity of hydroxyapatite (HA). Silicon-substituted HA (Ca(10)(PO(4))(6-x )(SiO(4))( x )(OH)(2-x ), Si-HA) composite coatings on a bioactive titanium substrate were prepared by using a vacuum-plasma spraying method. The surface structure was characterized by using XRD, SEM, XRF, EDS and FTIR. The bond strength of the coating was investigated and XRD patterns showed that Ti/Si-HA coatings were similar to patterns seen for HA. The only different XRD pattern was a slight trend toward a smaller angle direction with an increase in the molar ratio of silicon. FTIR spectra showed that the most notable effect of silicon substitution was that -OH group decreased as the silicon content increased. XRD and EDS elemental analysis indicated that the content of silicon in the coating was consistent with the silicon-substituted hydroxyapatite used in spraying. A bioactive TiO(2) coating was formed on an etched surface of Ti, and the etching might improve the bond strength of the coatings. The interaction of the Ti/Si-HA coating with human serum albumin (HSA) was much greater than that of the Ti/HA coating. This might suggest that the incorporation of silicon in HA can lead to significant improvements in the bioactive performance of HA.

  18. Freeze-drying of HI-6-loaded recombinant human serum albumin nanoparticles for improved storage stability.

    PubMed

    Dadparvar, Miriam; Wagner, Sylvia; Wien, Sascha; Worek, Franz; von Briesen, Hagen; Kreuter, Jörg

    2014-10-01

    Severe intoxications with organophosphates require the immediate administration of atropine in combination with acetyl cholinesterase (AChE) reactivators such as HI-6. Although this therapy regimen enables the treatment of peripheral symptoms, the blood-brain barrier (BBB) restricts the access of the hydrophilic antidotes to the central nervous system which could lead to a fatal respiratory arrest. Therefore, HI-6-loaded albumin nanoparticles were previously developed to enhance the transport across this barrier and were able to reactivate organophosphate-(OP)-inhibited AChE in an in vitro BBB model. Since HI-6 is known to be moisture-sensitive, the feasibility of freeze-drying of the HI-6-loaded nanoparticles was investigated in the present study using different cryo- and lyoprotectants at different concentrations. Trehalose and sucrose (3%, w/v)-containing formulations were superior to mannitol concerning the physicochemical parameters of the nanoparticles whereas trehalose-containing samples were subject of a prolonged storage stability study at temperatures between -20°C and +40°C for predetermined time intervals. Shelf-life computations of the freeze-dried HI-6 nanoparticle formulations revealed a shelf-life time of 18 months when stored at -20°C. The formulations' efficacy was proven in vitro by reactivation of OP-inhibited AChE after transport over a porcine brain capillary endothelial cell layer model.

  19. Effects of the Infusion of 4% or 20% Human Serum Albumin on the Skeletal Muscle Microcirculation in Endotoxemic Rats

    PubMed Central

    Damiani, Elisa; Ince, Can; Orlando, Fiorenza; Pierpaoli, Elisa; Cirioni, Oscar; Giacometti, Andrea; Mocchegiani, Federico; Pelaia, Paolo; Provinciali, Mauro; Donati, Abele

    2016-01-01

    Background Sepsis-induced microcirculatory alterations contribute to tissue hypoxia and organ dysfunction. In addition to its plasma volume expanding activity, human serum albumin (HSA) has anti-oxidant and anti-inflammatory properties and may have a protective role in the microcirculation during sepsis. The concentration of HSA infused may influence these effects. We compared the microcirculatory effects of the infusion of 4% and 20% HSA in an experimental model of sepsis. Methods Adult male Wistar rats were equipped with arterial and venous catheters and received an intravenous infusion of lipopolysaccharide (LPS, serotype O127:B8, 10 mg/kg over 30 minutes) or vehicle (SHAM, n = 6). Two hours later, endotoxemic animals were randomized to receive 10 mL/kg of either 4% HSA (LPS+4%HSA, n = 6), 20% HSA (LPS+20%HSA, n = 6) or 0.9% NaCl (LPS+0.9%NaCl, n = 6). No fluids were given to an additional 6 animals (LPS). Vessel density and perfusion were assessed in the skeletal muscle microcirculation with sidestream dark field videomicroscopy at baseline (t0), 2 hours after LPS injection (t1), after HSA infusion (t2) and 1 hour later (t3). The mean arterial pressure (MAP) and heart rate were recorded. Serum endothelin-1 was measured at t2. Results MAP was stable over time in all groups. The microcirculatory parameters were significantly altered in endotoxemic animals at t1. The infusion of both 4% and 20% HSA similarly increased the perfused vessel density and blood flow velocity and decreased the flow heterogeneity to control values. Microvascular perfusion was preserved in the LPS+20%HSA group at t3, whereas alterations reappeared in the LPS+4%HSA group. Conclusions In a rat model of normotensive endotoxemia, the infusion of 4% or 20% HSA produced a similar acute improvement in the microvascular perfusion in otherwise unresuscitated animals. PMID:26942605

  20. Analysis of glipizide binding to normal and glycated human serum albumin by high-performance affinity chromatography.

    PubMed

    Matsuda, Ryan; Li, Zhao; Zheng, Xiwei; Hage, David S

    2015-07-01

    In diabetes, the elevated levels of glucose in the bloodstream can result in the nonenzymatic glycation of proteins such as human serum albumin (HSA). This type of modification has been shown to affect the interactions of some drugs with HSA, including several sulfonylurea drugs that are used to treat type II diabetes. This study used high-performance affinity chromatography (HPAC) to examine the interactions of glipizide (i.e., a second-generation sulfonylurea drug) with normal HSA or HSA that contained various levels of in vitro glycation. Frontal analysis indicated that glipizide was interacting with both normal and glycated HSA through two general groups of sites: a set of relatively strong interactions and a set of weaker interactions with average association equilibrium constants at pH 7.4 and 37 °C in the range of 2.4-6.0 × 10(5) and 1.7-3.7 × 10(4) M(-1), respectively. Zonal elution competition studies revealed that glipizide was interacting at both Sudlow sites I and II, which were estimated to have affinities of 3.2-3.9 × 10(5) and 1.1-1.4 × 10(4) M(-1). Allosteric effects were also noted to occur for this drug between the tamoxifen site and the binding of R-warfarin at Sudlow site I. Up to an 18% decrease in the affinity for glipizide was observed at Sudlow site I ongoing from normal HSA to glycated HSA, while up to a 27% increase was noted at Sudlow site II. This information should be useful in indicating how HPAC can be used to investigate other drugs that have complex interactions with proteins. These results should also be valuable in providing a better understanding of how glycation may affect drug-protein interactions and the serum transport of drugs such as glipizide during diabetes. PMID:25912461

  1. Interaction of a tyrosine kinase inhibitor, vandetanib with human serum albumin as studied by fluorescence quenching and molecular docking.

    PubMed

    Kabir, Md Zahirul; Feroz, Shevin R; Mukarram, Abdul Kadir; Alias, Zazali; Mohamad, Saharuddin B; Tayyab, Saad

    2016-08-01

    Interaction of a tyrosine kinase inhibitor, vandetanib (VDB), with the major transport protein in the human blood circulation, human serum albumin (HSA), was investigated using fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular docking analysis. The binding constant of the VDB-HSA system, as determined by fluorescence quenching titration method was found in the range, 8.92-6.89 × 10(3 )M(-1) at three different temperatures, suggesting moderate binding affinity. Furthermore, decrease in the binding constant with increasing temperature revealed involvement of static quenching mechanism, thus affirming the formation of the VDB-HSA complex. Thermodynamic analysis of the binding reaction between VDB and HSA yielded positive ΔS (52.76 J mol(-1) K(-1)) and negative ΔH (-6.57 kJ mol(-1)) values, which suggested involvement of hydrophobic interactions and hydrogen bonding in stabilizing the VDB-HSA complex. Far-UV and near-UV CD spectral results suggested alterations in both secondary and tertiary structures of HSA upon VDB-binding. Three-dimensional fluorescence spectral results also showed significant microenvironmental changes around the Trp residue of HSA consequent to the complex formation. Use of site-specific marker ligands, such as phenylbutazone (site I marker) and diazepam (site II marker) in competitive ligand displacement experiments indicated location of the VDB binding site on HSA as Sudlow's site I (subdomain IIA), which was further established by molecular docking results. Presence of some common metal ions, such as Ca(2+), Zn(2+), Cu(2+), Ba(2+), Mg(2+), and Mn(2+) in the reaction mixture produced smaller but significant alterations in the binding affinity of VDB to HSA.

  2. The interaction of human serum albumin with selected lanthanide and actinide ions: Binding affinities, protein unfolding and conformational changes.

    PubMed

    Ali, Manjoor; Kumar, Amit; Kumar, Mukesh; Pandey, Badri N

    2016-04-01

    Human serum albumin (HSA), the most abundant soluble protein in blood plays critical roles in transportation of biomolecules and maintenance of osmotic pressure. In view of increasing applications of lanthanides- and actinides-based materials in nuclear energy, space, industries and medical applications, the risk of exposure with these metal ions is a growing concern for human health. In present study, binding interaction of actinides/lanthanides [thorium: Th(IV), uranium: U(VI), lanthanum: La(III), cerium: Ce(III) and (IV)] with HSA and its structural consequences have been investigated. Ultraviolet-visible, Fourier transform-infrared, Raman, Fluorescence and Circular dichroism spectroscopic techniques were applied to study the site of metal ions interaction, binding affinity determination and the effect of metal ions on protein unfolding and HSA conformation. Results showed that these metal ions interacted with carbonyl (CO..:)/amide(N..-H) groups and induced exposure of aromatic residues of HSA. The fluorescence analysis indicated that the actinide binding altered the microenvironment around Trp214 in the subdomain IIA. Binding affinity of U(VI) to HSA was slightly higher than that of Th(IV). Actinides and Ce(IV) altered the secondary conformation of HSA with a significant decrease of α-helix and an increase of β-sheet, turn and random coil structures, indicating a partial unfolding of HSA. A correlation was observed between metal ion's ability to alter HSA conformation and protein unfolding. Both cationic effects and coordination ability of metal ions seemed to determine the consequences of their interaction with HSA. Present study improves our understanding about the protein interaction of these heavy ions and their impact on its secondary structure. In addition, binding characteristics may have important implications for the development of rational antidote for the medical management of health effects of actinides and lanthanides.

  3. The interaction of human serum albumin with selected lanthanide and actinide ions: Binding affinities, protein unfolding and conformational changes.

    PubMed

    Ali, Manjoor; Kumar, Amit; Kumar, Mukesh; Pandey, Badri N

    2016-04-01

    Human serum albumin (HSA), the most abundant soluble protein in blood plays critical roles in transportation of biomolecules and maintenance of osmotic pressure. In view of increasing applications of lanthanides- and actinides-based materials in nuclear energy, space, industries and medical applications, the risk of exposure with these metal ions is a growing concern for human health. In present study, binding interaction of actinides/lanthanides [thorium: Th(IV), uranium: U(VI), lanthanum: La(III), cerium: Ce(III) and (IV)] with HSA and its structural consequences have been investigated. Ultraviolet-visible, Fourier transform-infrared, Raman, Fluorescence and Circular dichroism spectroscopic techniques were applied to study the site of metal ions interaction, binding affinity determination and the effect of metal ions on protein unfolding and HSA conformation. Results showed that these metal ions interacted with carbonyl (CO..:)/amide(N..-H) groups and induced exposure of aromatic residues of HSA. The fluorescence analysis indicated that the actinide binding altered the microenvironment around Trp214 in the subdomain IIA. Binding affinity of U(VI) to HSA was slightly higher than that of Th(IV). Actinides and Ce(IV) altered the secondary conformation of HSA with a significant decrease of α-helix and an increase of β-sheet, turn and random coil structures, indicating a partial unfolding of HSA. A correlation was observed between metal ion's ability to alter HSA conformation and protein unfolding. Both cationic effects and coordination ability of metal ions seemed to determine the consequences of their interaction with HSA. Present study improves our understanding about the protein interaction of these heavy ions and their impact on its secondary structure. In addition, binding characteristics may have important implications for the development of rational antidote for the medical management of health effects of actinides and lanthanides. PMID:26821345

  4. The potential use of lapatinib-loaded human serum albumin nanoparticles in the treatment of triple-negative breast cancer.

    PubMed

    Wan, Xu; Zheng, Xiaoyao; Pang, Xiaoying; Zhang, Zheming; Jing, Tao; Xu, Wei; Zhang, Qizhi

    2015-04-30

    Triple-negative breast cancer (TNBC) is an aggressive cancer with limited treatment options. However, the shared feature of epidermal growth factor receptor (EGFR) expression in TNBC offers the opportunity for targeted molecular therapy for this breast cancer subtype. Previous studies have indicated that lapatinib, a selective small-molecular dual-tyrosine kinase inhibitor of HER2 and EGFR, is effective in reducing cancer progression and metastasis, indicating that it might be a candidate for TNBC treatment. However, its poor water solubility, low and variable oral absorption, and large daily dose all limit the clinical use of lapatinib. In this study, we developed human serum albumin (HSA) nanoparticles loaded with lapatinib for intravenous administration to overcome these disadvantages and enhance its efficacy against TNBC. 4T1 cells (a murine TNBC cells) were selected as the cell model because their growth and metastatic spread are very close to those of human breast cancer cells. Lapatinib-loaded HSA nanoparticles (LHNPs) were prepared by Nab technology. LHNPs displayed cytotoxicity similar to the free drug but exhibited superior capacity to induce early apoptosis in 4T1 monolayer cells. Importantly, LHNPs showed improved penetration and inhibition effects in tumor spheroids compared to lapatinib solution (LS). Pharmacokinetic investigations revealed that HSA nanoparticles (i.v.) effectively increased the accumulation of lapatinib in tumor tissue at 2.38 and 16.6 times the level of LS (i.v.) and Tykerb (p.o.), respectively. Consequently, it had markedly better suppression effects both on primary breast cancer and lung metastasis in tumor-bearing mice compared to the commercial drug Tykerb. The improved anti-tumor efficacy of LHNPs may be partly attributed to its close binding to SPARC, which is widely present in the extracellular matrix of tumor tissue. These results demonstrated that LHNPs might be a promising anti-tumor agent for TNBC.

  5. The molecular mechanism of the neutral-to-base transition of human serum albumin. Acid/base titration and proton nuclear magnetic resonance studies on a large peptic and a large tryptic fragment of albumin.

    PubMed

    Bos, O J; Labro, J F; Fischer, M J; Wilting, J; Janssen, L H

    1989-01-15

    In order to obtain a better understanding of the neutral-to-base (N-B) transition of human serum albumin, we performed acid/base titration experiments and 500-MHz 1H NMR experiments on albumin and on a large peptic (residues 1-387) and large tryptic (residues 198-585) fragment of albumin. The acid/base titration experiments revealed that Ca2+ ions induce a downward pK shift of several histidine residues of the peptic (P46) fragment and of albumin. By contrast, Ca2+ has very little influence on the pK of histidine residues of the tryptic (T45) fragment. In albumin, the pH-dependent His C-2 proton resonances, observed with 1H NMR experiments, have been allotted the numbers 1-17. It proved possible to locate these resonances in the P46 and the T45 fragments. A correspondence was found between the number of histidines detected by the acid/base titration and by the 1H NMR experiments. The results of the experiments lead us to conclude that in domain 1 at least the histidines corresponding to the His C-2 proton resonances 1-5 play a dominant role in the N-B transition. The Cu2+-binding histidine residue 3 (resonance 8) of the albumin molecule is not involved in the N-B transition. In addition, we were able to assign His C-2 proton resonance 9 to histidine 464 of the albumin molecule. The role of the N-B transition in the transport and cellular uptake mechanisms of endogenous and exogenous compounds is discussed.

  6. (PCG) Protein Crystal Growth Horse Serum Albumin

    NASA Technical Reports Server (NTRS)

    1995-01-01

    Horse Serum Albumin crystals grown during the USML-1 (STS-50) mission's Protein Crystal Growth Glovebox Experiment. These crystals were grown using a vapor diffusion technique at 22 degrees C. The crystals were allowed to grow for nine days while in orbit. Crystals of 1.0 mm in length were produced. The most abundant blood serum protein, regulates blood pressure and transports ions, metabolites, and therapeutic drugs. Principal Investigator was Edward Meehan.

  7. Combined image guided monitoring the pharmacokinetics of rapamycin loaded human serum albumin nanoparticles with a split luciferase reporter

    NASA Astrophysics Data System (ADS)

    Wang, Fu; Yang, Kai; Wang, Zhe; Ma, Ying; Gutkind, J. Silvio; Hida, Naoki; Niu, Gang; Tian, Jie

    2016-02-01

    Imaging guided techniques have been increasingly employed to investigate the pharmacokinetics (PK) and biodistribution of nanoparticle based drug delivery systems. In most cases, however, the PK profiles of drugs could vary significantly from those of drug delivery carriers upon administration in the blood circulation, which complicates the interpretation of image findings. Herein we applied a genetically encoded luciferase reporter in conjunction with near infrared (NIR) fluorophores to investigate the respective PK profiles of a drug and its carrier in a biodegradable drug delivery system. In this system, a prototype hydrophobic agent, rapamycin (Rapa), was encapsulated into human serum albumin (HSA) to form HSA Rapa nanoparticles, which were then labeled with Cy5 fluorophore to facilitate the fluorescence imaging of HSA carrier. Meanwhile, we employed transgenetic HN12 cells that were modified with a split luciferase reporter, whose bioluminescence function is regulated by Rapa, to reflect the PK profile of the encapsulated agent. It was interesting to discover that there existed an obvious inconsistency of PK behaviors between HSA carrier and rapamycin in vitro and in vivo through near infrared fluorescence imaging (NIFRI) and bioluminescence imaging (BLI) after treatment with Cy5 labeled HSA Rapa. Nevertheless, HSA Rapa nanoparticles manifested favorable in vivo PK and tumor suppression efficacy in a follow-up therapeutic study. The developed strategy of combining a molecular reporter and a fluorophore in this study could be extended to other drug delivery systems to provide profound insights for non-invasive real-time evaluation of PK profiles of drug-loaded nanoparticles in pre-clinical studies.Imaging guided techniques have been increasingly employed to investigate the pharmacokinetics (PK) and biodistribution of nanoparticle based drug delivery systems. In most cases, however, the PK profiles of drugs could vary significantly from those of drug delivery

  8. Synergistic Effect of Human Serum Albumin and Fullerene on Gd-DO3A for Tumor-Targeting Imaging.

    PubMed

    Zhang, Ying; Zou, Toujun; Guan, Mirong; Zhen, Mingming; Chen, Daiqin; Guan, Xiangping; Han, Hongbin; Wang, Chunru; Shu, Chunying

    2016-05-11

    A macromolecular magnetic resonance imaging (MRI) contrast agent was successfully synthesized by conjugating the gadolinium/1,4,7,10-tetraazacyclododecane-1,4,7-tetracetic acid complex (Gd-DO3A) with 6,6-phenyl-C61 butyric acid (PC61BA) and upon further modification with human serum albumin (HSA). The final product, PC61BA-(Gd-DO3A)/HSA, has a high stability and exhibits a much higher relaxivity (r1 = 89.1 mM(-1) s(-1) at 0.5 T, 300 K) than Gd-DO3A (r1 = 4.7 mM(-1) s(-1)) does under the same condition, producing the synergistic positive effect of HSA and C60 on the relaxivity of Gd-DO3A. The in vivo MR images of PC61BA-(Gd-DO3A)/HSA-treated tumor-bearing mice show strong signal enhancement for the tumor area due to the enhanced permeability and retention effect. The maximum accumulation of PC61BA-(Gd-DO3A)/HSA at the tumor site was achieved at 4 h postinjection, which may guide surgery. The results from the hematology and histological observations indicate that PC61BA-(Gd-DO3A)/HSA has no obvious toxicity in vivo. These unique properties of PC61BA-(Gd-DO3A)/HSA enable them to be highly efficient for tumor-targeting MRI in vivo, possibly providing a good solution for tumor diagnosis.

  9. Ligand Binding to the FA3-FA4 Cleft Inhibits the Esterase-Like Activity of Human Serum Albumin

    PubMed Central

    Ascenzi, Paolo; Leboffe, Loris; di Masi, Alessandra; Trezza, Viviana; Fanali, Gabriella; Gioia, Magda; Coletta, Massimo; Fasano, Mauro

    2015-01-01

    The hydrolysis of 4-nitrophenyl esters of hexanoate (NphOHe) and decanoate (NphODe) by human serum albumin (HSA) at Tyr411, located at the FA3-FA4 site, has been investigated between pH 5.8 and 9.5, at 22.0°C. Values of Ks, k+2, and k+2/Ks obtained at [HSA] ≥ 5×[NphOXx] and [NphOXx] ≥ 5×[HSA] (Xx is NphOHe or NphODe) match very well each other; moreover, the deacylation step turns out to be the rate limiting step in catalysis (i.e., k+3 << k+2). The pH dependence of the kinetic parameters for the hydrolysis of NphOHe and NphODe can be described by the acidic pKa-shift of a single amino acid residue, which varies from 8.9 in the free HSA to 7.6 and 7.0 in the HSA:NphOHe and HSA:NphODe complex, respectively; the pK>a-shift appears to be correlated to the length of the fatty acid tail of the substrate. The inhibition of the HSA-Tyr411-catalyzed hydrolysis of NphOHe, NphODe, and 4-nitrophenyl myristate (NphOMy) by five inhibitors (i.e., diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol) has been investigated at pH 7.5 and 22.0°C, resulting competitive. The affinity of diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol for HSA reflects the selectivity of the FA3-FA4 cleft. Under conditions where Tyr411 is not acylated, the molar fraction of diazepam, diflunisal, ibuprofen, and 3-indoxyl-sulfate bound to HSA is higher than 0.9 whereas the molar fraction of propofol bound to HSA is ca. 0.5. PMID:25790235

  10. Multiple-probe analysis of folding and unfolding pathways of human serum albumin. Evidence for a framework mechanism of folding.

    PubMed

    Santra, Manas Kumar; Banerjee, Abhijit; Krishnakumar, Shyam Sundar; Rahaman, Obaidur; Panda, Dulal

    2004-05-01

    The changes in the far-UV CD signal, intrinsic tryptophan fluorescence and bilirubin absorbance showed that the guanidine hydrochloride (GdnHCl)-induced unfolding of a multidomain protein, human serum albumin (HSA), followed a two-state process. However, using environment sensitive Nile red fluorescence, the unfolding and folding pathways of HSA were found to follow a three-state process and an intermediate was detected in the range 0.25-1.5 m GdnHCl. The intermediate state displayed 45% higher fluorescence intensity than that of the native state. The increase in the Nile red fluorescence was found to be due to an increase in the quantum yield of the HSA-bound Nile red. Low concentrations of GdnHCl neither altered the binding affinity of Nile red to HSA nor induced the aggregation of HSA. In addition, the secondary structure of HSA was not perturbed during the first unfolding transition (<1.5 m GdnHCl); however, the secondary structure was completely lost during the second transition. The data together showed that the half maximal loss of the tertiary structure occurred at a lower GdnHCl concentration than the loss of the secondary structure. Further kinetic studies of the refolding process of HSA using multiple spectroscopic techniques showed that the folding occurred in two phases, a burst phase followed by a slow phase. An intermediate with native-like secondary structure but only a partial tertiary structure was found to form in the burst phase of refolding. Then, the intermediate slowly folded into the native state. An analysis of the refolding data suggested that the folding of HSA could be best explained by the framework model.

  11. Stereo-Selectivity of Human Serum Albumin to Enantiomeric and Isoelectronic Pollutants Dissected by Spectroscopy, Calorimetry and Bioinformatics

    PubMed Central

    Ahmad, Ejaz; Rabbani, Gulam; Zaidi, Nida; Singh, Saurabh; Rehan, Mohd; Khan, Mohd Moin; Rahman, Shah Kamranur; Quadri, Zainuddin; Shadab, Mohd.; Ashraf, Mohd Tashfeen; Subbarao, Naidu; Bhat, Rajiv; Khan, Rizwan Hasan

    2011-01-01

    1–naphthol (1N), 2–naphthol (2N) and 8–quinolinol (8H) are general water pollutants. 1N and 2N are the configurational enantiomers and 8H is isoelectronic to 1N and 2N. These pollutants when ingested are transported in the blood by proteins like human serum albumin (HSA). Binding of these pollutants to HSA has been explored to elucidate the specific selectivity of molecular recognition by this multiligand binding protein. The association constants (Kb) of these pollutants to HSA were moderate (104–105 M−1). The proximity of the ligands to HSA is also revealed by their average binding distance, r, which is estimated to be in the range of 4.39–5.37 nm. The binding free energy (ΔG) in each case remains effectively the same for each site because of enthalpy–entropy compensation (EEC). The difference observed between ΔCpexp and ΔCpcalc are suggested to be caused by binding–induced flexibility changes in the HSA. Efforts are also made to elaborate the differences observed in binding isotherms obtained through multiple approaches of calorimetry, spectroscopy and bioinformatics. We suggest that difference in dissociation constants of pollutants by calorimetry, spectroscopic and computational approaches could correspond to occurrence of different set of populations of pollutants having different molecular characteristics in ground state and excited state. Furthermore, our observation of enhanced binding of pollutants (2N and 8H) in the presence of hemin signifies that ligands like hemin may enhance the storage period of these pollutants in blood that may even facilitate the ill effects of these pollutants. PMID:22073150

  12. Ligand binding to the FA3-FA4 cleft inhibits the esterase-like activity of human serum albumin.

    PubMed

    Ascenzi, Paolo; Leboffe, Loris; di Masi, Alessandra; Trezza, Viviana; Fanali, Gabriella; Gioia, Magda; Coletta, Massimo; Fasano, Mauro

    2015-01-01

    The hydrolysis of 4-nitrophenyl esters of hexanoate (NphOHe) and decanoate (NphODe) by human serum albumin (HSA) at Tyr411, located at the FA3-FA4 site, has been investigated between pH 5.8 and 9.5, at 22.0°C. Values of Ks, k+2, and k+2/Ks obtained at [HSA] ≥ 5×[NphOXx] and [NphOXx] ≥ 5×[HSA] (Xx is NphOHe or NphODe) match very well each other; moreover, the deacylation step turns out to be the rate limiting step in catalysis (i.e., k+3 < k+2). The pH dependence of the kinetic parameters for the hydrolysis of NphOHe and NphODe can be described by the acidic pKa-shift of a single amino acid residue, which varies from 8.9 in the free HSA to 7.6 and 7.0 in the HSA:NphOHe and HSA:NphODe complex, respectively; the pK>a-shift appears to be correlated to the length of the fatty acid tail of the substrate. The inhibition of the HSA-Tyr411-catalyzed hydrolysis of NphOHe, NphODe, and 4-nitrophenyl myristate (NphOMy) by five inhibitors (i.e., diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol) has been investigated at pH 7.5 and 22.0°C, resulting competitive. The affinity of diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol for HSA reflects the selectivity of the FA3-FA4 cleft. Under conditions where Tyr411 is not acylated, the molar fraction of diazepam, diflunisal, ibuprofen, and 3-indoxyl-sulfate bound to HSA is higher than 0.9 whereas the molar fraction of propofol bound to HSA is ca. 0.5. PMID:25790235

  13. Dynamics of Loop 1 of Domain I in Human Serum Albumin WhenDissolved in Ionic Liquids

    SciTech Connect

    Page, Taylor; Kraut, Nadine; Page, Phillip; Baker, Gary A; Bright, Frank

    2009-01-01

    We report on the rotational reorientation dynamics associated with loop 1 of domain I within a large multidomain protein (human serum albumin, HSA) when it is dissolved in binary mixtures of ionic liquid (1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([C4mim][Tf2N]), 1-butyl-3-methylimidazolium tetrafluoroborate ([C4mim][BF4]), or 1-butyl-3-methylimidazolium hexafluorophosphate ([C4mim][PF6])) and distilled deionized water (ddH2O) as a function of temperature and water loading. In IL/2% ddH2O (v/v) mixtures, loop 1 of domain I is more significantly denatured in comparison to the protein dissolved in aqueous solutions containing strong chemical denaturants (e.g., 8 M guanidine HCl (Gu HCl) or urea). As water loading increases, there is evidence for progressive refolding of loop 1 of domain I followed by recoupling with domains I, II, and III in the [C4mim][BF4]/ddH2O mixtures at 20 C. Above 30% (v/v) water, where domain I appears refolded, the Ac reporter molecule s semiangle steadily decreases from 35 to 20 with increasing water loading. From the perspective of domain I in HSA, this behavior is similar to the effects of dilution from 4 to 0 M Gu HCl in aqueous solution. Overall, these results lend insight into the tangle of biocatalytic and structural/dynamical mechanisms that enzymes may undergo in ionic liquid-based systems. It will be particularly motivating to extend this work to include enzyme-attuned ionic liquids shown to improve biocatalytic performance beyond that possible in the native (predominantly aqueous) setting.

  14. Preparation and in vitro investigation of antigastric cancer activities of carvacrol-loaded human serum albumin nanoparticles.

    PubMed

    Maryam, Keshavarzi; Shakeri, Shahryar; Kiani, Keyhaneh

    2015-10-01

    In this study, carvacrol-loaded human serum albumin (HSA) nanoparticles were developed and characterised. Nanoparticles were prepared by desolvation and emulsion/desolvation methods. Encapsulation efficiency (EE%) and loading capacity (LC%) of nanoparticles prepared by desolvation method were 48.4 and 45.1%, respectively. Carvacrol-loaded nanoparticles had 132±42 nm in diameter with monomodal distribution. Carvacrol-loaded nanoparticles which is prepared by emulsion/desolvation method had EE% and LC% of 32 and 32.3%, respectively, and 230±38 nm in size. The release of carvacrol from nanoparticles was monitored in phosphate-buffered saline (pH=7.4), 100 rpm at 37°C for 10 days. About 21.4% of carvacrol was released after 3 h from nanoparticles that were prepared by desolvation method. In emulsion/desolvation method, 26.8% of total carvacrol was released during 3 h of incubation. Cytotoxicity effect of loaded carvacrol was assessed by 3-[4, 5 dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) test on gastric cancer cells line (AGS). Cell line was exposed to the free carvacrol, unloaded and carvacrol-loaded nanoparticles for 48 h. The half maximal inhibitory concentration (IC50) for free carvacrol, unloaded and carvacrol-loaded HSA nanoparticles were 30, 1070 and 120 µg/ml, respectively. In conclusion, the results of this study showed applications of HSA nanoparticles for entrapment of carvacrol and antigastric cancer activity. Moreover, loading of carvacrol in combination with chemotherapy agents into the HSA nanoparticles may treat cancer cells better than single drug loaded nanoparticles. PMID:26435283

  15. Comprehensive Multispectroscopic Analysis on the Interaction and Corona Formation of Human Serum Albumin with Gold/Silver Alloy Nanoparticles.

    PubMed

    Selva Sharma, Arumugam; Ilanchelian, Malaichamy

    2015-07-30

    In the present investigation, we have systematically studied the binding mechanism of model protein human serum albumin (HSA) with gold/silver alloy nanoparticles (Au/Ag NPs) using multiple spectroscopic techniques. Absorption spectral studies of Au/Ag NPs in the presence of increasing concentrations of HSA resulted in a slight red shift of the surface plasmon resonance band (SPR) of Au/Ag NPs, suggesting changes in the refractive index around the nanoparticle surface owing to the adsorption of HSA. The results from high-resolution transmission electron microscopy (HR-TEM), dynamic light scattering (DLS), and zeta potential analysis substantiated the formation of a dense layer of HSA on the surface of Au/Ag NPs. The formation of a ground-state complex between HSA and Au/Ag NPs was evident from the outcome of the steady-state emission titration experiments of the HSA-Au/Ag NPs system. The binding parameters computed from corrected emission quenching data revealed that HSA exhibited a significant binding affinity toward Au/Ag NPs. The identical fluorescence lifetime values of HSA and HSA-Au/Ag NPs from time-resolved fluorescence spectroscopic analysis further authenticated the findings of steady-state emission measurements. The formation of HSA corona on the Au/Ag NPs surface was established on the basis of experimental quenching data and theoretical values. The occurrence of partial unfolding of HSA upon its interaction with the Au/Ag NPs surface was established by using an extrinsic fluorophore 1-anilino-8-naphthalenesulfonic acid (ANS). Absorption, Fourier transform infrared (FT-IR), Raman, circular dichroism (CD), and excitation-emission matrix (3D) spectral studies were also carried out to explore Au/Ag NPs-induced tertiary and secondary conformational changes of HSA. The influence of Au/Ag NPs on the esterase-like activity of HSA was established by probing the hydrolysis of p-nitrophenyl acetate. PMID:26106942

  16. Structure of the methyl orange-binding site on human serum albumin and its color-change mechanism.

    PubMed

    Ito, Shigenori; Yamamoto, Daisuke

    2015-01-01

    The goal in this study was to clarify the color-change mechanisms of methyl orange (MO) bound to human serum albumin (HSA) and the structure of the binding site. The absorbance of the MOHSA complex was measured at 560 nm in solutions of varying pH (pH 2.4-6.6). The obtained pH-dependent experimental data were consistent with the data calculated using the Henderson-Hasselbalch equation and pKa values (3.8, MO; 1.4, carboxyl group). The extent of the binding of MO to an HSA molecule was determined to be 1-4 by performing surface plasmon resonance analysis. Furthermore, the binding of MO to HSA was inhibited by warfarin. A fitting model of MO to HSA was created to evaluate these results based on PDB data (warfarin-HSA complex: 2BXD) and protein-structure analysis. The color-change mechanism of the MO-HSA complex appears to be as follows: the dissociated sulfo group of MO binds to Arg218/Lys444 sidechains through electrostatic interaction in the warfarin-binding site, and, subsequently, the color change occurs through a proton exchange between the diazenyl group and the γ-carboxyl group of Glu292. The color-changed MO is fixed in the warfarin-binding site. These results could support the development of a reliable dye-binding method and of a new method for staining diverse tissues that is based on a validated mechanism. PMID:26299483

  17. Resonance energy transfer, pH-induced folded states and the molecular interaction of human serum albumin and icariin.

    PubMed

    Cheng, Xiao-Xia; Fan, Xiao-Yang; Jiang, Feng-Lei; Liu, Yi; Lei, Ke-Lin

    2015-11-01

    Icariin is a flavonol glycoside with a wide range of pharmacological and biological activities. The pharmacological and biological functions of flavonoid compounds mainly originate from their binding to proteins. The mode of interaction of icariin with human serum albumin (HSA) has been characterized by fluorescence spectroscopy and far- and near-UV circular dichroism (CD) spectroscopy under different pH conditions. Fluorescence quenching studies showed that the binding affinity of icariin with HSA in the buffer solution at different pH values is: Ka (pH 4.5) > Ka (pH 3.5) > Ka (pH 9.0) > Ka (pH 7.0). Red-edge excitation shift (REES) studies revealed that pH had an obvious effect on the mobility of the tryptophan microenvironment and the addition of icariin made the REES effect more distinct. The static quenching mechanism and number of binding sites (n ≈ 1) were obtained from fluorescence data at three temperatures (298, 304 and 310 K). Both ∆H(0) < 0 and ∆Ѕ(0) < 0 suggested that hydrogen bonding and van der Waal's interaction were major driving forces in the binding mechanism, and this was also confirmed by the molecular simulation results. The distance r between the donor (HSA) and the acceptor (icariin) was calculated based on Förster non-radiation energy transfer theory. We found that pH had little impact on the energy transfer between HSA and icariin. Far- and near-UV CD spectroscopy studies further indicated the influence of pH on the complexation process and the alteration in the protein conformation upon binding.

  18. Studies of the interaction between demeclocycline and human serum albumin by multi-spectroscopic and molecular docking methods

    NASA Astrophysics Data System (ADS)

    Dong, Chengyu; Ma, Shuying; Liu, Ying

    2013-02-01

    This study was designed to examine the interaction of demeclocycline (DMCTC) with human serum albumin (HSA) by multi-spectroscopic and molecular docking methods. The inner filter effect was corrected before we calculated the binding parameters. Fluorescence and UV-vis spectroscopy revealed that DMCTC induced the fluorescence quenching of HSA though a static quenching procedure. Thermodynamic analysis by Van Hoff equation found enthalpy change (ΔH) and entropy change (ΔS) were -53.01 kJ mol-1 and -65.13 J mol-1 K-1, respectively, which indicated hydrogen bond and van der Waals force were the predominant force in the binding process. According to fluorescence resonance energy transfer (FRET), the specific binding distances between Trp-214 (donor) and DMCTC (acceptor) were 3.18 nm. Through site marker competitive experiments, subdomain IIA of HSA has been assigned to possess the high-affinity binding site of DMCTC. The three dimensional fluorescence showed that the conformation of HSA was changed after its complexation with DMCTC, and the alternations of protein secondary structure were quantitatively calculated from FT-IR with reduction of α-helices content about 4.8%, β-sheet from 30.3% to 21.6% and with increases of β-turn from 15.6% to 22.2%. Furthermore, the binding details between DMCTC and HSA were further confirmed by molecular docking studies, which revealed that DMCTC was bound at subdomain IIA through multiple interactions, such as hydrophobic effect, polar forces and π-π interactions. Moreover, the coexist metal ions such as Al3+, Fe3+, Cu2+, Cr3+ and Cd2+ can decrease the binding constants of DMCTC-HSA.

  19. Interaction of different polyphenols with bovine serum albumin (BSA) and human salivary alpha-amylase (HSA) by fluorescence quenching.

    PubMed

    Soares, Susana; Mateus, Nuno; Freitas, Victor de

    2007-08-01

    Phenolic compounds are responsible for major organoleptic characteristics of plant-derived food and beverages; these substances have received much attention, given that the major function of these compounds is their antioxidant ability. In the context of this study, our major aim was study the binding of several phenolic compounds such as (+)-catechin, (-)-epicatechin, (-)-epicatechin gallate, malvidin-3-glucoside, tannic acid, procyanidin B4, procyanidin B2 gallate, and procyanidin oligomers to different proteins (bovine serum albumin and human alpha-amylase) by fluorescence quenching of protein intrinsic fluorescence. From the spectra obtained, the Stern-Volmer, the apparent static, and the bimolecular quenching constants were calculated. The structure of polyphenols revealed to significantly affect the binding/quenching process; in general, the binding affinity increased with the molecular weight of polyphenol compounds and in the presence of galloyl groups. For catechin monomer and procyanidin dimer B4, the K(SV) was 14,100 and 13,800 M(-1), respectively, and for galloyl derivatives, the K(SV) was 19,500 and 21,900 M(-1), respectively. Tannic acid was shown to be the major quenching molecule for both proteins. However, comparing different proteins, the same polyphenol showed different quenching effects, which are suggested to be related to the three-dimensional structure of the proteins studied. For (+)-catechin and BSA, the K(SV) was 8700 M(-1), and with alpha-amylase, it was 14,100 M(-1); for tannic acid, the K(SV) was 10,0548 and 11,0674 M(-1), respectively. From the results obtained, besides the main binding analysis performed, we conclude that this technique is more sensitive than thought because we can detect several interactions that have not been proven by other methods, namely, nephelometry. Overall, fluorescence quenching has proven to be a very sensitive technique with many potentialities to analyze the interaction between polyphenols and proteins.

  20. Evaluation of enantioselective binding of propanocaine to human serum albumin by ultrafiltration and electrokinetic chromatography under intermediate precision conditions.

    PubMed

    Martínez-Gómez, María Amparo; Escuder-Gilabert, Laura; Villanueva-Camañas, Rosa María; Sagrado, Salvador; Medina-Hernández, María José

    2012-03-15

    Stereoselectivity in protein binding can have a significant effect on the pharmacokinetic and pharmacodynamic properties of chiral drugs. In this paper, the enantioselective binding of propanocaine (PRO) enantiomers to human serum albumin (HSA), the most relevant plasmatic protein in view of stereoselectivity, has been evaluated by incubation and ultrafiltration of racemic PRO-HSA mixtures and chiral analysis of the bound and unbound fractions by electrokinetic chromatography using HSA as chiral selector. Experimental conditions for the separation of PRO enantiomers using HSA as chiral selector and electrokinetic chromatography have been optimised. Affinity constants and protein binding in percentage (PB) were obtained for both enantiomers of PRO, as well as the enantioselectivity (ES) to HSA. Data were obtained in two independent working sessions (days). The influence of the session and fraction processed factors were examined. A univariate direct-estimation approach was used facilitating outliers' identification and statistical comparison. Non-linear fitting of data was used to verify the stoichiometry and affinity estimations obtained by the direct approach. Robust statistics were applied to obtain reliable estimations of uncertainty, accounting for the factors (day and processed fraction), thus representing intermediate precision conditions. Mimicking in vivo experimental conditions, information unapproachable by in vivo experiments was obtained for PRO enantiomers interacting with HSA. For the first (E1) and the second (E2) eluted PRO enantiomers the results were: 1:1 stoichiometry, medium affinity constants, logK(E1)=3.20±0.16 and log K(E2)=3.40±0.14, medium protein binding percentage, PB=48.7 and 60.1% for E1 and E2, respectively, and moderate but significant enantioselectivity, ES=K(E2)/K(E1)=1.5±0.3.

  1. Targeting naproxen coupled to human serum albumin to nonparenchymal cells reduces endotoxin-induced mortality in rats with biliary cirrhosis.

    PubMed

    Albrecht, C; Meijer, D K; Lebbe, C; Sägesser, H; Melgert, B N; Poelstra, K; Reichen, J

    1997-12-01

    Endotoxin is thought to play a major role in cirrhotic liver disease. Cyclo-oxygenase inhibitors were shown to be partially protective against endotoxin but cannot be used in cirrhotic patients because of renal side-effects. We argued that administration of naproxen (NAP) linked to human serum albumin (HSA), which results in specific delivery of NAP to endothelial cells (EC) and Kupffer cells (KC) and exhibited hepatoprotective effects against lipopolysaccharide (LPS) in vitro, could protect cirrhotic rats from LPS toxicity while preserving renal function. The studies were performed in rats rendered cirrhotic by bile duct ligation (BDL); animals received LPS (Escherichia coli, 800 microg/kg) intravenously. Five groups were studied: LPS alone, rats pretreated with a conventional dose of NAP (50 mg/kg), NAP-HSA (22 mg/kg), NAP equimolar to NAP-HSA (1.5 mg/kg), or the HSA carrier. LPS induced significant mortality (55%); this was not affected by equimolar NAP (57%) but accentuated by conventional NAP (88%). In contrast, NAP-HSA provided significant protection (9%; P < .05). After conventional NAP treatment, significant renal toxicity was observed as evidenced by a marked reduction in sodium excretion (LPS vs. NAP-HSA vs. NAP [50 mg/kg] 33 +/- 22 vs. 50 +/- 39 vs. 4 +/- 3 micromol/h; P < .05). Renal prostaglandin E2 (PGE2) excretion was reduced by NAP in all groups, but most markedly at the conventional dosage (LPS vs. NAP-HSA vs. NAP [50 mg/kg] 132 +/- 115 vs. 39 +/- 19 vs. 9 +/- 8 ng/mL; P < .05). Successful targeting was evidenced by a significant hepatic enrichment of NAP in the NAP-HSA group compared with the equimolar untargeted group (30.16 +/- 9.33 vs. 1.13 +/- 1.95 nmol/g liver). Thus, targeting NAP to EC/KC results in improved survival, higher efficacy, and sparing of renal function in cirrhotic rats.

  2. Simultaneous determination of four local anesthetics by CE with ECL and study on interaction between procainamide and human serum albumin.

    PubMed

    Duan, Hong-Bing; Cao, Jun-Tao; Yang, Jiu-Jun; Wang, Hui; Liu, Yan-Ming

    2016-07-01

    A new method of capillary electrophoresis (CE) coupled with tris(2, 2'-bipyridyl) ruthenium(II) electrochemiluminescence (ECL) detection has been developed to detect four local anesthetics procainamide (PAH), tetracaine (TCH), proparacaine (PCH) and cinchocaine (CIN) simultaneously. An europium (III)-doped prussian blue analogue film (Eu-PB) modified platinum electrode was prepared and applied to improve the detection sensitivity. The parameters including additives, concentration and pH of the running buffer, separation voltage and detection potential that affect CE separation and ECL detection were optimized in detail. The four local anesthetics were baseline separated and detected within 10min under the optimized conditions. The detection limits (LOD) of PAH, TCH, PCH and CIN are 5.5×10(-8), 9.6×10(-8), 2.5×10(-8) and 3.5×10(-8)molL(-1) (S/N=3), respectively. RSDs of the migration time for four analytes range from 1.2% to 2.5% within intraday and from 2.4% to 4.9% in interday, RSDs of the peak area for four analytes are from 1.7% to 3.3% within intraday and from 2.2% to 5.6% in interday, respectively. The limits of quantitation (LOQ) (S/N=10) for PAH, TCH, PCH and CIN in human urine sample are 5.9×10(-7), 9.2×10(-7), 8.3×10(-7) and 5.0×10(-7)molL(-1), separately. The recoveries (n=3) of four analytes in human urine are from 87.6% to 107.7% with less than 5.9% in RSDs. The developed method was used to determine four local anesthetics in human urine samples and investigate the interaction between PAH and human serum albumin (HSA). The number of binding sites and the binding constant of PAH with HSA were calculated to be 1.03 and 2.4×10(4)Lmol(-1), respectively.

  3. Simultaneous determination of four local anesthetics by CE with ECL and study on interaction between procainamide and human serum albumin.

    PubMed

    Duan, Hong-Bing; Cao, Jun-Tao; Yang, Jiu-Jun; Wang, Hui; Liu, Yan-Ming

    2016-07-01

    A new method of capillary electrophoresis (CE) coupled with tris(2, 2'-bipyridyl) ruthenium(II) electrochemiluminescence (ECL) detection has been developed to detect four local anesthetics procainamide (PAH), tetracaine (TCH), proparacaine (PCH) and cinchocaine (CIN) simultaneously. An europium (III)-doped prussian blue analogue film (Eu-PB) modified platinum electrode was prepared and applied to improve the detection sensitivity. The parameters including additives, concentration and pH of the running buffer, separation voltage and detection potential that affect CE separation and ECL detection were optimized in detail. The four local anesthetics were baseline separated and detected within 10min under the optimized conditions. The detection limits (LOD) of PAH, TCH, PCH and CIN are 5.5×10(-8), 9.6×10(-8), 2.5×10(-8) and 3.5×10(-8)molL(-1) (S/N=3), respectively. RSDs of the migration time for four analytes range from 1.2% to 2.5% within intraday and from 2.4% to 4.9% in interday, RSDs of the peak area for four analytes are from 1.7% to 3.3% within intraday and from 2.2% to 5.6% in interday, respectively. The limits of quantitation (LOQ) (S/N=10) for PAH, TCH, PCH and CIN in human urine sample are 5.9×10(-7), 9.2×10(-7), 8.3×10(-7) and 5.0×10(-7)molL(-1), separately. The recoveries (n=3) of four analytes in human urine are from 87.6% to 107.7% with less than 5.9% in RSDs. The developed method was used to determine four local anesthetics in human urine samples and investigate the interaction between PAH and human serum albumin (HSA). The number of binding sites and the binding constant of PAH with HSA were calculated to be 1.03 and 2.4×10(4)Lmol(-1), respectively. PMID:27154684

  4. Investigation of binding mechanism of novel 8-substituted coumarin derivatives with human serum albumin and α-1-glycoprotein.

    PubMed

    Yeggoni, Daniel Pushpa Raju; Manidhar, Darla Mark; Suresh Reddy, Cirandur; Subramanyam, Rajagopal

    2016-09-01

    Coumarin molecules have biological activities possessing lipid-controlling activity, anti-hepatitis C activity, anti-diabetic, anti-Parkinson activity, and anti-cancer activity. Here, we have presented an inclusive study on the interaction of 8-substituted-7-hydroxy coumarin derivatives (Umb-1/Umb-2) with α-1-glycoprotein (AGP) and human serum albumin (HSA) which are the major carrier proteins in the human blood plasma. Binding constants obtained from fluorescence emission data were found to be KUmb-1=3.1 ± .01 × 10(4) M(-1), KUmb-2 = 7 ± .01 × 10(4) M(-1), which corresponds to -6.1 and -6.5 kcal/mol of free energy for Umb-1 and Umb-2, respectively, suggesting that these derivatives bind strongly to HSA. Also these molecules bind to AGP with binding constants of KUmb-1-AGP=3.1 ± .01 × 10(3) M(-1) and KUmb-2-AGP = 4.6 ± .01 × 10(3) M(-1). Further, the distance, r between the donor (HSA) and acceptor (Umb-1/Umb-2) was calculated based on the Forster's theory of non-radiation energy transfer and the values were observed to be 1.14 and 1.29 nm in Umb-1-HSA and Umb-2-HSA system, respectively. The protein secondary structure of HSA was partially unfolded upon binding of Umb-1 and Umb-2. Furthermore, site displacement experiments with lidocaine, phenylbutazone (IIA), and ibuprofen (IIIA) proves that Umb derivatives significantly bind to subdomain IIIA of HSA which is further supported by docking studies. Furthermore, Umb-1 binds to LYS402 with one hydrogen bond distance of 2.8 Å and Umb-2 binds to GLU354 with one hydrogen bond at a distance of 2.0 Å. Moreover, these molecules are stabilized by hydrophobic interactions and hydrogen bond between the hydroxyl groups of carbon-3 of coumarin derivatives.

  5. Depletion of human serum albumin in embryo culture media for in vitro fertilization using monolithic columns with immobilized antibodies.

    PubMed

    Tarasova, Irina A; Lobas, Anna A; Černigoj, Urh; Solovyeva, Elizaveta M; Mahlberg, Barbara; Ivanov, Mark V; Panić-Janković, Tanja; Nagy, Zoltan; Pridatchenko, Marina L; Pungor, Andras; Nemec, Blaž; Vidic, Urška; Gašperšič, Jernej; Krajnc, Nika Lendero; Vidič, Jana; Gorshkov, Mikhail V; Mitulović, Goran

    2016-09-01

    Affinity depletion of abundant proteins such as HSA is an important stage in routine sample preparation prior to MS/MS analysis of biological samples with high range of concentrations. Due to the charge competition effects in electrospray ion source that results in discrimination of the low-abundance species, as well as limited dynamic range of MS/MS, restricted typically by three orders of magnitude, the identification of low-abundance proteins becomes a challenge unless the sample is depleted from high-concentration compounds. This dictates a need for developing efficient separation technologies allowing fast and automated protein depletion. In this study, we performed evaluation of a novel immunoaffinity-based Convective Interaction Media analytical columns (CIMac) depletion column with specificity to HSA (CIMac-αHSA). Because of the convective flow-through channels, the polymethacrylate CIMac monoliths afford flow rate independent binding capacity and resolution that results in relatively short analysis time compared with traditional chromatographic supports. Seppro IgY14 depletion kit was used as a benchmark to control the results of depletion. Bottom-up proteomic approach followed by label-free quantitation using normalized spectral indexes were employed for protein quantification in G1/G2 and cleavage/blastocyst in vitro fertilization culture media widely utilized in clinics for embryo growth in vitro. The results revealed approximately equal HSA level of 100 ± 25% in albumin-enriched fractions relative to the nondepleted samples for both CIMac-αHSA column and Seppro kit. In the albumin-free fractions concentrated 5.5-fold by volume, serum albumin was identified at the levels of 5-30% and 20-30% for the CIMac-αHSA and Seppro IgY14 spin columns, respectively. PMID:27122488

  6. Depletion of human serum albumin in embryo culture media for in vitro fertilization using monolithic columns with immobilized antibodies.

    PubMed

    Tarasova, Irina A; Lobas, Anna A; Černigoj, Urh; Solovyeva, Elizaveta M; Mahlberg, Barbara; Ivanov, Mark V; Panić-Janković, Tanja; Nagy, Zoltan; Pridatchenko, Marina L; Pungor, Andras; Nemec, Blaž; Vidic, Urška; Gašperšič, Jernej; Krajnc, Nika Lendero; Vidič, Jana; Gorshkov, Mikhail V; Mitulović, Goran

    2016-09-01

    Affinity depletion of abundant proteins such as HSA is an important stage in routine sample preparation prior to MS/MS analysis of biological samples with high range of concentrations. Due to the charge competition effects in electrospray ion source that results in discrimination of the low-abundance species, as well as limited dynamic range of MS/MS, restricted typically by three orders of magnitude, the identification of low-abundance proteins becomes a challenge unless the sample is depleted from high-concentration compounds. This dictates a need for developing efficient separation technologies allowing fast and automated protein depletion. In this study, we performed evaluation of a novel immunoaffinity-based Convective Interaction Media analytical columns (CIMac) depletion column with specificity to HSA (CIMac-αHSA). Because of the convective flow-through channels, the polymethacrylate CIMac monoliths afford flow rate independent binding capacity and resolution that results in relatively short analysis time compared with traditional chromatographic supports. Seppro IgY14 depletion kit was used as a benchmark to control the results of depletion. Bottom-up proteomic approach followed by label-free quantitation using normalized spectral indexes were employed for protein quantification in G1/G2 and cleavage/blastocyst in vitro fertilization culture media widely utilized in clinics for embryo growth in vitro. The results revealed approximately equal HSA level of 100 ± 25% in albumin-enriched fractions relative to the nondepleted samples for both CIMac-αHSA column and Seppro kit. In the albumin-free fractions concentrated 5.5-fold by volume, serum albumin was identified at the levels of 5-30% and 20-30% for the CIMac-αHSA and Seppro IgY14 spin columns, respectively.

  7. Comparative analysis the binding affinity of mycophenolic sodium and meprednisone with human serum albumin: Insight by NMR relaxation data and docking simulation.

    PubMed

    Ma, Xiaoli; He, Jiawei; Yan, Jin; Wang, Qing; Li, Hui

    2016-03-25

    Mycophenolic sodium is an immunosuppressive agent that is always combined administration with corticosteroid in clinical practice. Considering the distribution and side-effect of the drug may change when co-administrated drug exist, this paper comparatively analyzed the binding ability of mycophenolic sodium and meprednisone toward human serum albumin by nuclear magnetic resonance relaxation data and docking simulation. The nuclear magnetic resonance approach was based on the analysis of proton selective and non-selective relaxation rate enhancement of the ligand in the absence and presence of macromolecules. The contribution of the bound ligand fraction to the observed relaxation rate in relation to protein concentration allowed the calculation of the affinity index. This approach allowed the comparison of the binding affinity of mycophenolic sodium and meprednisone. Molecular modeling was operated to simulate the binding model of ligand and albumin through Autodock 4.2.5. Competitive binding of mycophenolic sodium and meprednisone was further conducted through fluorescence spectroscopy. PMID:26892221

  8. Multispectroscopic and docking studies on the binding of chlorogenic acid isomers to human serum albumin: Effects of esteryl position on affinity.

    PubMed

    Tang, Bin; Huang, Yanmei; Ma, Xiangling; Liao, Xiaoxiang; Wang, Qing; Xiong, Xinnuo; Li, Hui

    2016-12-01

    Structural differences among various dietary polyphenols affect their absorption, metabolism, and bioactivities. In this work, chlorogenic acid (CA) and its two positional isomers, neochlorogenic acid (NCA) and cryptochlorogenic acid (CCA), were investigated for their binding reactions with human serum albumin (HSA) using fluorescence, ultraviolet-visible, Fourier transform infrared and circular dichroism spectroscopies, as well as molecular docking. All three isomers were bound to HSA at Sudlow's site I and affected the protein secondary structure. CCA presented the strongest ability of hydrogen-bond formation, and both CA and NCA generated more electrostatic interactions with HSA. The albumin-binding capacity of these compounds decreased in the order CCA>NCA>CA. The compound with 4-esteryl structure showed higher binding affinity and larger conformational changes to HSA than that with 3- or 5-esteryl structures. These comparative studies on structure-affinity relationship contributed to the structural modification and design of phenolic food additives or new polyphenol-like drugs. PMID:27374553

  9. Effects of Gold Salt Speciation and Structure of Human and Bovine Serum Albumin on the Synthesis and Stability of Gold Nanostructures

    NASA Astrophysics Data System (ADS)

    Miranda, Érica; Tofanello, Aryane; Brito, Adrianne; Lopes, David; Giacomelli, Fernando; Albuquerque, Lindomar; Costa, Fanny; Ferreira, Fabio; Araujo-Chaves, Juliana; de Castro, Carlos; Nantes, Iseli

    2016-03-01

    The present study aimed to investigate the influence of albumin structure and gold speciation on the synthesis of gold nanoparticles (GNPs). The strategy of synthesis was the addition of HAuCl4 solutions at different pH values (3-12) to solutions of human and bovine serum albumins (HSA and BSA) at the same corresponding pH values. Different pH values influence the GNP synthesis due to gold speciation. Besides the inherent effect of pH on the native structure of albumins, the use N-ethylmaleimide (NEM)-treated and heat-denaturated forms of HSA and BSA provided additional insights about the influence of protein structure, net charge, and thiol group approachability on the GNP synthesis. NEM treatment, heating, and the extreme values of pH promoted loss of the native albumin structure. The formation of GNPs indicated by the appearance of surface plasmon resonance (SPR) bands became detectable from fifteen days of the synthesis processes that were carried out with native, NEM-treated and heat-denaturated forms of HSA and BSA, exclusively at pH 6 and 7. After two months of incubation, SPR band was also detected for all synthesis carried out at pH 8.0. The mean values of the hydrodynamic radius (RH) were 24 and 34 nm for GNPs synthesized with native HSA and BSA, respectively. X-ray diffraction (XRD) revealed crystallites of 13 nm. RH, XRD, and zeta potential values were consistent with GNP capping by the albumins. However, the GNPs produced with NEM-treated and heat-denaturated albumins exhibited loss of protein capping by lowering the ionic strength. This result suggests a significant contribution of non-electrostatic interactions of albumins with the GNP surface, in these conditions. The denaturation of proteins exposes hydrophobic groups to the solvent, and these groups could interact with the gold surface. In these conditions, the thiol blockage or oxidation, the latter probably favored upon heating, impaired the formation of a stable capping by thiol coordination

  10. Superior serum half life of albumin tagged TNF ligands

    SciTech Connect

    Mueller, Nicole; Schneider, Britta; Pfizenmaier, Klaus; Wajant, Harald

    2010-06-11

    Due to their immune stimulating and apoptosis inducing properties, ligands of the TNF family attract increasing interest as therapeutic proteins. A general limitation of in vivo applications of recombinant soluble TNF ligands is their notoriously rapid clearance from circulation. To improve the serum half life of the TNF family members TNF, TWEAK and TRAIL, we genetically fused soluble variants of these molecules to human serum albumin (HSA). The serum albumin-TNF ligand fusion proteins were found to be of similar bioactivity as the corresponding HSA-less counterparts. Upon intravenous injection (i.v.), serum half life of HSA-TNF ligand fusion proteins, as determined by ELISA, was around 15 h as compared to approximately 1 h for all of the recombinant control TNF ligands without HSA domain. Moreover, serum samples collected 6 or 24 h after i.v. injection still contained high TNF ligand bioactivity, demonstrating that there is only limited degradation/inactivation of circulating HSA-TNF ligand fusion proteins in vivo. In a xenotransplantation model, significantly less of the HSA-TRAIL fusion protein compared to the respective control TRAIL protein was required to achieve inhibition of tumor growth indicating that the increased half life of HSA-TNF ligand fusion proteins translates into better therapeutic action in vivo. In conclusion, our data suggest that genetic fusion to serum albumin is a powerful and generally applicable mean to improve bioavailability and in vivo activity of TNF ligands.

  11. Development of an HPLC assay to study the effect of endogenous and exogenous substances on heat-induced aggregation of human serum albumin.

    PubMed

    Saso, L; Casini, M L; Valentini, G; Mattei, E; Panzironi, C; Silvestrini, B

    1998-03-01

    The mechanism of action of non-steroidal anti-inflammatory drugs which are used in high doses in chronic inflammatory conditions is not clearly understood. Their known protein-stabilizing properties could play a significant role. The inhibition of cyclooxygenase may not be essential for their anti-rheumatic activity, since compounds with strong anti-denaturant properties and devoid of anti-inflammatory activity were shown to be effective in an experimental model of rheumatoid arthritis. Hence, to develop new anti-rheumatic drugs it is essential that a simple in vitro method to evaluate the anti-denaturant activity of endogenous and exogenous compounds is available. We developed a new assay, using gel permeation high performance liquid chromatography, to study the effect of endogenous and exogenous compounds on heat-induced aggregation of human serum albumin in conditions in which protein precipitation does not occur. Non-steroidal anti-inflammatory drugs like diclofenac, ibuprofen and naproxen inhibited the aggregation of albumin at low concentrations (EC50 10(-4)-10(-5) mol/l) comparable to those active in a classical turbidimetric method, whereas the effect of weak stabilizers, like sodium cloride and formic, fumaric, maleic, malonic, and succinic acid (EC50 10(-1)-10(-2) mol/l in the Mizushima test) was not detectable. Furthermore, the HPLC assay allowed the examination of a number of coloured substances, including bilirubin, which appeared to be a strong stabilizer of its physiological carrier, albumin. These data could be clinically relevant, since the drugs examined are used at very high doses in rheumatoid arthritis and related conditions, with plasma levels that could cause significant stabilization of serum albumin and perhaps other proteins.

  12. In-vitro study on the competitive binding of diflunisal and uraemic toxins to serum albumin and human plasma using a potentiometric ion-probe technique.

    PubMed

    Davilas, A; Koupparis, M; Macheras, P; Valsami, G

    2006-11-01

    The competitive binding of diflunisal and three well-known uraemic toxins (3-indoxyl sulfate, indole-3-acetic acid and hippuric acid) to bovine serum albumin (BSA), human serum albumin (HSA) and human plasma was studied by direct potentiometry. The method used the potentiometric drug ion-probe technique with a home-made ion sensor (electrode) selective to the drug anion. The site-oriented Scatchard model was used to describe the binding of diflunisal to BSA, HSA and human plasma, while the general competitive binding model was used to calculate the binding parameters of the three uraemic toxins to BSA. Diflunisal binding parameters, number of binding sites, n(i) and association constants for each class of binding site, K(i), were calculated in the absence and presence of uraemic toxins. Although diflunisal exhibits high binding affinity for site I of HSA and the three uraemic toxins bind primarily to site II, strong interaction was observed between the drug and the three toxins, which were found to affect the binding of diflunisal on its primary class of binding sites on both BSA and HSA molecules and on human plasma. These results are strong evidence that the decreased binding of diflunisal that occurs in uraemic plasma may not be solely attributed to the lower albumin concentration observed in many patients with renal failure. The uraemic toxins that accumulate in uraemic plasma may displace the drug from its specific binding sites on plasma proteins, resulting in increased free drug plasma concentration in uraemic patients. PMID:17132209

  13. Effects of Gold Salt Speciation and Structure of Human and Bovine Serum Albumins on the Synthesis and Stability of Gold Nanostructures

    PubMed Central

    Miranda, Érica G. A.; Tofanello, Aryane; Brito, Adrianne M. M.; Lopes, David M.; Albuquerque, Lindomar J. C.; de Castro, Carlos E.; Costa, Fanny N.; Giacomelli, Fernando C.; Ferreira, Fabio F.; Araújo-Chaves, Juliana C.; Nantes, Iseli L.

    2016-01-01

    The present study aimed to investigate the influence of albumin structure and gold speciation on the synthesis of gold nanoparticles (GNPs). The strategy of synthesis was the addition of HAuCl4 solutions at different pH values (3–12) to solutions of human and bovine serum albumins (HSA and BSA) at the same corresponding pH values. Different pH values influence the GNP synthesis due to gold speciation. Besides the inherent effect of pH on the native structure of albumins, the use N-ethylmaleimide (NEM)-treated and heat-denaturated forms of HSA and BSA provided additional insights about the influence of protein structure, net charge, and thiol group approachability on the GNP synthesis. NEM treatment, heating, and the extreme values of pH promoted loss of the native albumin structure. The formation of GNPs indicated by the appearance of surface plasmon resonance (SPR) bands became detectable from 15 days of the synthesis processes that were carried out with native, NEM-treated and heat-denaturated forms of HSA and BSA, exclusively at pH 6 and 7. After 2 months of incubation, SPR band was also detected for all synthesis carried out at pH 8.0. The mean values of the hydrodynamic radius (RH) were 24 and 34 nm for GNPs synthesized with native HSA and BSA, respectively. X-ray diffraction (XRD) revealed crystallites of 13 nm. RH, XRD, and zeta potential values were consistent with GNP capping by the albumins. However, the GNPs produced with NEM-treated and heat-denaturated albumins exhibited loss of protein capping by lowering the ionic strength. This result suggests a significant contribution of non-electrostatic interactions of albumins with the GNP surface, in these conditions. The denaturation of proteins exposes hydrophobic groups to the solvent, and these groups could interact with the gold surface. In these conditions, the thiol blockage or oxidation, the latter probably favored upon heating, impaired the formation of a stable capping by thiol coordination with

  14. Effects of Gold Salt Speciation and Structure of Human and Bovine Serum Albumins on the Synthesis and Stability of Gold Nanostructures.

    PubMed

    Miranda, Érica G A; Tofanello, Aryane; Brito, Adrianne M M; Lopes, David M; Albuquerque, Lindomar J C; de Castro, Carlos E; Costa, Fanny N; Giacomelli, Fernando C; Ferreira, Fabio F; Araújo-Chaves, Juliana C; Nantes, Iseli L

    2016-01-01

    The present study aimed to investigate the influence of albumin structure and gold speciation on the synthesis of gold nanoparticles (GNPs). The strategy of synthesis was the addition of HAuCl4 solutions at different pH values (3-12) to solutions of human and bovine serum albumins (HSA and BSA) at the same corresponding pH values. Different pH values influence the GNP synthesis due to gold speciation. Besides the inherent effect of pH on the native structure of albumins, the use N-ethylmaleimide (NEM)-treated and heat-denaturated forms of HSA and BSA provided additional insights about the influence of protein structure, net charge, and thiol group approachability on the GNP synthesis. NEM treatment, heating, and the extreme values of pH promoted loss of the native albumin structure. The formation of GNPs indicated by the appearance of surface plasmon resonance (SPR) bands became detectable from 15 days of the synthesis processes that were carried out with native, NEM-treated and heat-denaturated forms of HSA and BSA, exclusively at pH 6 and 7. After 2 months of incubation, SPR band was also detected for all synthesis carried out at pH 8.0. The mean values of the hydrodynamic radius (RH) were 24 and 34 nm for GNPs synthesized with native HSA and BSA, respectively. X-ray diffraction (XRD) revealed crystallites of 13 nm. RH, XRD, and zeta potential values were consistent with GNP capping by the albumins. However, the GNPs produced with NEM-treated and heat-denaturated albumins exhibited loss of protein capping by lowering the ionic strength. This result suggests a significant contribution of non-electrostatic interactions of albumins with the GNP surface, in these conditions. The denaturation of proteins exposes hydrophobic groups to the solvent, and these groups could interact with the gold surface. In these conditions, the thiol blockage or oxidation, the latter probably favored upon heating, impaired the formation of a stable capping by thiol coordination with the

  15. Effects of Gold Salt Speciation and Structure of Human and Bovine Serum Albumins on the Synthesis and Stability of Gold Nanostructures.

    PubMed

    Miranda, Érica G A; Tofanello, Aryane; Brito, Adrianne M M; Lopes, David M; Albuquerque, Lindomar J C; de Castro, Carlos E; Costa, Fanny N; Giacomelli, Fernando C; Ferreira, Fabio F; Araújo-Chaves, Juliana C; Nantes, Iseli L

    2016-01-01

    The present study aimed to investigate the influence of albumin structure and gold speciation on the synthesis of gold nanoparticles (GNPs). The strategy of synthesis was the addition of HAuCl4 solutions at different pH values (3-12) to solutions of human and bovine serum albumins (HSA and BSA) at the same corresponding pH values. Different pH values influence the GNP synthesis due to gold speciation. Besides the inherent effect of pH on the native structure of albumins, the use N-ethylmaleimide (NEM)-treated and heat-denaturated forms of HSA and BSA provided additional insights about the influence of protein structure, net charge, and thiol group approachability on the GNP synthesis. NEM treatment, heating, and the extreme values of pH promoted loss of the native albumin structure. The formation of GNPs indicated by the appearance of surface plasmon resonance (SPR) bands became detectable from 15 days of the synthesis processes that were carried out with native, NEM-treated and heat-denaturated forms of HSA and BSA, exclusively at pH 6 and 7. After 2 months of incubation, SPR band was also detected for all synthesis carried out at pH 8.0. The mean values of the hydrodynamic radius (RH) were 24 and 34 nm for GNPs synthesized with native HSA and BSA, respectively. X-ray diffraction (XRD) revealed crystallites of 13 nm. RH, XRD, and zeta potential values were consistent with GNP capping by the albumins. However, the GNPs produced with NEM-treated and heat-denaturated albumins exhibited loss of protein capping by lowering the ionic strength. This result suggests a significant contribution of non-electrostatic interactions of albumins with the GNP surface, in these conditions. The denaturation of proteins exposes hydrophobic groups to the solvent, and these groups could interact with the gold surface. In these conditions, the thiol blockage or oxidation, the latter probably favored upon heating, impaired the formation of a stable capping by thiol coordination with the

  16. Acetylcholinesterase Reactivators (HI-6, Obidoxime, Trimedoxime, K027, K075, K127, K203, K282): Structural Evaluation of Human Serum Albumin Binding and Absorption Kinetics

    PubMed Central

    Zemek, Filip; Zdarova, Jana Karasova; Sepsova, Vendula; Kuca, Kamil

    2013-01-01

    Acetylcholinesterase (AChE) reactivators (oximes) are compounds predominantly targeting the active site of the enzyme. Toxic effects of organophosphates nerve agents (OPNAs) are primarily related to their covalent binding to AChE and butyrylcholinesterase (BChE), critical detoxification enzymes in the blood and in the central nervous system (CNS). After exposure to OPNAs, accumulation of acetylcholine (ACh) overstimulates receptors and blocks neuromuscular junction transmission resulting in CNS toxicity. Current efforts at treatments for OPNA exposure are focused on non-quaternary reactivators, monoisonitrosoacetone oximes (MINA), and diacylmonoxime reactivators (DAM). However, so far only quaternary oximes have been approved for use in cases of OPNA intoxication. Five acetylcholinesterase reactivator candidates (K027, K075, K127, K203, K282) are presented here, together with pharmacokinetic data (plasma concentration, human serum albumin binding potency). Pharmacokinetic curves based on intramuscular application of the tested compounds are given, with binding information and an evaluation of structural relationships. Human Serum Albumin (HSA) binding studies have not yet been performed on any acetylcholinesterase reactivators, and correlations between structure, concentration curves and binding are vital for further development. HSA bindings of the tested compounds were 1% (HI-6), 7% (obidoxime), 6% (trimedoxime), and 5%, 10%, 4%, 15%, and 12% for K027, K075, K127, K203, and K282, respectively. PMID:23917882

  17. Molecular modeling and spectroscopic studies on binding of 2,6-bis[4-(4-amino-2-trifluoromethylphenoxy)benzoyl] pyridine to human serum albumin

    NASA Astrophysics Data System (ADS)

    He, Wen-ying; Chen, Hui-juan; Sheng, Fen-ling; Yao, Xiao-jun

    2009-10-01

    BAFP (2,6-bis[4-(4-amino-2-trifluoromethylphenoxy)benzoyl] pyridine), a synthesized polyimide compound, was exploited for the first time to analyze its interaction with human serum albumin (HSA) by molecular modeling, fluorescence and Fourier transform infrared attenuated total reflection spectroscopy (FTIR ATR) with drug concentrations of 3.3 × 10 -6 to 3.0 × 10 -5 mol L -1. Molecular docking was performed to reveal the possible binding mode. The results suggested that BAFP can strongly bind to human serum albumin (HSA) and the primary binding site of BAFP is located in site II of HSA, which is supported by the results from the competitive experiment. The binding constants for the interaction of BAFP with HSA have been evaluated from relevant fluorescence data at different temperatures (296, 303, 310 and 308 K). The alterations of the protein secondary structure in the presence of BAFP in aqueous solution were quantitatively calculated by the evidences from FTIR ATR spectroscopes. The binding process was exothermic and spontaneous, as indicated by the thermodynamic analyses, and the major part of the binding energy is hydrophobic interaction, which is also in good agreement with the results of molecule modeling study. The enthalpy change Δ H0, the free energy change Δ G0 and the entropy change Δ S0 of 296 K were calculated to be -7.75, -27.68 kJ mol -1 and 67.33 J mol -1 K -1, respectively.

  18. Study of human serum albumin structure by dynamic light scattering: two types of reactions under different pH and interaction with physiologically active compounds

    NASA Astrophysics Data System (ADS)

    Luik, A. I.; Naboka, Yu. N.; Mogilevich, S. E.; Hushcha, T. O.; Mischenko, N. I.

    1998-09-01

    The effect of pH and binding of ten physiologically active compounds (isoproterenol, yohimbine, propranolol, clonidine, phenylephrine, carbachol, tripeptide fMLP, diphenhydramine, chlorpromazine and atropine) on the molecular structure of human serum albumin (HSA) has been studied using the dynamic light scattering. It was found that albumin globule has the most compact configuration (Stokes diameter 59-62 Å) at physiological pH 7.4. The changes in pH, both increase to 8.0 and decrease to 5.4, result in the growth of globule size to 72-81 Å. At acidic shift of pH an additional peak arises in the correlation spectra caused by the light scattering on the structures with the Stokes diameters of 29-37 Å. Those conform to the sizes of the albumin subdomains. The indicated peak is not displayed at basic shift of pH. The interaction with propranolol, clonidine, phenylephrine, carbachol and tripeptide fMLP which hinder adenylate cyclase (AdC) and activate Ca-polyphosphoinositide (Ca-PPI) signaling system of a cell initiates structural rearrangements similar to acidic transitions. Isoproterenol, yohimbine diphenhydramine, chlorpromazine and atropine, which activate AdC and hinder Ca-PPI, cause conformational changes of HSA similar to basic transitions.

  19. SDS-binding assay based on tyrosine fluorescence as a tool to determine binding properties of human serum albumin in blood plasma

    NASA Astrophysics Data System (ADS)

    Zhdanova, Nadezda; Shirshin, Evgeny; Fadeev, Victor; Priezzhev, Alexander

    2016-04-01

    Among all plasma proteins human serum albumin (HSA) is the most studied one as it is the main transport protein and can bind a wide variety of ligands especially fatty acids (FAs). The concentration of FAs bound to HSA in human blood plasma differs by three times under abnormal conditions (fasting, physical exercises or in case of social important diseases). In the present study a surfactant sodium dodecyl sulfate (SDS) was used to simulate FAs binding to HSA. It was shown that the increase of Tyr fluorescence of human blood plasma due to SDS addition can be completely explained by HSA-SDS complex formation. Binding parameters of SDS-HSA complex (average number of sites and apparent constant of complex formation) were determined from titration curves based on tyrosine (Tyr) fluorescence.

  20. Probing the binding of an endocrine disrupting compound-Bisphenol F to human serum albumin: Insights into the interactions of harmful chemicals with functional biomacromolecules

    NASA Astrophysics Data System (ADS)

    Pan, Fang; Xu, Tianci; Yang, Lijun; Jiang, Xiaoqing; Zhang, Lei

    2014-11-01

    Bisphenol F (BPF) as an endocrine disrupting compounds (EDCs) pollutant in the environment poses a great threat to human health. To evaluate the toxicity of BPF at the protein level, the effects of BPF on human serum albumin (HSA) were investigated at three temperatures 283, 298, and 308 K by multiple spectroscopic techniques. The experimental results showed that BPF effectively quenched the intrinsic fluorescence of HSA via static quenching. The number of binding sites, the binding constant, the thermodynamic parameters and the binding subdomain were measured, and indicated that BPF could spontaneously bind with HSA on subdomain IIA through H-bond and van der Waals interactions. Furthermore, the conformation of HSA was demonstrably changed in the presence of BPF. The work provides accurate and full basic data for clarifying the binding mechanisms of BPF with HSA in vivo and is helpful for understanding its effect on protein function during its transportation and distribution in blood.

  1. Photooxidation of Tryptophan and Tyrosine Residues in Human Serum Albumin Sensitized by Pterin: A Model for Globular Protein Photodamage in Skin.

    PubMed

    Reid, Lara O; Roman, Ernesto A; Thomas, Andrés H; Dántola, M Laura

    2016-08-30

    Human serum albumin (HSA) is the most abundant protein in the circulatory system. Oxidized albumin was identified in the skin of patients suffering from vitiligo, a depigmentation disorder in which the protection against ultraviolet (UV) radiation fails because of the lack of melanin. Oxidized pterins, efficient photosensitizers under UV-A irradiation, accumulate in the skin affected by vitiligo. In this work, we have investigated the ability of pterin (Ptr), the parent compound of oxidized pterins, to induce structural and chemical changes in HSA under UV-A irradiation. Our results showed that Ptr is able to photoinduce oxidation of the protein in at least two amino acid residues: tryptophan (Trp) and tyrosine (Tyr). HSA undergoes oligomerization, yielding protein structures whose molecular weight increases with irradiation time. The protein cross-linking, due to the formation of dimers of Tyr, does not significantly affect the secondary and tertiary structures of HSA. Trp is consumed in the photosensitized process, and N-formylkynurenine was identified as one of its oxidation products. The photosensitization of HSA takes place via a purely dynamic process, which involves the triplet excited state of Ptr. The results presented in this work suggest that protein photodamage mediated by endogenous photosensitizers can significantly contribute to the harmful effects of UV-A radiation on the human skin. PMID:27500308

  2. Characterization of the binding of metoprolol tartrate and guaifenesin drugs to human serum albumin and human hemoglobin proteins by fluorescence and circular dichroism spectroscopy.

    PubMed

    Duman, Osman; Tunç, Sibel; Kancı Bozoğlan, Bahar

    2013-07-01

    The interactions of metoprolol tartrate (MPT) and guaifenesin (GF) drugs with human serum albumin (HSA) and human hemoglobin (HMG) proteins at pH 7.4 were studied by fluorescence and circular dichroism (CD) spectroscopy. Drugs quenched the fluorescence spectra of HSA and HMG proteins through a static quenching mechanism. For each protein-drug system, the values of Stern-Volmer quenching constant, bimolecular quenching constant, binding constant and number of binding site on the protein molecules were determined at 288.15, 298.15, 310.15 and 318.15 K. It was found that the binding constants of HSA-MPT and HSA-GF systems were smaller than those of HMG-MPT and HMG-GF systems. For both drugs, the affinity of HMG was much higher than that of HSA. An increase in temperature caused a negative effect on the binding reactions. The number of binding site on blood proteins for MPT and GF drugs was approximately one. Thermodynamic parameters showed that MPT interacted with HSA through electrostatic attraction forces. However, hydrogen bonds and van der Waals forces were the main interaction forces in the formation of HSA-GF, HMG-MPT and HMG-GF complexes. The binding processes between protein and drug molecules were exothermic and spontaneous owing to negative ∆H and ∆G values, respectively. The values of binding distance between protein and drug molecules were calculated from Förster resonance energy transfer theory. It was found from CD analysis that the bindings of MPT and GF drugs to HSA and HMG proteins altered the secondary structure of HSA and HMG proteins. PMID:23471625

  3. Esophagectomy-related thoracic duct injury detected by lymphoscintigraphy with 99mTc-diethylenetriamine pentaacetic acid-human serum albumin: report of a case.

    PubMed

    Tsuda, Yasuo; Morita, Masaru; Saeki, Hiroshi; Ando, Koji; Ida, Satoshi; Kimura, Yasue; Oki, Eiji; Ohga, Takefumi; Kusumoto, Tetsuya; Abe, Koichiro; Baba, Shingo; Isoda, Takuro; Maehara, Yoshihiko

    2015-04-01

    Chylothorax is an uncommon but potentially life-threatening complication of esophagectomy. A 72-year-old man underwent thoracoscopy-assisted subtotal esophagectomy and reconstruction with a gastric tube, through a retrosternal route, after preoperative chemoradiotherapy. Chylothorax was detected after starting enteral feeding on postoperative day (POD) 7. Despite conservative therapy such as fasting, total parenteral nutrition, and octreotide administration, massive fluid drainage continued. On POD 19, lymphoscintigraphy with (99m)Tc-diethylenetriamine pentaacetic acid-human serum albumin (HSA-D) was performed and the site of leakage was detected at the level of the fourth thoracic vertebra. On POD 23, the thoracic duct was ligated, following which the volume of chylothorax decreased. Lymphoscintigraphy 12 days after the reoperation showed no leakage from the thoracic duct. We recommend lymphoscintigraphy with (99m)Tc-HSA-D for locating the chyle leakage site and helping decide about the operative indication.

  4. Human serum albumin supported lipid patterns for the targeted recognition of microspheres coated by membrane based on ss-DNA hybridization

    SciTech Connect

    Zhang Xiaoming; He Qiang; Duan Li; Li Junbai . E-mail: jbli@iccas.ac.cn

    2006-10-27

    Human serum albumin (HSA) patterns have been successfully fabricated for the deposition of lipid bilayer, 1,2-dimyristoyl-sglycerophosphate (DMPA), by making use of the micro-contact printing ({mu}CP) technique and liposome fusion. Confocal laser scanning microscopy (CLSM) results indicate that lipid bilayer has been assembled in HSA patterns with a good stability. Such well-defined lipid patterns formed on HSA surface create possibility to incorporate specific components like channels or receptors for specific recognition. In view of this, microspheres coated with lipid membranes were immobilized in HSA-supported lipid patterns via the hybridization of complementary ss-DNAs. This procedure enables to transfer solid materials to a soft surface through a specific recognition.

  5. Investigation of the binding sites and orientation of caffeine on human serum albumin by surface-enhanced Raman scattering and molecular docking

    NASA Astrophysics Data System (ADS)

    Wang, Weinan; Zhang, Wei; Duan, Yaokai; Jiang, Yong; Zhang, Liangren; Zhao, Bing; Tu, Pengfei

    2013-11-01

    Fluorescence, normal Raman and surface-enhanced Raman scattering (SERS) were introduced to explore the absorptive geometry of caffeine on Human Serum Albumin (HSA) at physiological condition. The molecular docking was also employed to make a better understanding of the interaction between caffeine and HSA as well as to elucidate the detailed information of the major binding site. The results showed that caffeine could bind to HSA via the hydrophobic force of aromatic stacking and the main binding group on caffeine could be the pyrimidine ring. In addition, a consecutive set of changes in the orientation of caffeine molecule had been demonstrated during the process of caffeine binding to HSA, and the primary binding site was considered to be a hydrophobic cavity formed by Leu198, Lys199, Ser202, Phe211, Trp214, Val344, Ser454 and Leu481 in domain II.

  6. Binding studies of L-tryptophan to human serum albumin with nanogold-structured sensor by piezoelectric quartz crystal impedance analysis.

    PubMed

    Long, Yumei; Yao, Shouzhuo; Chen, Jinhua

    2011-12-01

    Nanogold-modified sensor was constructed and applied to study the binding of L-tryptophan to human serum albumin (HSA) in situ by piezoelectric quartz crystal impedance (PQCI) analysis. It was interesting that the as-prepared nanogold modified sensor was more sensitive and biocompatible than bare gold electrode. The frequency changes due to protein adsorption on the nanogold-modified sensor might be described as a sum of two exponential functions and detailed explanation was given. Additionally, the kinetics of the binding process was also investigated. The binding constant (K) and the number of binding site (n) for the binding process without competitor are fitted to be 1.07 x 10(4) (mol l(-1))(-1) s(-1) and 1.13, respectively, and 2.24 x 10(3) (mol l-(1))(-1) s(-1) and 1.18, respectively for the binding process with competitor.

  7. Human serum albumin nanoparticles as an efficient noscapine drug delivery system for potential use in breast cancer: preparation and in vitro analysis.

    PubMed

    Sebak, Safaa; Mirzaei, Maryam; Malhotra, Meenakshi; Kulamarva, Arun; Prakash, Satya

    2010-01-01

    Drug delivery systems such as nanoparticles can provide enhanced efficacy for anticancer agents. Noscapine, a widely used cough suppressant for decades has recently been shown to cause significant inhibition and regression of tumor volumes without any detectable toxicity in cells or tissues. Nanoparticles made of human serum albumin (HSA) represent promising strategy for targeted drug delivery to tumor cells by enhancing the drug's bioavailability and distribution, and reducing the body's response towards drug resistance. In the present study, we report for the first time the incorporation and delivery of noscapine-loaded HSA nanoparticles to tumor cells. The nanoparticles were designed and optimized to achieve a particle size in the range of 150-300 nm with a drug-loading efficiency of 85%-96%. The nanoparticles were evaluated in vitro for their anticancer activity and efficacy on breast cancer cells. PMID:20957217

  8. Study on the Mechanism of Interaction Between Tubeimoside I and Human Serum Albumin at Different Temperatures by Three-Dimensional Fluorescence Spectrum

    NASA Astrophysics Data System (ADS)

    Lin, Xiaogang; Li, Wenchao; Ye, Changbin; Liu, Zhiyuan

    2015-06-01

    Tubeimoside (TBMS), the bulb of Bolbostemma paniculatum (Maxim.) Franquet (Cucurbitaceae), is one of the traditional Chinese medicines often used for the treatment of tumors as well as for detoxication. Tubeimoside I (TBMS I) is one of the main active ingredients of TBMS, the mechanism of action of which remains unknown. Human serum albumin (HSA) is the most abundant carrier protein in blood circulation. Three-dimensional (3D) fluorescence spectra and the excitation-emission matrix of interaction between TBMS I and HSA were measured at different temperatures. The results showed that HSA fluorescence was quenched by TBMS I through a static quenching mechanism. Also, the HSA fluorescence was quenched with the temperature increase from 283 K to 353 K. 3D spectral results revealed the changes in the secondary structure of HSA upon interaction with TBMS I.

  9. Analysis of drug-protein interactions by high-performance affinity chromatography: interactions of sulfonylurea drugs with normal and glycated human serum albumin.

    PubMed

    Matsuda, Ryan; Anguizola, Jeanethe; Hoy, Krina S; Hage, David S

    2015-01-01

    High-performance affinity chromatography (HPAC) is a type of liquid chromatography that has seen growing use as a tool for the study of drug-protein interactions. This report describes how HPAC can be used to provide information on the number of binding sites, equilibrium constants, and changes in binding that can occur during drug-protein interactions. This approach will be illustrated through recent data that have been obtained by HPAC for the binding of sulfonylurea drugs and other solutes to the protein human serum albumin (HSA), and especially to forms of this protein that have been modified by non-enzymatic glycation. The theory and use of both frontal analysis and zonal elution competition studies in such work will be discussed. Various practical aspects of these experiments will be presented, as well as factors to consider in the extension of these methods to other drugs and proteins or additional types of biological interactions. PMID:25749961

  10. Trajectories of Serum Albumin Predict Survival of Peritoneal Dialysis Patients

    PubMed Central

    Chiu, Ping-Fang; Tsai, Chun-Chieh; Wu, Chia-Lin; Yang, Tse-Yen; Liou, Hung-Hsiang; Chen, Hung-Lin; Kor, Chew-Teng; Chang, Chia-Chu; Chang, Horng-Rong

    2016-01-01

    Abstract Although initial serum albumin level is highly associated with overall and cardiovascular mortality in peritoneal dialysis (PD) patients, we consider that the dynamic change and trend of albumin after initiation of PD are also essential. We enrolled patients who received PD for more than 3 months from January 1999 to March 2014. We categorized these patients into 2 groups by the difference in serum albumin level (Δalbumin = difference between peak with initial albumin level = peak albumin level − initial albumin level) after PD. The patients with Δalbumin < 0.2 g/dL (median level) were considered as group A (n, number = 238) and those with Δalbumin ≥ 0.2 g/dL were considered as group B (n = 278). Further, we stratified these patients into quartiles: Q1 Δalbumin < −0.2 g/dL; Q2, −0.2 ≦∼ <0.2 g/dL; Q3, 0.2 ≦∼ <0.6 g/dL; and Q4, ≥0.6 g/dL. Regression analysis was performed to determine the correlation of initial albumin and Δalbumin. Group A patients presented with higher levels of serum albumin (3.71 ± 0.54 vs 3.04 ± 0.55 g/dL; P < 0.001) and hematocrit as well as better initial residual renal function. However, those in group A had lower serum albumin increment and downward-sloped trends after dialysis. In contrast, the albumin trend was upward sloped and the increment of albumin was remarkable in group B, despite the high prevalence of cardiovascular diseases and diabetes. Overtime, group A patients had poorer survival and experienced more frequent and longer hospitalizations. Group Q1 patients with least albumin increment had worst survival. Group Q4 patients with lowest initial albumin also had poor survival. Age, diabetes, cardiovascular diseases, BMI, initial albumin, and Δalbumin could affect patient outcomes independently. Regression analysis showed a better outcome can be obtained if the initial albumin level is at least above 3.15 g/dL. (Initial albumin level

  11. Extraction and purification of recombinant human serum albumin from Pichia pastoris broths using aqueous two-phase system combined with hydrophobic interaction chromatography.

    PubMed

    Dong, Yuesheng; Zhang, Fan; Wang, Zhiming; Du, Li; Hao, Aiyu; Jiang, Bo; Tian, Mingyu; Li, Qiao; Jia, Qian; Wang, Shicong; Xiu, Zhilong

    2012-07-01

    Recombinant human serum albumin (rHSA) is considered as an alternative of human serum albumin and used to treat patients with severe burn, shock or blood loss. However, separation and purification of rHSA are difficult and have become the bottle neck in industrial production. In this study, ethanol/K₂HPO₄ aqueous two-phase system (ATPS) and hydrophobic interaction chromatography (HIC) were integrated to provide a new approach for the extraction and purification of rHSA from high density fermentation broth. Using a 0.01-73 L ATPS scale, the extraction of rHSA from the fermentation broth attained an average recovery of 100.4%. At the same time, 99.8% of cells and 87.2% of polysaccharides as well as some other protein impurities were also removed. The activity of proteinase A in the broth was also remarkably decreased. The purified rHSA appeared as a single band on reduced SDS-PAGE gel, and it had a purity of 99.1% as determined by HPLC. It was essentially identical to the plasma-derived HSA in terms of molecular weight and circular dichroism spectrum. The total recovery of rHSA was 75.2%, which was 1.1-2.0 times higher than that obtained from conventional processes. Residual polysaccharide was reduced to 1.61 μg/mg rHSA and the degree of coloring was lower than that of plasma-derived HSA. The procedure employed in this study has the advantages of simple operation, shorter time, low energy consumption and high yield, and it could produce rHSA with high purity. It is therefore suitable in the production of rHSA and other biological products produced by high-density fermentation. PMID:22658659

  12. Photochemiluminescent detection of antiradical activity. VI. Antioxidant characteristics of human blood plasma, low density lipoprotein, serum albumin and amino acids during in vitro oxidation.

    PubMed

    Popov, I; Lewin, G

    1999-01-01

    During investigation of the mechanism of therapeutic efficacy of UV (254 nm)-irradiated autologous blood, the antioxidant capacity (AC) of plasma, serum albumin, 20 amino acids, uric acid, ascorbic acid and alpha-tocopherol was measured after irradiation in vitro. AC of plasma demonstrated a biphasic time course (decrease to in a minimum (greater zero) followed by an increase). AC of uric acid, ascorbic acid and alpha-tocopherol diminished under irradiation to zero. In contrast, albumin and seven amino acids developed AC following irradiation. During the Cu(+2)-induced oxidation of low-density lipoprotein (LDL), the AC measured in a system for water-soluble substances appeared, after a lag-phase, to be comparable to that of the conjugated diene formation; AC of LDL measured in a system for lipid-soluble substances demonstrated a biphasic course. Our results suggest that: (a) the oxidized products of some amino acids and proteins acquire antiradical properties at the same time that they are being produced; (b) the biphasic character of changes of antioxidant properties of blood plasma during UV irradiation represents the overlapping of two processes: disappearing of antioxidants and acquisition of AC by (lipo)proteins; and (c) the measurement of AC of blood plasma (lipo)proteins can possibly serve to detect the existence and extent of oxidative stress in human organism.

  13. Study of the cross-reaction between rabbit anti-bovine serum albumin antibodies and equine serum albumin

    PubMed Central

    Rangel, H.

    1965-01-01

    Cross-reactions between bovine serum albumin and equine serum albumin were studied using heterologous soluble complexes and specifically purified cross-reacting antibody. Experiments with soluble complexes showed that homologous antigen can displace heterologous antigen specifically bound to antibody but heterologous antigen cannot displace homologous antigen. On gel precipitation tests a specific precipitation resulted when heterologous soluble complex reacted with homologous antigen. By using equine serum albumin conjugated to polyaminopolystyrene the cross-reacting antibodies from anti-bovine serum albumin imune sera could be isolated. These are divalent 7S, γ-globulin antibodies. A figure of cross-reaction was obtained when these purified antibodies were tested by double diffusion in agar with bovine and equine serum albumins. The results obtained both with soluble complexes and with purified antibody support the view that cross-reacting antibody is more avid for the homologous than for the heterologous antigen. ImagesFIG. 3FIG. 4FIG. 5 PMID:14245318

  14. Crystals of Serum Albumin for Use in Genetic Engineering and Rational Drug Design

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C. (Inventor)

    1996-01-01

    Serum albumin crystal forms have been produced which exhibit superior x-ray diffraction quality. The crystals are produced from both recombinant and wild-type human serum albumin, canine, and baboon serum albumin and allow the performance of drug-binding studies as well as genetic engineering studies. The crystals are grown from solutions of polyethylene glycol or ammonium sulphate within prescribed limits during growth times from one to several weeks and include the following space groups: P2(sub 1), C2, P1.

  15. 4-alkoxyethoxy-N-octadecyl-1,8-naphthalimide fluorescent sensor for human serum albumin and other major blood proteins: design, synthesis and solvent effect.

    PubMed

    Wei, Song; Sun, Yang; Tan, Chunlei; Yan, Sen; Guo, Ping; Hu, Xiaoyun; Fan, Jun

    2013-01-01

    A series of 4-alkoxyethoxy-N-octadecyl-1,8-naphthalimides with intense blue fluorescence were designed and synthesized as polarity and spectrofluorimetric probes for the determination of proteins. In solvents of different polarities, the Stokes shifts of two dyes increased with increasing solvent polarity and fluorescence quantum yields decreased significantly, suggesting that electronic transiting from ground to excited states was π-π(*) in character. Dipole moment changes were estimated from solvent-dependent Stokes shift data using a solvatochromic method based on bulk solvent polarity functions and the microscopic solvent polarity parameter (E(T)N). These results were generally consistent with semi-empirical molecular orbital calculations and were found to be quite reliable based on the fact that the correlation of the solvatochromic Stokes shifts with E(T)N was superior to that obtained using bulk solvent polarity functions. Fluorescence data revealed that the fluorescence quenching of human serum albumin (HSA) by dyes was the result of the formation of a Dye-HSA complex. The method was applied to the determination of total proteins (HSA + immunoglobulins) in human serum samples and results were in good agreement with those reported by the research institute.

  16. Effects of serum-isolated vs synthetic bilirubin-albumin complexes on dye-binding methods for estimating serum albumin.

    PubMed

    Ihara, H; Nakamura, H; Aoki, Y; Aoki, T; Yoshida, M

    1991-07-01

    Bush and Reed reported (Clin Chem 1987;33:821-3) that the reaction of albumin with bromcresol purple but not with bromcresol green underestimated the concentration of albumin in synthetically obtained bilirubin-albumin (Bd) by 29%. Their unproven assumption was that chemically synthesized Bd behaved in a manner indistinguishable from the natural Bd in icteric serum. Here we verify that Bd, whether synthetically obtained or isolated from serum, causes an underestimation of albumin in the bromcresol purple but not in the bromcresol green method. The molar ratio of Bd from either source to underestimated albumin approximates 1.0, suggesting that one molecule of Bd would react equivalently to a molecule of albumin in the bromcresol purple method. This underestimation might falsely suggest hypoalbuminemia in patients with increased serum Bd. PMID:1855300

  17. Concentration-dependent reversible self-oligomerization of serum albumins through intermolecular β-sheet formation.

    PubMed

    Bhattacharya, Arpan; Prajapati, Roopali; Chatterjee, Surajit; Mukherjee, Tushar Kanti

    2014-12-16

    Proteins inside a cell remain in highly crowded environments, and this often affects their structure and activity. However, most of the earlier studies involving serum albumins were performed under dilute conditions, which lack biological relevance. The effect of protein-protein interactions on the structure and properties of serum albumins at physiological conditions have not yet been explored. Here, we report for the first time the effect of protein-protein and protein-crowder interactions on the structure and stability of two homologous serum albumins, namely, human serum albumin (HSA) and bovine serum albumin (BSA), at physiological conditions by using spectroscopic techniques and scanning electron microscopy (SEM). Concentration-dependent self-oligomerization and subsequent structural alteration of serum albumins have been explored by means of fluorescence and circular dichroism spectroscopy at pH 7.4. The excitation wavelength (λex) dependence of the intrinsic fluorescence and the corresponding excitation spectra at each emission wavelength indicate the presence of various ground state oligomers of serum albumins in the concentration range 10-150 μM. Circular dichroism and thioflavin T binding assay revealed formation of intermolecular β-sheet rich interfaces at high protein concentration. Excellent correlations have been observed between β-sheet content of both the albumins and fluorescence enhancement of ThT with protein concentrations. SEM images at a concentration of 150 μM revealed large dispersed self-oligomeric states with sizes vary from 330 to 924 nm and 260 to 520 nm for BSA and HSA, respectively. The self-oligomerization of serum albumins is found to be a reversible process; upon dilution, these oligomers dissociate into a native monomeric state. It has also been observed that synthetic macromolecular crowder polyethylene glycol (PEG 200) stabilizes the self-associated state of both the albumins which is contrary to expectations that the

  18. Granulomatous interstitial pneumonia in a miniature swine associated with repeated intravenous injections of Tc-99m human serum albumin: concise communication

    SciTech Connect

    Whinnery, J.E.; Young, J.T.

    1980-03-01

    Albumin lung-scanning agents have a proven high degree of safety, with the only contraindication to their use being allergic hypersensitivity. We have used these agents to investigate the physiologic effects of high G/sub z/ acceleratory forces on pulmonary perfusion using the miniature swine. Multiple doses of human macroaggregated albumin and human-albumin microspheres were given to a miniature swine at various levels of centrifugal acceleration over a 6-wk period. The dosages given were the same per kilogram as those used for routine clinical human studies. The animal subsequently died from a severe granulomatous interstitial pneumonia. The granulomatous lesions suggest that the pathogenesis may have involved a cell-mediated delayed hypersensitivity. This interstitial pneumonia may represent the end point in a chronic hypersensitivity response to the human-albumin lung-scanning agents.

  19. A novel exendin-4 human serum albumin fusion protein, E2HSA, with an extended half-life and good glucoregulatory effect in healthy rhesus monkeys.

    PubMed

    Zhang, Ling; Wang, Lin; Meng, Zhiyun; Gan, Hui; Gu, Ruolan; Wu, Zhuona; Gao, Lei; Zhu, Xiaoxia; Sun, Wenzhong; Li, Jian; Zheng, Ying; Dou, Guifang

    2014-03-01

    Glucagon-like peptide-1 (GLP-1) has attracted considerable research interest in terms of the treatment of type 2 diabetes due to their multiple glucoregulatory functions. However, the short half-life, rapid inactivation by dipeptidyl peptidase-IV (DPP-IV) and excretion, limits the therapeutic potential of the native incretin hormone. Therefore, efforts are being made to develop the long-acting incretin mimetics via modifying its structure. Here we report a novel recombinant exendin-4 human serum albumin fusion protein E2HSA with HSA molecule extends their circulatory half-life in vivo while still retaining exendin-4 biological activity and therapeutic properties. In vitro comparisons of E2HSA and exendin-4 showed similar insulinotropic activity on rat pancreatic islets and GLP-1R-dependent biological activity on RIN-m5F cells, although E2HSA was less potent than exendin-4. E2HSA had a terminal elimation half-life of approximate 54 h in healthy rhesus monkeys. Furthermore, E2HSA could reduce postprandial glucose excursion and control fasting glucose level, dose-dependent suppress food intake. Improvement in glucose-dependent insulin secretion and control serum glucose excursions were observed during hyperglycemic clamp test (18 h) and oral glucose tolerance test (42 h) respectively. Thus the improved physiological characterization of E2HSA make it a new potent anti-diabetic drug for type 2 diabetes therapy.

  20. Subchronic toxicity study in vivo and allergenicity study in vitro for genetically modified rice that expresses pharmaceutical protein (human serum albumin).

    PubMed

    Sheng, Yao; Qi, Xiaozhe; Liu, Yifei; Guo, Mingzhang; Chen, Siyuan; He, Xiaoyun; Huang, Kunlun; Xu, Wentao

    2014-10-01

    Genetically modified (GM) crops that express pharmaceutical proteins have become an important focus of recent genetic engineering research. Food safety assessment is necessary for the commercial development of these crops. Subchronic toxicity study in vivo and allergenicity study in vitro were designed to evaluate the food safety of the rice variety expressing human serum albumin (HSA). Animals were fed rodent diets containing 12.5%, 25.0% and 50.0% GM or non-GM rice for 90 days. The composition analysis of the GM rice demonstrated several significant differences. However, most of the differences remained within the ranges reported in the literature. In the animal study, a range of indexes including clinical observation, feed efficiency, hematology, serum chemistry, organ weights and histopathology were examined. Random changes unrelated to the GM rice exposure, within the range of historical control values and not associated with any signs of illness were observed. The results of heat stability and in vitro digestion of HSA indicated no evidence of potential allergenicity of the protein. Overall, the results of these studies suggest that the GM rice appears to be safe as a dietary ingredient when it is used at up to 50% in the diet on a subchronic basis.

  1. Lapatinib-loaded human serum albumin nanoparticles for the prevention and treatment of triple-negative breast cancer metastasis to the brain

    PubMed Central

    Wan, Xu; Zheng, Xiaoyao; Pang, Xiaoyin; Pang, Zhiqing; Zhao, Jingjing; Zhang, Zheming; Jiang, Tao; Xu, Wei; Zhang, Qizhi; Jiang, Xinguo

    2016-01-01

    Brain metastasis from triple-negative breast cancer (TNBC) has continued to lack effective clinical treatments until present. However, the feature of epidermal growth factor receptor (EGFR) frequently overexpressed in TNBC offers the opportunity to employ lapatinib, a dual-tyrosine kinase inhibitor of human epidermal growth factor receptor-2 (HER2) and EGFR, in the treatment of brain metastasis of TNBC. Unfortunately, the low oral bioavailability of lapatinib and drug efflux by blood-brain barrier have resulted in low drug delivery efficiency into the brain and limited therapeutic effects for patients with brain metastasis in clinical trials. To overcome such disadvantages, we developed lapatinib-loaded human serum albumin (HSA) nanoparticles, named LHNPs, by modified nanoparticle albumin-bound (Nab) technology. LHNPs had a core-shell structure and the new HSA/phosphatidylcholine sheath made LHNPs stable in bloodstream. Compared to free lapatinib, LHNPs could inhibit the adhesion, migration and invasion ability of high brain-metastatic 4T1 cells more effectively in vitro. Tissue distribution following intravenous administration revealed that LHNPs (i.v., 10 mg/kg) achieved increased delivery to the metastatic brain at 5.43 and 4.36 times the levels of Tykerb (p.o., 100 mg/kg) and lapatinib solution (LS, i.v., 10 mg/kg), respectively. Compared to the marketed Tykerb group, LHNPs had markedly better inhibition effects on brain micrometastasis and significantly extended the median survival time of 4T1 brain metastatic mice in consequence. The improved anti-tumor efficacy of LHNPs could be partly ascribed to down-regulating metastasis-related proteins. Therefore, these results clearly indicated that LHNPs could become a promising candidate for clinical applications against brain metastasis of TNBC. PMID:27086917

  2. Serum albumin analysis for type II diabetes detection using surface-enhanced Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Lin, Jinyong; Cao, Gang; Lin, Juqiang; Liu, Nenrong; Liao, Fadian; Ruan, Qiuyong; Wu, Shanshan; Huang, Zufang; Li, Ling; Chen, Rong

    2014-09-01

    Surface-enhanced Raman scattering (SERS) spectroscopy combined with membrane electrophoresis (ME) was firstly employed to detect albumin variation in type II diabetic development. Albumin was first purified from human serum by ME and then mixed with silver nanoparticles to perform SERS spectral analysis. SERS spectra were obtained from blood albumin samples of 20 diabetic patients and 19 healthy volunteers. Subtle but discernible changes in the acquired mean spectra of the two groups were observed. Tentative assignment of albumin SERS bands indicated specific structural changes of albumin molecule with diabetic development. Meanwhile, PCA-LDA diagnostic algorithms were employed to classify the two kinds of albumin SERS spectra, yielding the diagnostic sensitivity of 90% and specificity of 94.7%. The results from this exploratory study demonstrated that the EM-SERS method in combination with multivariate statistical analysis has great potential for the label-free detection of albumin variation for improving type II diabetes screening.

  3. Human serum albumin-coated gold nanoparticles for selective extraction of lysozyme from real-world samples prior to capillary electrophoresis.

    PubMed

    Yeh, Pei-Rong; Tseng, Wei-Lung

    2012-12-14

    This study describes the use of human serum albumin (HSA)-modified gold nanoparticles (HSA-AuNPs) for the selective extraction and enrichment of high-pI protein, lysozyme (Lyz) prior to analysis by capillary electrophoresis (CE) with UV detection. HSA-AuNPs are capable of extracting Lyz from a complex matrix because a HSA capping layer not only stabilizes gold nanoparticles in a high-salt environment but also exhibits strong electrostatic attraction with Lyz under neutral pH condition. Efficient separation of Lyz and other high-pI proteins has been successfully achieved by the filling of cationic polyelectrolyte, poly(diallydimethylammonium chloride) (PDDAC), to the background electrolyte. After capturing Lyz with HSA-AuNPs, PDDAC-filled CE can be directly used for the analysis of the extracted Lyz without the addition of the releasing agent into the extractor. The extraction efficiency relied on the pH of the solution and the concentration of HSA-AuNPs. Under optimal extraction conditions, the limit of detection at a signal-to-noise ratio of 3 for Lyz was down to 8 nM. The combination of HSA-AuNP extraction and PDDAC-filled CE has been applied the analyses of Lyz in hen egg white, human milk, and human tear. Also, this NP-based extraction can be coupled to matrix-assisted desorption/ionization time-of-flight mass spectrometry and sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

  4. Investigation of bovine serum albumin glycation by THz spectroscopy

    NASA Astrophysics Data System (ADS)

    Cherkasova, Olga P.; Nazarov, Maxim M.; Shkurinov, Alexander P.

    2016-04-01

    Protein glycation is accelerated under hyperglycemic conditions resulting to loss in the structure and biological functions of proteins. The transmission THz spectroscopy has been used for measuring of bovine serum albumin glycation dynamics. It was found that amplitude of albumin THz absorption depends on type of sugars and incubation time.

  5. Study on Serum Albumin in Third Trimester of Pregnancy.

    PubMed

    Sufrin, S; Nessa, A; Islam, M T; Das, R K; Rahman, M H

    2015-07-01

    Various hormones can cause marked changes in pregnant woman's appearance. Decreased level of serum albumin occurs in third trimester of pregnancy, which may be associated with increased maternal and infant mortality and morbidity. So, this study was carried out to evaluate and assess the level of serum albumin in third trimester of pregnancy. This cross-sectional study was carried out in the Department of Physiology Mymensingh Medical College, Mymensingh. This study enrolled 100 pregnant women of third trimester of pregnancy and 100 aged matched non-pregnant women from Mymensingh district. In this study serum albumin level in study group were 33.41 ± 4.62gm/l and in control group were 37.09 ± 4.21 gm/l, which was statistically decreased. The lower level of serum albumin in third trimester of pregnancy is the major concern of development of physiological edema during pregnancy and may be associated with pre-eclampsia.

  6. The Five-To-Six-Coordination Transition of Ferric Human Serum Heme-Albumin Is Allosterically-Modulated by Ibuprofen and Warfarin: A Combined XAS and MD Study

    PubMed Central

    Bionducci, Monica; Fanali, Gabriella; Meli, Massimiliano; Colombo, Giorgio; Fasano, Mauro; Ascenzi, Paolo; Mobilio, Settimio

    2014-01-01

    Human serum albumin (HSA) is involved physiologically in heme scavenging; in turn, heme-albumin (HSA-heme-Fe) displays globin-like properties. Here, the allosteric effect of ibuprofen and warfarin on the local atomic structure around the ferric heme-Fe (heme-Fe(III)) atom of HSA-heme-Fe (HSA-heme-Fe(III)) has been probed by Fe-K edge X-ray absorption spectroscopy (XAS). The quantitative analysis of the Fe-K edge extended X-ray absorption fine structure (EXAFS) signals and modeling of the near edge (XANES) spectral features demonstrated that warfarin and ibuprofen binding modify the local structure of the heme-Fe(III). Combined XAS data analysis and targeted molecular dynamics (MD) simulations provided atomic resolution insights of protein structural rearrangements required to accommodate the heme-Fe(III) upon ibuprofen and warfarin binding. In the absence of drugs, the heme-Fe(III) atom is penta-coordinated having distorted 4+1 configuration made by the nitrogen atoms of the porphyrin ring and the oxygen phenoxy atom of the Tyr161 residue. MD simulations show that ibuprofen and warfarin association to the secondary fatty acid (FA) binding site 2 (FA2) induces a reorientation of domain I of HSA-heme-Fe(III), this leads to the redirection of the His146 residue providing an additional bond to the heme-Fe(III) atom, providing the 5+1 configuration. The comparison of Fe-K edge XANES spectra calculated using MD structures with those obtained experimentally confirms the reliability of the proposed structural model. As a whole, combining XAS and MD simulations it has been possible to provide a reliable model of the heme-Fe(III) atom coordination state and to understand the complex allosteric transition occurring in HSA-heme-Fe(III) upon ibuprofen and warfarin binding. PMID:25153171

  7. Human serum albumin-stabilized gold nanoclusters act as an electron transfer bridge supporting specific electrocatalysis of bilirubin useful for biosensing applications.

    PubMed

    Santhosh, Mallesh; Chinnadayyala, Somasekhar R; Singh, Naveen K; Goswami, Pranab

    2016-10-01

    Human serum albumin (HSA)-stabilized Au18 nanoclusters (AuNCs) were synthesized and chemically immobilized on an Indium tin oxide (ITO) plate. The assembly process was characterized by advanced electrochemical and spectroscopic techniques. The bare ITO electrode generated three irreversible oxidation peaks, whereas the HSA-AuNC-modified electrode produced a pair of redox peaks for bilirubin at a formal potential of 0.27V (vs. Ag/AgCl). However, the native HSA protein immobilized on the ITO electrode failed to produce any redox peak for bilirubin. The results indicate that the AuNCs present in HSA act as electron transfer bridge between bilirubin and the ITO plate. Docking studies of AuNC with HSA revealed that the best docked structure of the nanocluster is located around the vicinity of the bilirubin binding site, with an orientation that allows specific oxidation. When the HSA-AuNC-modified electrode was employed for the detection of bilirubin using chronoamperometry at 0.3V (vs. Ag/AgCl), a steady-state current response against bilirubin in the range of 0.2μM to 7μM, with a sensitivity of 0.34μAμM(-1) and limit of detection of 86.32nM at S/N 3, was obtained. The bioelectrode was successfully applied to measure the bilirubin content in spiked serum samples. The results indicate the feasibility of using HSA-AuNC as a biorecognition element for the detection of serum bilirubin levels using an electrochemical technique.

  8. Simultaneous determination of estrogens (ethinylestradiol and norgestimate) concentrations in human and bovine serum albumin by use of fluorescence spectroscopy and multivariate regression analysis.

    PubMed

    Hordge, LaQuana N; McDaniel, Kiara L; Jones, Derick D; Fakayode, Sayo O

    2016-05-15

    The endocrine disruption property of estrogens necessitates the immediate need for effective monitoring and development of analytical protocols for their analyses in biological and human specimens. This study explores the first combined utility of a steady-state fluorescence spectroscopy and multivariate partial-least-square (PLS) regression analysis for the simultaneous determination of two estrogens (17α-ethinylestradiol (EE) and norgestimate (NOR)) concentrations in bovine serum albumin (BSA) and human serum albumin (HSA) samples. The influence of EE and NOR concentrations and temperature on the emission spectra of EE-HSA EE-BSA, NOR-HSA, and NOR-BSA complexes was also investigated. The binding of EE with HSA and BSA resulted in increase in emission characteristics of HSA and BSA and a significant blue spectra shift. In contrast, the interaction of NOR with HSA and BSA quenched the emission characteristics of HSA and BSA. The observed emission spectral shifts preclude the effective use of traditional univariate regression analysis of fluorescent data for the determination of EE and NOR concentrations in HSA and BSA samples. Multivariate partial-least-squares (PLS) regression analysis was utilized to correlate the changes in emission spectra with EE and NOR concentrations in HSA and BSA samples. The figures-of-merit of the developed PLS regression models were excellent, with limits of detection as low as 1.6×10(-8) M for EE and 2.4×10(-7) M for NOR and good linearity (R(2)>0.994985). The PLS models correctly predicted EE and NOR concentrations in independent validation HSA and BSA samples with a root-mean-square-percent-relative-error (RMS%RE) of less than 6.0% at physiological condition. On the contrary, the use of univariate regression resulted in poor predictions of EE and NOR in HSA and BSA samples, with RMS%RE larger than 40% at physiological conditions. High accuracy, low sensitivity, simplicity, low-cost with no prior analyte extraction or separation

  9. Mass Spectrometric Characterization of Human Serum Albumin Adducts Formed with N-Oxidized Metabolites of 2-Amino-1-methylphenylimidazo[4,5-b]pyridine in Human Plasma and Hepatocytes.

    PubMed

    Wang, Yi; Peng, Lijuan; Bellamri, Medjda; Langouët, Sophie; Turesky, Robert J

    2015-05-18

    2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a carcinogenic heterocyclic aromatic amine formed in cooked meats, is metabolically activated to electrophilic intermediates that form covalent adducts with DNA and protein. We previously identified an adduct of PhIP formed at the Cys(34) residue of human serum albumin following reaction of albumin with the genotoxic metabolite 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP). The major adducted peptide recovered from a tryptic/chymotryptic digest was identified as the missed-cleavage peptide LQQC*([SO2PhIP])PFEDHVK, a [cysteine-S-yl-PhIP]-S-dioxide linked adduct. In this investigation, we have characterized the albumin adduction products of N-sulfooxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-sulfooxy-PhIP), which is thought to be a major genotoxic metabolite of PhIP formed in vivo. Targeted and data-dependent scanning methods showed that N-sulfooxy-PhIP adducted to the Cys(34) of albumin in human plasma to form LQQC*([SO2PhIP])PFEDHVK at levels that were 8-10-fold greater than the adduct levels formed with N-(acetyloxy)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-acetoxy-PhIP) or HONH-PhIP. We also discovered that N-sulfooxy-PhIP forms an adduct at the sole tryptophan (Trp(214)) residue of albumin in the sequence AW*([PhIP])AVAR. However, stable adducts of PhIP with albumin were not detected in human hepatocytes. Instead, PhIP and 2-amino-1-methyl-6-(5-hydroxy)phenylimidazo[4,5-b]pyridine (5-HO-PhIP), a solvolysis product of the proposed nitrenium ion of PhIP, were recovered during the proteolysis, suggesting a labile sulfenamide linkage had formed between an N-oxidized intermediate of PhIP and Cys(34) of albumin. A stable adduct was formed at the Tyr(411) residue of albumin in hepatocytes and identified as a deaminated product of PhIP, Y(*[desaminoPhIP])TK, where the 4-HO-tyrosine group bound to the C-2 imidazole atom of PhIP. PMID:25815793

  10. Ghrelin binding to serum albumin and its biological impact.

    PubMed

    Lufrano, Daniela; Trejo, Sebastián A; Llovera, Ramiro E; Salgueiro, Mariano; Fernandez, Gimena; Martínez Damonte, Valentina; González Flecha, F Luis; Raingo, Jesica; Ermácora, Mario R; Perelló, Mario

    2016-11-15

    Ghrelin is an octanoylated peptide hormone that plays a key role in the regulation of the body weight and glucose homeostasis. In plasma, ghrelin circulates bound to larger proteins whose identities are partially established. Here, we used size exclusion chromatography, mass spectrometry and isothermal titration microcalorimetry to show that ghrelin interacts with serum albumin. Furthermore, we found that such interaction displays an estimated dissociation constant (KD) in the micromolar range and involves albumin fatty-acid binding sites as well as the octanoyl moiety of ghrelin. Notably, albumin-ghrelin interaction reduces the spontaneous deacylation of the hormone. Both in vitro experiments-assessing ghrelin ability to inhibit calcium channels-and in vivo studies-evaluating ghrelin orexigenic effects-indicate that the binding to albumin affects the bioactivity of the hormone. In conclusion, our results suggest that ghrelin binds to serum albumin and that this interaction impacts on the biological activity of the hormone. PMID:27431015

  11. Exploring the interaction between Salvia miltiorrhiza and human serum albumin: Insights from herb-drug interaction reports, computational analysis and experimental studies.

    PubMed

    Shao, Xin; Ai, Ni; Xu, Donghang; Fan, Xiaohui

    2016-05-15

    Human serum albumin (HSA) binding is one of important pharmacokinetic properties of drug, which is closely related to in vivo distribution and may ultimately influence its clinical efficacy. Compared to conventional drug, limited information on this transportation process is available for medicinal herbs, which significantly hampers our understanding on their pharmacological effects, particularly when herbs and drug are co-administrated as polytherapy to the ailment. Several lines of evidence suggest the existence of Salvia miltiorrhiza-Warfarin interaction. Since Warfarin is highly HSA bound in the plasma with selectivity to site I, it is critical to evaluate the possibility of HSA-related herb-drug interaction. Herein an integrated approach was employed to analyze the binding of chemicals identified in S. miltiorrhiza to HSA. Molecular docking simulations revealed filtering criteria for HSA site I compounds that include docking score and key molecular determinants for binding. For eight representative ingredients from the herb, their affinity and specificity to HSA site I was measured and confirmed fluorometrically, which helps to improve the knowledge of interaction mechanisms between this herb and HSA. Our results indicated that several compounds in S. miltiorrhiza were capable of decreasing the binding constant of Warfarin to HSA site I significantly, which may increase free drug concentration in vivo, contributing to the herb-drug interaction observed clinically. Furthermore, the significance of HSA mediated herb-drug interactions was further implied by manual mining on the published literatures on S. miltiorrhiza.

  12. Studies on the interaction between promethazine and human serum albumin in the presence of flavonoids by spectroscopic and molecular modeling techniques.

    PubMed

    He, Ling-Ling; Wang, Zhi-Xin; Wang, Yong-Xia; Liu, Xian-Ping; Yang, Yan-Jie; Gao, Yan-Ping; Wang, Xin; Liu, Bin; Wang, Xin

    2016-09-01

    Fluorescence, absorption, time-correlated single photon counting (TCSPC), and circular dichroism (CD) spectroscopic techniques as well as molecular modeling methods were used to study the binding characterization of promethazine (PMT) to human serum albumin (HSA) and the influence of flavonoids, rutin and baicalin, on their affinity. The results indicated that the fluorescence quenching mechanism of HSA by PMT is a static quenching due to the formation of complex. The reaction was spontaneous and mainly mediated by hydrogen bonds and hydrophobic interactions. The binding distance between the tryptophan residue of HSA and PMT is less than 8nm, which indicated that the energy transfer from the tryptophan residue of HSA to PMT occurred. The binding site of PMT on HSA was located in sites I and the presence of PMT can cause the conformational changes of HSA. There was the competitive binding to HSA between PMT and flavonoids because of the overlap of binding sites in HSA. The flavonoids could decrease the association constant and increase the binding distance. In addition, their synergistic effect can further change the conformation of HSA. The decrease in the affinities of PMT binding to HSA in the presence of flavonoids may lead to the increase of free drug in blood, which would affect the transportation or disposition of drug and evoke an adverse or toxic effect. Hence, rationalising dosage and diet regimens should be taken into account in clinical application of PMT.

  13. Spectral and molecular modeling studies on the influence of β-cyclodextrin and its derivatives on aripiprazole-human serum albumin binding.

    PubMed

    Yan, Jin; Wu, Di; Ma, Xiaoli; Wang, Lili; Xu, Kailin; Li, Hui

    2015-10-20

    The binding mechanism of aripiprazole (APZ) with human serum albumin (HSA) in the absence and presence of three cyclodextrins (CyDs) (β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and (2,6-di-O-methyl)-β-cyclodextrin) was studied by fluorescence, ultraviolet-visible absorption, nuclear magnetic resonance, and circular dichroism (CD) spectroscopy. The CD results revealed some degree of recovery of refolding caused by APZ after the addition of CyD. The Stern-Volmer quenching constant and binding constant of the APZ-HSA interaction were smaller in the presence of the three CyDs. The ultraviolet-visible absorption results indicated that APZ formed 1:1 complex with the three CyDs. The (1)H NMR spectra of CyD showed chemical shift and resolution loss of proton after the addition of HSA. Molecular modeling studies showed that both APZ and CyD bind to HSA. The process was initiated through inclusion of free APZ molecules by CyD and the increase in steric hindrance of CyD-HSA binding.

  14. The increased binding affinity of curcumin with human serum albumin in the presence of rutin and baicalin: A potential for drug delivery system

    NASA Astrophysics Data System (ADS)

    Liu, Bing-Mi; Zhang, Jun; Hao, Ai-Jun; Xu, Liang; Wang, Dan; Ji, Hui; Sun, Shi-Jie; Chen, Bo-Qi; Liu, Bin

    2016-02-01

    The impacts of rutin and baicalin on the interaction of curcumin (CU) with human serum albumin (HSA) were investigated by fluorescence and circular dichroism (CD) spectroscopies under imitated physiological conditions. The results showed that the fluorescence quenching of HSA by CU was a simultaneous static and dynamic quenching process, irrespective of the presence or absence of flavonoids. The binding constants between CU and HSA in the absence and presence of rutin and baicalin were 2.268 × 105 M- 1, 3.062 × 105 M- 1, and 3.271 × 105 M- 1, indicating that the binding affinity was increased in the case of two flavonoids. Furthermore, the binding distance determined according to Förster's theory was decreased in the presence of flavonoids. Combined with the fact that flavonoids and CU have the same binding site (site I), it can be concluded that they may simultaneously bind in different regions in site I, and formed a ternary complex of flavonoid-HSA-CU. Meanwhile, the results of fluorescence quenching, CD and three-dimensional fluorescence spectra revealed that flavonoids further strengthened the microenvironmental and conformational changes of HSA induced by CU binding. Therefore, it is possible to develop a novel complex involving CU, flavonoid and HSA for CU delivery. The work may provide some valuable information in terms of improving the poor bioavailabiliy of CU.

  15. Interface potential sensing from adsorption of human serum albumin (HSA) on carbon nanotube (CNT) monitored by zero current potentiometry for HSA determination.

    PubMed

    Wang, Huan; Wu, Yi; Song, Jun-Feng

    2015-10-15

    In this work, the adsorption of human serum albumin (HSA) on the bare multiwall carbon nanotube (MWNT) was investigated by a new electrochemical method, termed as zero current potentiometry. For this, a MWNT strip was prepared by directly adhering MWNTs on the transparent adhesive tape surface. Moreover, when HSA adsorbed onto MWNT at the MWNT/solution interface, an interface potential Ψ yielded. The interface potential Ψ as the zero current potential Ezcp simply related to it was monitored by zero current potentiometry. The relationship between the zero current potential Ezcp, the HSA concentration and others was established in simple stoichiometric relation. Based on this, both the adsorption of HSA on MWNT and the HSA determination can be studied. For the HSA determination, the theoretic conclusion consisted with experimental results. The zero current potential Ezcp was proportional to the HSA concentration in the range of 2.8 × 10(-8) - 3.4 × 10(-7)M with the limit of detection 2 × 10(-8)M. The linear regression equation was Ezcp/V (vs, SCE) = (0.159 ± 0.01) + (0.358 ± 0.02) × 10(6)CHSA (µM). This determination was fast, high sensitive and good selective.

  16. Endocytosis of a mannose-terminated glycoprotein and formaldehyde-treated human serum albumin in liver and kidney cells from fish (Salmo alpinus L.).

    PubMed

    Smedsrud, T; Dannevig, B H; Tolleshaug, H; Berg, T

    1984-01-01

    The uptake and degradation of a mannose-terminated glycoprotein, yeast invertase, in char (Salmo alpinus L.) tissue was studied after intravenously injection of the 125I-labelled protein. 125I-labelled formaldehyde-treated human serum albumin (fHSA) and native HSA was also injected for comparison. Labelled invertase was rapidly cleared from blood and at about the same rate as labelled fHSA (at 8 degrees C). Approximately 50% of the initial concentration remained in blood 15 min after the injection of the ligands. Acid soluble degradation products appeared in the circulation about 60 min after the injection of the proteins. 125I-labelled invertase was recovered in the liver, pronephros and kidney. The clearance of labelled invertase from blood and the uptake in the organs were inhibited by co-injection of excess unlabelled invertase. fHSA was taken up in the pronephros and kidney tissue, while HSA was not taken up in any organs. In vitro degradation of the labelled ligands was studied in isolated pronephros cells, which had taken up the proteins in vivo. The degradation of invertase in isolated cells was partly inhibited by ammonium chloride. Ammonium chloride and chloroquine inhibited degradation of fHSA, but not leupeptin. These results together suggest that invertase and fHSA were taken up in the organs described by the receptor-mediated endocytosis. The degradation was partly or wholly lysosomal. PMID:6500136

  17. Characterization of interactions between methoxatin disodium salt and human serum albumin by pressure-assisted capillary electrophoresis/frontal analysis and circular dichroism spectroscopy.

    PubMed

    Zhao, Lijuan; Chen, Dongying

    2015-01-01

    Pressure-assisted capillary electrophoresis (PACE)/frontal analysis (FA) and circular dichroism spectroscopy were utilized to investigate the interactions between methoxatin disodium salt (PQQ-2Na) and human serum albumin (HSA). With the PACE/FA method, sodium phosphate buffer solution (67 mm, pH 7.4) was used as the background electrolyte. Hydrodynamic injection at 50 mbar for 50 s and external pressure of 50 mbar were applied. The binding constant and the number of primary binding sites to HSA were obtained under fixed concentration of PQQ-2Na (100 µm) and increasing HSA concentration (0~475 µm). The thermodynamic parameters characterized the main acting forces of hydrophobic and electrostatic interactions. The displacement experiments using phenylbutazone and flurbiprofen as ligand markers suggested that the binding site was the Sudlow site I of the HSA molecule. Circular dichroism spectroscopy was further employed to evaluate the conformation changes of HSA under the interaction of PQQ-2Na. This work provides comprehensive information for understanding the interactions between PQQ-2Na and HSA.

  18. Studies on the binding of a carditionic agent to human serum albumin by two-dimensional correlation fluorescence spectroscopy and molecular modeling

    NASA Astrophysics Data System (ADS)

    Wang, Teng; Xiang, Bing-Ren; Li, Yue; Chen, Chang-Yun; Zhou, Xiao-Hua; Wang, Zhi-Mei; Dong, Ying; Wang, Ying; Fang, Hui-Sheng

    2009-03-01

    The binding of a novel carditionic agent (2-amino-4-chloro- N-(4-(6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)benzamide (ACPB)) to human serum albumin (HSA) under physiological conditions has been investigated by using UV/vis absorption, fluorescence, circular dichroism (CD) and Fourier transform infrared (FT-IR) in combination with protein-ligand docking study. It was observed that there was a prominent interaction between ACPB and HSA. The interaction was also confirmed by two-dimensional (2D) correction analysis based on the quenching perturbation on the fluorescence spectra and the order of the response of ACPB and HSA to the quenching perturbation was also elucidated based on Noda's rule. Fluorescence data revealed that the fluorescence quenching was a static quenching process and the binding constants were calculated to be 8.781 × 10 5, 7.310 × 10 5, and 5.358 × 10 5 M -1 at 290, 300, and 310 K, respectively. The thermodynamic parameters were calculated according to the Van't Hoff equation and the binding mode was determined. In addition, the alterations of protein secondary structure were qualitatively and quantitatively determined by the evidence from synchronous fluorescence, CD and FT-IR. Furthermore, docking studies that corroborate our experimental results revealed that the binding sites were located in subdomain IIA of HSA.

  19. A Rapid Study of Botanical Drug-Drug Interaction with Protein by Re-ligand Fishing using Human Serum Albumin-Functionalized Magnetic Nanoparticles.

    PubMed

    Qing, Lin-Sen; Xue, Ying; Ding, Li-Sheng; Liu, Yi-Ming; Liang, Jian; Liao, Xun

    2015-12-01

    A great many active constituents of botanical drugs bind to human serum albumin (HSA) reversibly with a dynamic balance between the free- and bound-forms in blood. The curative or side effect of a drug depends on its free-form level, which is always influenced by other drugs, combined dosed or multi-constituents of botanical drugs. This paper presented a rapid and convenient methodology to investigate the drug-drug interactions with HSA. The interaction of two steroidal saponins, dioscin and pseudo-protodioscin, from a botanical drug was studied for their equilibrium time and equilibrium amount by re-ligand fishing using HSA functionalized magnetic nanoparticles. A clear competitive situation was obtained by this method. The equilibrium was reached soon about 15 s at a ratio of 0.44: 1. Furthermore, the interaction of pseudo-protodioscin to total steroidal saponins from DAXXK was also studied. The operation procedures of this method were faster and more convenient compared with other methods reported. PMID:26882690

  20. Developing Anticancer Copper(II) Pro-drugs Based on the Nature of Cancer Cells and the Human Serum Albumin Carrier IIA Subdomain.

    PubMed

    Gou, Yi; Qi, Jinxu; Ajayi, Joshua-Paul; Zhang, Yao; Zhou, Zuping; Wu, Xiaoyang; Yang, Feng; Liang, Hong

    2015-10-01

    To synergistically enhance the selectivity and efficiency of anticancer copper drugs, we proposed and built a model to develop anticancer copper pro-drugs based on the nature of human serum albumin (HSA) IIA subdomain and cancer cells. Three copper(II) compounds of a 2-hydroxy-1-naphthaldehyde benzoyl hydrazone Schiff-base ligand in the presence pyridine, imidazole, or indazole ligands were synthesized (C1-C3). The structures of three HSA complexes revealed that the Cu compounds bind to the hydrophobic cavity in the HSA IIA subdomain. Among them, the pyridine and imidazole ligands of C1 and C2 are replaced by Lys199, and His242 directly coordinates with Cu(II). The indazole and Br ligands of C3 are replaced by Lys199 and His242, respectively. Compared with the Cu(II) compounds alone, the HSA complexes enhance cytotoxicity in MCF-7 cells approximately 3-5-fold, but do not raise cytotoxicity levels in normal cells in vitro through selectively accumulating in cancer cells to some extent. We find that the HSA complex has a stronger capacity for cell cycle arrest in the G2/M phase of MCF-7 by targeting cyclin-dependent kinase 1 (CDK1) and down-regulating the expression of CDK1 and cyclin B1. Moreover, the HSA complex promotes MCF-7 cell apoptosis possibly through the intrinsic reactive oxygen species (ROS) mediated mitochondrial pathway, accompanied by the regulation of Bcl-2 family proteins.

  1. Analysis of the interactions between human serum albumin/amphiphilic penicillin in different aqueous media: an isothermal titration calorimetry and dynamic light scattering study

    NASA Astrophysics Data System (ADS)

    Barbosa, Silvia; Taboada, Pablo; Mosquera, Victor

    2005-04-01

    The complexation process of the amphiphilic penicillins sodium cloxacillin and sodium dicloxacillin with the protein human serum albumin (HSA) in aqueous buffered solutions of pH 4.5 and 7.4 at 25 °C was investigated through isothermal titration calorimetry (ITC) and dynamic light scattering. ITC experiments were carried out in the very dilute regime and showed that although hydrophobic interactions are the leading forces for complexation, electrostatic interactions also play an important role. The possibility of the formation of hydrogen bonds is also deduced from experimental data. The thermodynamic quantities of the binding mechanism, i.e, the enthalpy, ΔHITCi, entropy, ΔSITCi, Gibbs energy, ΔGITCi, binding constant, KITCi and the number of binding sites, ni, were obtained. The binding was saturable and is characterised by Langmuir adsorption isotherms. From ITC data and following a theoretical model, the number of bound and free penicillin molecules was calculated. From Scatchard plots, KITCi and ni were obtained and compared with those from ITC data. The interaction potential between the HSA-penicillin complexes and their stability were determined at pH 7.4 from the dependence of the diffusion coefficients on protein concentration by application of the DLVO colloidal stability theory. The results indicate decreasing stability of the colloidal dispersion of the drug-protein complexes with increase in the concentration of added drug.

  2. [Study of heart region of interest setting method in the hepatic functional reserve index of (99m)tc-diethylenetriamine pentaacetic Acid-galactosyl human serum albumin].

    PubMed

    Takahashi, Yoshimasa; Akiyama, Masayuki; Saitou, Toru; Kato, Kyoichi; Nakazawa, Yasuo

    2014-08-01

    In this study we analyzed the influence of region of interest (ROI) selection on the uptake ratio of the liver to the liver plus heart at 15 min (LHL15) during (99m)Tc-galactosyl human serum albumin (GSA) scintigraphy and determined the optimal ROI by evaluating the individual effects of different ROIs in the heart on LHL15. Twenty patients were randomly selected from those who had undergone (99m)Tc-GSA scintigraphy GSA between April 2008 and June 2009. The liver body (L/B) ratio, liver uptake 15 min (LU15), and LHL15 were analyzed and compared among the following ROIs: entire heart, both ventricles, right ventricle, and left ventricle. There were significant differences in the L/B ratio and LU15 values among the different ROIs. However, LHL15 showed a tendency to shift toward a normal value when the size of the ROI was small (only the right or left ventricle), resulting in a lack of distinction between normal and abnormal LHL15 values. Furthermore, setting the entire heart as the ROI was difficult and reproducibility was low. Our results suggest that the use of both ventricles as the ROI provides optimal LHL15 values during (99m)Tc-GSA scintigraphy.

  3. Studies on the interaction between promethazine and human serum albumin in the presence of flavonoids by spectroscopic and molecular modeling techniques.

    PubMed

    He, Ling-Ling; Wang, Zhi-Xin; Wang, Yong-Xia; Liu, Xian-Ping; Yang, Yan-Jie; Gao, Yan-Ping; Wang, Xin; Liu, Bin; Wang, Xin

    2016-09-01

    Fluorescence, absorption, time-correlated single photon counting (TCSPC), and circular dichroism (CD) spectroscopic techniques as well as molecular modeling methods were used to study the binding characterization of promethazine (PMT) to human serum albumin (HSA) and the influence of flavonoids, rutin and baicalin, on their affinity. The results indicated that the fluorescence quenching mechanism of HSA by PMT is a static quenching due to the formation of complex. The reaction was spontaneous and mainly mediated by hydrogen bonds and hydrophobic interactions. The binding distance between the tryptophan residue of HSA and PMT is less than 8nm, which indicated that the energy transfer from the tryptophan residue of HSA to PMT occurred. The binding site of PMT on HSA was located in sites I and the presence of PMT can cause the conformational changes of HSA. There was the competitive binding to HSA between PMT and flavonoids because of the overlap of binding sites in HSA. The flavonoids could decrease the association constant and increase the binding distance. In addition, their synergistic effect can further change the conformation of HSA. The decrease in the affinities of PMT binding to HSA in the presence of flavonoids may lead to the increase of free drug in blood, which would affect the transportation or disposition of drug and evoke an adverse or toxic effect. Hence, rationalising dosage and diet regimens should be taken into account in clinical application of PMT. PMID:27315330

  4. New Site(s) of Methylglyoxal-Modified Human Serum Albumin, Identified by Multiple Reaction Monitoring, Alter Warfarin Binding and Prostaglandin Metabolism

    PubMed Central

    Kimzey, Michael J.; Yassine, Hussein N.; Riepel, Brent M.; Tsaprailis, George; Monks, Terrence J.; Lau, Serrine S.

    2010-01-01

    Methylglyoxal (MG) is a biologically reactive byproduct of glucose metabolism levels of which increase in diabetes. MG modification of protein generates neutral hydroimidazolone adducts on arginine residues which can alter functional active sites. We investigated the site-specificity of MG adduction to human serum albumin (HSA) using multiple reaction monitoring (MRM) of 13 MG modified tryptic peptides, each containing an internal arginine. Seven new sites for MG modification (R257 >R209 >R222 >R81 >R485 >R472 >R10) are described. Analysis of MG-treated HSA showed substantial R257 and R410 modification, with MG-modified R257 (at 100 μM MG) in drug site I causing significant inhibition of prostaglandin catalysis. The MG hydroimidazolone (MG-H1) adduct was modeled at R257, and molecular dynamics simulations and affinity docking revealed a decrease of 12.8–16.5 kcal/mol (S and R isomers, respectively) for warfarin binding in drug site I. Taken together, these results suggest that R257 is a likely site for MG modification in vivo, which may have functional consequences for prostaglandin metabolism and drug bioavailability. PMID:20934417

  5. Systematic investigation of the influence of CdTe QDs size on the toxic interaction with human serum albumin by fluorescence quenching method

    NASA Astrophysics Data System (ADS)

    Xiao, Jianbo; Bai, Yalong; Wang, Yuanfeng; Chen, Jingwen; Wei, Xinlin

    2010-06-01

    Quantum dots (QDs) are complementary tools to the organic fluorescent dyes used in biological system. Investigation of QDs biological toxicity has attracted great interest for their depth application. Here, the fluorescence quenching method was used to investigate the influence of CdTe QDs size on the toxic interaction with human serum albumin (HSA). Two aqueous-compatible CdTe QDs with maximum emission of 535 nm (green-emitting QDs, G-QDs, 2.04 nm) and 654 nm (red-emitting QDs, R-QDs, 3.79 nm) were tested. The fluorescence quenching results indicated that the quenching effect of QDs on HSA fluorescence depended on the size and the nature of quenching is not dynamic but probably static, resulting in forming QDs-HSA complexes. The binding constants and the number of binding sites between R-QDs and HSA were higher than those of G-QDs. The results illustrated that the size of CdTe quantum dots affected the affinity for HSA and the increasing size of QDs enhanced the affinity for HSA. The values of lg Ka are proportional to the number of binding sites ( n). This result confirms the method used here is suitable to study the toxic interaction between QDs and HSA.

  6. Analysis of the Interaction of Dp44mT with Human Serum Albumin and Calf Thymus DNA Using Molecular Docking and Spectroscopic Techniques

    PubMed Central

    Xu, Zhongjie; Liu, Youxun; Zhou, Sufeng; Fu, Yun; Li, Changzheng

    2016-01-01

    Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) exhibits significant antitumor activity. However, the mechanism of its pharmacological interaction with human serum albumin (HSA) and DNA remains poorly understood. Here, we aimed to elucidate the interactions of Dp44mT with HSA and DNA using MTT assays, spectroscopic methods, and molecular docking analysis. Our results indicated that addition of HSA at a ratio of 1:1 did not alter the cytotoxicity of Dp44mT, but did affect the cytotoxicity of the Dp44mT-Cu complex. Data from fluorescence quenching and UV-VIS absorbance measurements demonstrated that Dp44mT could bind to HSA with a moderate affinity (Ka = approximately 104 M−1). CD spectra revealed that Dp44mT could slightly disrupt the secondary structure of HSA. Dp44mT could also interact with Ct-DNA, but had a moderate binding constant (KEB = approximately 104 M−1). Docking studies indicated that the IB site of HSA, but not the IIA and IIIA sites, could be favorable for Dp44mT and that binding of Dp44mT to HSA involved hydrogen bonds and hydrophobic force, consistent with thermodynamic results from spectral investigations. Thus, the moderate binding affinity of Dp44mT with HSA and DNA partially contributed to its antitumor activity and may be preferable in drug design approaches. PMID:27376275

  7. Investigation of the effects of Zn2+, Ca2+ and Na+ ions on the interaction between zonisamide and human serum albumin (HSA) by spectroscopic methods

    NASA Astrophysics Data System (ADS)

    Shahabadi, Nahid; Khorshidi, Aref; Mohammadpour, Mahnaz

    2014-03-01

    In this work, The effect of three metal ions Zn2+, Ca2+ and Na1+ on the interaction between human serum albumin (HSA) and zonisamide (ZNS) was investigated employing fluorescence, ultraviolet-visible (UV-Vis) absorption and circular dichroism (CD) under simulated physiological conditions. Fluorescence spectroscopy revealed that these metallic ions and ZNS can quench the HSA fluorescence, and this quenching effect became more significant when both ion and drug are present together. It was found that the quenching mechanism is a combination of static quenching with nonradiative energy transfer. The binding sites number (n), the binding constant (Kb) and the spatial-distance (r) of ZNS with HSA without or with Zn2+, Ca2+ and Na1+ ions were calculated. The presence of Ca2+ and Na+ ions decreased the binding constants (Kb) and the number of binding sites (n) of ZNS-HSA complex. However, the presence of Zn2+ increased the affinity of ZNS for HSA largely. Changes in UV-Vis absorption and CD data are due to the microenvironment of amide moieties in HSA molecules.

  8. Protection against influenza A virus by vaccination with a recombinant fusion protein linking influenza M2e to human serum albumin (HSA).

    PubMed

    Mu, Xupeng; Hu, Kebang; Shen, Mohan; Kong, Ning; Fu, Changhao; Yan, Weiqun; Wei, Anhui

    2016-02-01

    The highly conserved extracellular domain of M2 protein (M2e) of influenza A viruses has limited immunogenicity on its own. Hence, aiming to enhance immunogenicity of M2e protein, optimal approaches remain to be established. In this study, we created recombinant fusion protein vaccines by linking M2e consensus sequence of influenza A viruses with C-terminal domain of human serum albumin (HSA). Then HSA/M2e recombinant fusion protein was studied. Our results showed that HSA/M2e could induce strong anti-M2e specific humoral immune responses in the established mice model. Administration of HSA/M2e with Freund's adjuvant resulted in a higher number of IFN-γ-producing cells compared to HSA/M2e or M2e peptide emulsified in Freund's adjuvant. Furthermore, HSA/M2e was able to reduce viral load in the mice lungs and provide significant protection against lethal challenge with an H1N1 or an H3N2 virus compared to controls. In conclusion, this study has demonstrated a potential vaccine that could provide protection in preventing the threat of influenza outbreak because of rapid variation of the influenza virus.

  9. ANALYSIS OF DRUG INTERACTIONS WITH MODIFIED PROTEINS BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY: BINDING OF GLIBENCLAMIDE TO NORMAL AND GLYCATED HUMAN SERUM ALBUMIN

    PubMed Central

    Matsuda, Ryan; Anguizola, Jeanethe; Joseph, K.S.; Hage, David S.

    2012-01-01

    High-performance affinity chromatography (HPAC) was used to examine the changes in binding that occur for the sulfonylurea drug glibenclamide with human serum albumin (HSA) at various stages of glycation for HSA. Frontal analysis on columns containing normal HSA or glycated HSA indicated glibenclamide was interacting through both high affinity sites (association equilibrium constant, Ka, 1.4–1.9 × 106 M−1 at pH 7.4 and 37°C) and lower affinity sites (Ka, 4.4–7.2 × 104 M−1). Competition studies were used to examine the effect of glycation at specific binding sites of HSA. An increase in affinity of 1.7- to 1.9-fold was seen at Sudlow site I with moderate to high levels of glycation. An even larger increase of 4.3- to 6.0-fold in affinity was noted at Sudlow site II for all of the tested samples of glycated HSA. A slight decrease in affinity may have occurred at the digitoxin site, but this change was not significant for any individual glycated HSA sample. These results illustrate how HPAC can be used as tool for examining the interactions of relatively non-polar drugs like glibenclamide with modified proteins and should lead to a more complete understanding of how glycation can alter the binding of drugs in blood. PMID:23092871

  10. CHARACTERIZATION OF INTERACTION KINETICS BETWEEN CHIRAL SOLUTES AND HUMAN SERUM ALBUMIN BY USING HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY AND PEAK PROFILING

    PubMed Central

    Tong, Zenghan; Hage, David S.

    2011-01-01

    Peak profiling and high-performance columns containing immobilized human serum albumin (HSA) were used to study the interaction kinetics of chiral solutes with this protein. This approach was tested using the phenytoin metabolites 5-(3-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) as model analytes. HSA columns provided some resolution of the enantiomers for each phenytoin metabolite, which made it possible to simultaneously conduct kinetic studies on each chiral form. The dissociation rate constants for these interactions were determined by using both the single flow rate and multiple flow rate peak profiling methods. Corrections for non-specific interactions with the support were also considered. The final estimates obtained at pH 7.4 and 37°C for the dissociation rate constants of these interactions were 8.2–9.6 s−1 for the two enantiomers of m-HPPH and 3.2–4.1 s−1 for the enantiomers of p-HPPH. These rate constants agreed with previous values that have been reported for other drugs and solutes that have similar affinities and binding regions on HSA. The approach used in this report was not limited to phenytoin metabolites or HSA but could be applied to a variety of other chiral solutes and proteins. This method could also be adopted for use in the rapid screening of drug-protein interactions. PMID:21872871

  11. NMR metabolomics profiling of blood plasma mimics shows that medium- and long-chain fatty acids differently release metabolites from human serum albumin

    NASA Astrophysics Data System (ADS)

    Jupin, M.; Michiels, P. J.; Girard, F. C.; Spraul, M.; Wijmenga, S. S.

    2014-02-01

    Metabolite profiling by NMR of body fluids is increasingly used to successfully differentiate patients from healthy individuals. Metabolites and their concentrations are direct reporters of body biochemistry. However, in blood plasma the NMR-detected free-metabolite concentrations are also strongly affected by interactions with the abundant plasma proteins, which have as of yet not been considered much in metabolic profiling. We previously reported that many of the common NMR-detected metabolites in blood plasma bind to human serum albumin (HSA) and many are released by fatty acids present in fatted HSA. HSA is the most abundant plasma protein and main transporter of endogenous and exogenous metabolites. Here, we show by NMR how the two most common fatty acids (FAs) in blood plasma - the long-chain FA, stearate (C18:0) and medium-chain FA, myristate (C14:0) - affect metabolite-HSA interaction. Of the set of 18 common NMR-detected metabolites, many are released by stearate and/or myristate, lactate appearing the most strongly affected. Myristate, but not stearate, reduces HSA-binding of phenylalanine and pyruvate. Citrate signals were NMR invisible in the presence of HSA. Only at high myristate-HSA mole ratios 11:1, is citrate sufficiently released to be detected. Finally, we find that limited dilution of blood-plasma mimics releases HSA-bound metabolites, a finding confirmed in real blood plasma samples. Based on these findings, we provide recommendations for NMR experiments for quantitative metabolite profiling.

  12. Exploring the interaction between Salvia miltiorrhiza and human serum albumin: Insights from herb-drug interaction reports, computational analysis and experimental studies

    NASA Astrophysics Data System (ADS)

    Shao, Xin; Ai, Ni; Xu, Donghang; Fan, Xiaohui

    2016-05-01

    Human serum albumin (HSA) binding is one of important pharmacokinetic properties of drug, which is closely related to in vivo distribution and may ultimately influence its clinical efficacy. Compared to conventional drug, limited information on this transportation process is available for medicinal herbs, which significantly hampers our understanding on their pharmacological effects, particularly when herbs and drug are co-administrated as polytherapy to the ailment. Several lines of evidence suggest the existence of Salvia miltiorrhiza-Warfarin interaction. Since Warfarin is highly HSA bound in the plasma with selectivity to site I, it is critical to evaluate the possibility of HSA-related herb-drug interaction. Herein an integrated approach was employed to analyze the binding of chemicals identified in S. miltiorrhiza to HSA. Molecular docking simulations revealed filtering criteria for HSA site I compounds that include docking score and key molecular determinants for binding. For eight representative ingredients from the herb, their affinity and specificity to HSA site I was measured and confirmed fluorometrically, which helps to improve the knowledge of interaction mechanisms between this herb and HSA. Our results indicated that several compounds in S. miltiorrhiza were capable of decreasing the binding constant of Warfarin to HSA site I significantly, which may increase free drug concentration in vivo, contributing to the herb-drug interaction observed clinically. Furthermore, the significance of HSA mediated herb-drug interactions was further implied by manual mining on the published literatures on S. miltiorrhiza.

  13. A toxic organic solvent-free technology for the preparation of PEGylated paclitaxel nanosuspension based on human serum albumin for effective cancer therapy

    PubMed Central

    Yin, Tingjie; Dong, Lihui; Cui, Bei; Wang, Lei; Yin, Lifang; Zhou, Jianping; Huo, Meirong

    2015-01-01

    Clinically, paclitaxel (PTX) is one of most commonly prescribed therapies against a wide range of solid neoplasms. Despite its success, the clinical applicability of PTX (Taxol®) is severely hampered by systemic toxicities induced by Cremophor EL. While attempts to bypass the need for Cremophor EL have been developed through platforms such as Abraxane™, nab™ relies heavily on the use of organic solvents, namely, chloroform. The toxicity introduced by residual chloroform poses a potential risk to patient health. To mitigate the toxicities of toxic organic solvent-based manufacture methods, we have designed a method for the formulation of PTX nanosuspensions (PTX-PEG [polyethylene glycol]-HSA [human serum albumin]) that eliminates the dependence on toxic organic solvents. Coined the solid-dispersion technology, this technique permits the dispersion of PTX into PEG skeleton without the use of organic solvents or Cremophor EL as a solubilizer. Once the PTX-PEG dispersion is complete, the dispersion can be formulated with HSA into nanosuspensions suitable for intravenous administration. Additionally, the incorporation of PEG permits the prolonged circulation through the steric stabilization effect. Finally, HSA-mediated targeting permits active receptor-mediated endocytosis for enhanced tumor uptake and reduced side effects. By eliminating the need for both Cremophor EL and organic solvents while simultaneously increasing antitumor efficacy, this method provides a superior alternative to currently accepted methods for PTX delivery. PMID:26715846

  14. Enhanced Anti-Tumoral Activity of Methotrexate-Human Serum Albumin Conjugated Nanoparticles by Targeting with Luteinizing Hormone-Releasing Hormone (LHRH) Peptide

    PubMed Central

    Taheri, Azade; Dinarvand, Rassoul; Atyabi, Fatemeh; Ahadi, Fatemeh; Nouri, Farank Salman; Ghahremani, Mohammad Hossein; Ostad, Seyed Nasser; Borougeni, Atefeh Taheri; Mansoori, Pooria

    2011-01-01

    Active targeting could increase the efficacy of anticancer drugs. Methotrexate-human serum albumin (MTX-HSA) conjugates, functionalized by luteinizing hormone-releasing hormone (LHRH) as targeting moieties, with the aim of specifically targeting the cancer cells, were prepared. Owing to the high expression of LHRH receptors in many cancer cells as compared to normal cells, LHRH was used as the targeting ligand in this study. LHRH was conjugated to MTX-HSA nanoparticles via a cross-linker. Three types of LHRH targeted nanoparticles with a mean particle size between 120–138 nm were prepared. The cytotoxicity of LHRH targeted and non-targeted nanoparticles were determined on the LHRH positive and negative cell lines. The internalization of the targeted and non-targeted nanoparticles in LHRH receptor positive and negative cells was investigated using flow cytometry analysis and fluorescence microscopy. The cytotoxicity of the LHRH targeted nanoparticles on the LHRH receptor positive cells were significantly more than non-targeted nanoparticles. LHRH targeted nanoparticles were also internalized by LHRH receptor positive cells significantly more than non-targeted nanoparticles. There were no significant differences between the uptake of targeted and non-targeted nanoparticles to the LHRH receptor negative cells. The active targeting procedure using LHRH targeted MTX-HSA nanoparticles could increase the anti-tumoral activity of MTX. PMID:21845098

  15. Enhanced laser thermal ablation for the in vitro treatment of liver cancer by specific delivery of multiwalled carbon nanotubes functionalized with human serum albumin.

    PubMed

    Iancu, Cornel; Mocan, Lucian; Bele, Constantin; Orza, Anamaria Ioana; Tabaran, Flaviu A; Catoi, Cornel; Stiufiuc, Rares; Stir, Ariana; Matea, Cristian; Iancu, Dana; Agoston-Coldea, Lucia; Zaharie, Florin; Mocan, Teodora

    2011-01-17

    The main goal of this investigation was to develop and test a new method of treatment for human hepatocellular carcinoma (HCC). We present a method of carbon nanotube-enhanced laser thermal ablation of HepG2 cells (human hepatocellular liver carcinoma cell line) based on a simple multiwalled carbon nanotube (MWCNT) carrier system, such as human serum albumin (HSA), and demonstrate its selective therapeutic efficacy compared with normal hepatocyte cells. Both HepG2 cells and hepatocytes were treated with HSA-MWCNTs at various concentrations and at various incubation times and further irradiated using a 2 W, 808 nm laser beam. Transmission electron, phase contrast, and confocal microscopy combined with immunochemical staining were used to demonstrate the selective internalization of HSA-MWCNTs via Gp60 receptors and the caveolin-mediated endocytosis inside HepG2 cells. The postirradiation apoptotic rate of HepG2 cells treated with HSA-MWCNTs ranged from 88.24% (for 50 mg/L) at 60 sec to 92.34% (for 50 mg/L) at 30 min. Significantly lower necrotic rates were obtained when human hepatocytes were treated with HSA-MWCNTs in a similar manner. Our results clearly show that HSA-MWCNTs selectively attach on the albondin (aka Gp60) receptor located on the HepG2 membrane, followed by an uptake through a caveolin-dependent endocytosis process. These unique results may represent a major step in liver cancer treatment using nanolocalized thermal ablation by laser heating.

  16. Determination of Supplier-to-Supplier and Lot-to-Lot Variability in Glycation of Recombinant Human Serum Albumin Expressed in Oryza sativa

    PubMed Central

    Frahm, Grant E.; Smith, Daryl G. S.; Kane, Anita; Lorbetskie, Barry; Cyr, Terry D.; Girard, Michel; Johnston, Michael J. W.

    2014-01-01

    The use of different expression systems to produce the same recombinant human protein can result in expression-dependent chemical modifications (CMs) leading to variability of structure, stability and immunogenicity. Of particular interest are recombinant human proteins expressed in plant-based systems, which have shown particularly high CM variability. In studies presented here, recombinant human serum albumins (rHSA) produced in Oryza sativa (Asian rice) (OsrHSA) from a number of suppliers have been extensively characterized and compared to plasma-derived HSA (pHSA) and rHSA expressed in yeast (Pichia pastoris and Saccharomyces cerevisiae). The heterogeneity of each sample was evaluated using size exclusion chromatography (SEC), reversed-phase high-performance liquid chromatography (RP-HPLC) and capillary electrophoresis (CE). Modifications of the samples were identified by liquid chromatography-mass spectrometry (LC-MS). The secondary and tertiary structure of the albumin samples were assessed with far U/V circular dichroism spectropolarimetry (far U/V CD) and fluorescence spectroscopy, respectively. Far U/V CD and fluorescence analyses were also used to assess thermal stability and drug binding. High molecular weight aggregates in OsrHSA samples were detected with SEC and supplier-to-supplier variability and, more critically, lot-to-lot variability in one manufactures supplied products were identified. LC-MS analysis identified a greater number of hexose-glycated arginine and lysine residues on OsrHSA compared to pHSA or rHSA expressed in yeast. This analysis also showed supplier-to-supplier and lot-to-lot variability in the degree of glycation at specific lysine and arginine residues for OsrHSA. Both the number of glycated residues and the degree of glycation correlated positively with the quantity of non-monomeric species and the chromatographic profiles of the samples. Tertiary structural changes were observed for most OsrHSA samples which correlated well

  17. Study of interactions between oppositely charged dendrigraft poly-L-lysine and human serum albumin by continuous frontal analysis capillary electrophoresis and fluorescence spectroscopy.

    PubMed

    Sisavath, Nicolas; Leclercq, Laurent; Le Saux, Thomas; Oukacine, Farid; Cottet, Hervé

    2013-05-10

    Dendrigraft poly-L-lysine (DGL) are biomacromolecules of great interest for many applications including antibacterial activity, drug delivery systems, gene therapy and production of antibodies. As human serum albumin (HSA) is the most abundant serum protein, the study of interactions between these two compounds is crucial for the use of DGL in drug or gene delivery systems. The present work aims at determining the number of binding sites and the corresponding successive equilibrium constants between DGL of generation 3 (G3) and HSA in physiological conditions. To meet this end, continuous frontal analysis capillary electrophoresis (FACCE) and fluorescence spectroscopic methods were implemented and compared. FACCE was performed on a polycationic modified capillary in combination with a co-pressure that allowed for selectively introducing the free G3 from the G3/HSA mixtures. FACCE studies demonstrated that HSA has 2 binding sites with DGL G3 with the following successive constants K1=31.2×10(3) M(-1) and K2=30.6×10(3) M(-1). For a 1 g/L concentration in G3 and assuming a plasmatic HSA concentration of 40g/L, these binding constants lead to only 5% free DGL in the medium. It was also shown that the interactions between G3 and HSA corresponded to a model of cooperative sites. These results are in good agreement with the presence of two negatively charged domains in the HSA. Good fitting of the fluorescence spectroscopy data was obtained using the equilibrium constants derived from FACCE. Nevertheless, due to the high number of fitting parameters, it was difficult to fit the fluorescence spectroscopic data independently of the results obtained by FACCE.

  18. Investigation of the binding of cis/trans-[MCl4(1H-indazole)(NO)](-) (M = Ru, Os) complexes to human serum albumin.

    PubMed

    Dömötör, Orsolya; Rathgeb, Anna; Kuhn, Paul-Steffen; Popović-Bijelić, Ana; Bačić, Goran; Enyedy, Eva Anna; Arion, Vladimir B

    2016-06-01

    Overall binding affinity of sodium or indazolium cis/trans-[MCl4(1H-indazole)(NO)] (M = Ru, Os) complexes towards human serum albumin (HSA) and high molecular mass components of the blood serum was monitored by ultrafiltration. HSA was found to be mainly responsible for the binding of the studied ruthenium and osmium complexes. In other words, this protein can provide a depot for the compounds and can affect their biodistribution and transport processes. In order to elucidate the HSA binding sites tryptophan fluorescence quenching studies and displacement reactions with the established site markers warfarin and dansylglycine were performed. Conditional stability constants for the binding to sites I and II on HSA were computed showing that the studied ruthenium and osmium complexes are able to bind into both sites with moderately strong affinity (logK' = 4.4-5.1). Site I is slightly more favored over site II for all complexes. No significant differences in the HSA binding properties were found for these metal complexes demonstrating negligible influence of the type of counterion (sodium vs indazolium), the metal ion center identity (Ru vs. Os) or the position of the nitrosyl group on the binding event. Electron paramagnetic resonance spin labeling of HSA revealed that indazolium trans-[RuCl4(1H-indazole)(NO)] and long-chain fatty acids show competitive binding to HSA. Moreover, this complex has a higher affinity for site I, but when present in excess, it is able to bind to site II as well, and displace fatty acids. PMID:26908285

  19. A novel exendin-4 human serum albumin fusion protein, E2HSA, with an extended half-life and good glucoregulatory effect in healthy rhesus monkeys

    SciTech Connect

    Zhang, Ling; Wang, Lin; Meng, Zhiyun; Gan, Hui; Gu, Ruolan; Wu, Zhuona; Gao, Lei; Zhu, Xiaoxia; Sun, Wenzhong; Li, Jian; Zheng, Ying; Dou, Guifang

    2014-03-07

    Highlights: • E2HSA has an extended half-life and good plasma stability. • E2HSA could improve glucose-dependent insulin secretion. • E2HSA has excellent glucoregulatory effects in vivo. • E2HSA could potentially be used as a new long-acting GLP-1 receptor agonist for type 2 diabetes management. - Abstract: Glucagon-like peptide-1 (GLP-1) has attracted considerable research interest in terms of the treatment of type 2 diabetes due to their multiple glucoregulatory functions. However, the short half-life, rapid inactivation by dipeptidyl peptidase-IV (DPP-IV) and excretion, limits the therapeutic potential of the native incretin hormone. Therefore, efforts are being made to develop the long-acting incretin mimetics via modifying its structure. Here we report a novel recombinant exendin-4 human serum albumin fusion protein E2HSA with HSA molecule extends their circulatory half-life in vivo while still retaining exendin-4 biological activity and therapeutic properties. In vitro comparisons of E2HSA and exendin-4 showed similar insulinotropic activity on rat pancreatic islets and GLP-1R-dependent biological activity on RIN-m5F cells, although E2HSA was less potent than exendin-4. E2HSA had a terminal elimation half-life of approximate 54 h in healthy rhesus monkeys. Furthermore, E2HSA could reduce postprandial glucose excursion and control fasting glucose level, dose-dependent suppress food intake. Improvement in glucose-dependent insulin secretion and control serum glucose excursions were observed during hyperglycemic clamp test (18 h) and oral glucose tolerance test (42 h) respectively. Thus the improved physiological characterization of E2HSA make it a new potent anti-diabetic drug for type 2 diabetes therapy.

  20. An aqueous extract from toad skin prevents gelatinase activities derived from fetal serum albumin and serum-free culture medium of human breast carcinoma MDA-MB-231 cells.

    PubMed

    Nakata, Munehiro; Kawaguchi, Shota; Oikawa, Ayami; Inamura, Akito; Nomoto, Shunki; Miyai, Hirokazu; Nonaka, Tomomi; Ichimi, Saeko; Fujita-Yamaguchi, Yoko; Luo, Chuan; Gao, Bo; Tang, Wei

    2015-12-01

    An aqueous extract from toad skin, cinobufacini, has been known to possess anticancer ability. The present study examined effect of toad skin extract on activity of gelatinases including matrix metalloproteinases-2 and -9 which play an important role in invasion of carcinoma cells. Gelatinase activities derived from fetal serum albumin and culture medium of human breast carcinoma cell line MDA-MB-231 were significantly prevented in the presence of toad skin extract. The inhibitory activity was found in water-soluble fraction of the extract prepared by the Bligh & Dyer method but not in CHCl(3)-soluble lipid fraction. These results suggest that an aqueous extract from toad skin contains a water-soluble substance possessing a potent ability to prevent gelatinase activity. In conclusion, the water-soluble substance in toad skin extract cinobufacini may be able to regulate cancer cell migration accelerated by matrix metalloproteinases.

  1. Complexation of serum albumins and triton X-100: Quenching of tryptophan fluorescence and analysis of the rotational diffusion of complexes

    NASA Astrophysics Data System (ADS)

    Vlasova, I. M.; Vlasov, A. A.; Saletskii, A. M.

    2016-07-01

    The polarized and nonpolarized fluorescence of bovine serum albumin and human serum albumin in Triton X-100 solutions is studied at different pH values. Analysis of the constants of fluorescence quenching for BSA and HSA after adding Triton X-100 and the hydrodynamic radii of BSA/HSA-detergent complexes show that the most effective complexation between both serum albumins and Triton X-100 occurs at pH 5.0, which lies near the isoelectric points of the proteins. Complexation between albumin and Triton X-100 affects the fluorescence of the Trp-214 residing in the hydrophobic pockets of both BSA and HSA.

  2. Hyperglycemia induced structural and functional changes in human serum albumin of diabetic patients: a physico-chemical study.

    PubMed

    Neelofar, Km; Arif, Zarina; Alam, Khursheed; Ahmad, Jamal

    2016-07-19

    Structural and functional changes in albumin are of particular interest as numerous studies in vivo have reported a strong involvement of glycated-HSA in the development and progression of chronic diabetic complications. Non-enzymatic addition of glucose molecules to a protein induces structural changes in it. These changes depend on the degree of glycation. In this study, conformational changes in glycated-HSA and its antioxidant capacity were evaluated. HSA was purified from diabetic patients with/without CKD and healthy subjects. Glycation induced an increase in the molecular mass of HSA as determined by mass spectroscopy. Further secondary and tertiary structural changes were observed by UV, circular dichroism (CD) spectroscopy, Fourier transform infrared spectroscopy (FTIR), tryptophan and 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence. The mean α-helix content was found to be 59.46% for normal HSA and it was reduced down to 45.63% in HSA isolated from diabetic patients without CKD and to 37.48% in CKD-HSA. FTIR analysis showed Amide I and Amide II band shifting in HSA of diabetic patients without and with CKD. These findings indicate the secondary structure changes in glycated HSA. The tertiary structure is also affected by in vivo glycation as confirmed by intrinsic fluorescence and ANS fluorescence results. Consequently, these structural changes associated with glycation provoked a reduction in the free thiol group and a strong increment of protein carbonyl contents and the fructosamine level in glycated HSA. Antioxidant activity was evaluated by a RBC hemolysis test. The result indicates that the free radical scavenging capacities of HSA were decreased in diabetic patients with or without CKD. Our study revealed that structural and functional features of glycated HSA, isolated from diabetic patients with and without CKD were significantly different from the HSA isolated from non-diabetic subjects. Moreover these changes were more prominent in HSA

  3. Serum Albumin Levels and Economic Status in Japanese Older Adults

    PubMed Central

    Ota, Asami; Kondo, Naoki; Murayama, Nobuko; Tanabe, Naohito; Shobugawa, Yugo; Kondo, Katsunori

    2016-01-01

    Background Low serum albumin levels are associated with aging and medical conditions such as cancer, liver dysfunction, inflammation, and malnutrition and might be an independent predictor of long-term mortality in healthy older populations. We tested the hypothesis that economic status is associated with serum albumin levels and explained by nutritional and health status in Japanese older adults. Design We performed a cross-sectional analysis using data from the Japan Gerontological Evaluation study (JAGES). The study participants were 6528 functionally independent residents (3189 men and 3339 women) aged ≥65 years living in four municipalities in Aichi prefecture. We used household income as an indicator of economic status. Multiple linear regression was used to compare serum albumin levels in relation to household income, which was classified as low, middle, and high. Additionally, mediation by nutritional and health-related factors was analyzed in multivariable models. Results With the middle-income group as reference, participants with low incomes had a significantly lower serum albumin level, even after adjustment for sex, age, residential area, education, marital status, and household structure. The estimated mean difference was −0.17 g/L (95% confidence interval, −0.33 to −0.01 g/L). The relation between serum albumin level and low income became statistically insignificant when “body mass index”, “consumption of meat or fish”, “self-rated health”, “presence of medical conditions”, “hyperlipidemia”, or “respiratory disease “was included in the model. Conclusion Serum albumin levels were lower in Japanese older adults with low economic status. The decrease in albumin levels appears to be mediated by nutrition and health-related factors with low household incomes. Future studies are needed to reveal the existence of other pathways. PMID:27276092

  4. Raman, SERS, and induced circular dichroism techniques as a probe of pharmaceuticals in their interactions with the human serum albumin and p-glycoprotein

    NASA Astrophysics Data System (ADS)

    Fleury, Fabrice; Ianoul, Anatoli I.; Baggetto, Loris; Jardillier, Jean-Claude; Alix, Alain J.; Nabiev, Igor R.

    1999-04-01

    Camptothecin (CPT) derivatives are the well known inhibitors of the human DNA topoisomerase (topo) I. Two of them, irinotecan and topotecan, are just in the clinics; 9-amino- CPT is on the stage II of clinical trials, and the active search for new derivatives is now in progress. Stability of the CPT derivatives on their way to the target and resistance of cancer cells to these drugs present the crucial problem of the chemotherapy. Human serum albumin (HSA) is the mediator of transport and metabolism of numerous pharmaceuticals in the blood and P-glycoprotein (P- gp) plays a crucial role of the mediator of the multidrug resistance (MDR) of the cancer cells. This paper present the result of analysis of molecular interactions of some drugs of CPT family with the HSA and P-gp. Induced circular dichroism (CD) and Raman techniques have been applied for monitoring molecular interaction of drugs with HSA as well as to identify the conformational transition of the protein induced by the drug binding. Drug molecular determinants responsible for interaction have been identified and their binding sites within the HSA have been localized. New cancer cells lines exhibiting an extremely high level of MDR resistance have been established and were shown to contain the P-gp overproduced in the quantities of 35 percent from the all membrane proteins. The membrane fractions of these cells with the controls presented by the membranes of the parental membrane proteins. The membrane fractions of these cells with the controls presented by the membranes of the parental sensitive cells may be used as a model system for spectroscopic analysis of the specific pharmaceuticals/P-gp interactions.

  5. Exploring the binding mechanism of ondansetron hydrochloride to serum albumins: spectroscopic approach.

    PubMed

    B, Sandhya; Hegde, Ashwini H; K C, Ramesh; J, Seetharamappa

    2012-02-01

    The mechanism of interaction of ondansetron hydrochloride (OND) to serum albumins [bovine serum albumin (BSA) and human serum albumin (HSA)] was studied for the first time employing fluorimetric, circular dichroism, FTIR and UV-vis absorption techniques under the simulated physiological conditions. Fluorimetric results were utilized to investigate the binding and conformational characteristics of protein upon interaction with varying concentrations of the drug. Higher binding constant values revealed the strong interaction between the drug and protein while the number of binding sites close to unity indicated single class of binding site for OND in protein. Thermodynamic results revealed that both hydrogen bond and hydrophobic interactions played a major role in stabilizing drug-protein complex. Site marker competitive experiments indicated that the OND bound to albumins at subdomin II A (Sudlow's site I). Further, the binding distance between OND and serum albumin was calculated based on the Förster's theory of non-radioactive energy transfer and found to be 2.30 and 3.41 nm, respectively for OND-BSA and OND-HSA. The circular dichroism data revealed that the presence of OND decreased the α-helix content of serum albumins. 3D-fluorescence results also indicated the conformational changes in protein upon interaction with OND. Further, the effects of some cations have been investigated in the interaction of drug to protein.

  6. Structural studies on serum albumins under green light irradiation.

    PubMed

    Comorosan, Sorin; Polosan, Silviu; Popescu, Irinel; Ionescu, Elena; Mitrica, Radu; Cristache, Ligia; State, Alina Elena

    2010-10-01

    This paper presents two new experimental results: the protective effect of green light (GL) on ultraviolet (UV) denaturation of proteins, and the effect of GL on protein macromolecular structures. The protective effect of GL was revealed on two serum albumins, bovine (BSA) and human (HSA), and recorded by electrophoresis, absorption, and circular dichroism spectra. The effect of GL irradiation on protein structure was recorded by using fluorescence spectroscopy and electrophoresis. These new effects were modeled by quantum-chemistry computation using Gaussian 03 W, leading to good fit between theoretical and experimental absorption and circular dichroism spectra. A mechanism for these phenomena is suggested, based on a double-photon absorption process. This nonlinear effect may lead to generation of long-lived Rydberg macromolecular systems, capable of long-range interactions. These newly suggested systems, with macroscopic quantum coherence behaviors, may block the UV denaturation processes. PMID:20473754

  7. PRODUCTION OF UNIFORMLY SIZED SERUM ALBUMIN AND DEXTROSE MICROBUBBLES

    PubMed Central

    Borrelli, Michael J.; O’Brien, William D.; Bernock, Laura J.; Williams, Heather R.; Hamilton, Eric; Wu, Jonah; Oelze, Michael L.; Culp, William C.

    2011-01-01

    Uniformly-sized preparations with average microbubble (MB) diameters from 1 µm to 7 µm were produced reliably by sonicating decafluorobutane-saturated solutions of serum albumin and dextrose. Detailed protocols for producing and size-separating the MBs are presented, along with the effects that changing each production parameter (serum albumin concentration, sonication power, sonication time, etc.) had on MB size distribution and acoustic stability. These protocols can be used to produce MBs for experimental applications or serve as templates for developing new protocols that yield MBs with physical and acoustic properties better suited to specific applications. Size stability and ultrasonic performance quality control tests were developed to assure that successive MB preparations perform identically and to distinguish the physical and acoustic properties of identically sized MBs produced with different serum albumin-dextrose formulations and sonication parameters. MBs can be stored at 5°C for protracted periods (2 weeks to one year depending on formulation). PMID:21689961

  8. Strong interactions with polyethylenimine-coated human serum albumin nanoparticles (PEI-HSA NPs) alter α-synuclein conformation and aggregation kinetics

    NASA Astrophysics Data System (ADS)

    Mohammad-Beigi, Hossein; Shojaosadati, Seyed Abbas; Marvian, Amir Tayaranian; Pedersen, Jannik Nedergaard; Klausen, Lasse Hyldgaard; Christiansen, Gunna; Pedersen, Jan Skov; Dong, Mingdong; Morshedi, Dina; Otzen, Daniel E.

    2015-11-01

    The interaction between nanoparticles (NPs) and the small intrinsically disordered protein α-synuclein (αSN), whose aggregation is central in the development of Parkinson's disease, is of great relevance in biomedical applications of NPs as drug carriers. Here we showed using a combination of different techniques that αSN interacts strongly with positively charged polyethylenimine-coated human serum albumin (PEI-HSA) NPs, leading to a significant alteration in the αSN secondary structure. In contrast, the weak interactions of αSN with HSA NPs allowed αSN to remain unfolded. These different levels of interactions had different effects on αSN aggregation. While the weakly interacting HSA NPs did not alter the aggregation kinetic parameters of αSN, the rate of primary nucleation increased in the presence of PEI-HSA NPs. The aggregation rate changed in a PEI-HSA NP-concentration dependent and size independent manner and led to fibrils which were covered with small aggregates. Furthermore, PEI-HSA NPs reduced the level of membrane-perturbing oligomers and reduced oligomer toxicity in cell assays, highlighting a potential role for NPs in reducing αSN pathogenicity in vivo. Collectively, our results highlight the fact that a simple modification of NPs can strongly modulate interactions with target proteins, which may have important and positive implications in NP safety.The interaction between nanoparticles (NPs) and the small intrinsically disordered protein α-synuclein (αSN), whose aggregation is central in the development of Parkinson's disease, is of great relevance in biomedical applications of NPs as drug carriers. Here we showed using a combination of different techniques that αSN interacts strongly with positively charged polyethylenimine-coated human serum albumin (PEI-HSA) NPs, leading to a significant alteration in the αSN secondary structure. In contrast, the weak interactions of αSN with HSA NPs allowed αSN to remain unfolded. These different

  9. Combination of UV-vis spectroscopy and chemometrics to understand protein-nanomaterial conjugate: a case study on human serum albumin and gold nanoparticles.

    PubMed

    Wang, Yong; Ni, Yongnian

    2014-02-01

    Study of the interactions between proteins and nanomaterials is of great importance for understanding of protein nanoconjugate. In this work, we choose human serum albumin (HSA) and citrate-capped gold nanoparticles (AuNPs) as a model of protein and nanomaterial, and combine UV-vis spectroscopy with multivariate curve resolution by an alternating least squares (MCR-ALS) algorithm to present a new and efficient method for comparatively comprehensive study of evolution of protein nanoconjugate. UV-vis spectroscopy coupled with MCR-ALS allows qualitative and quantitative extraction of the distribution diagrams, spectra and kinetic profiles of absorbing pure species (AuNPs and AuNPs-HSA conjugate are herein identified) and undetectable species (HSA) from spectral data. The response profiles recovered are converted into the desired thermodynamic, kinetic and structural parameters describing the protein nanoconjugate evolution. Analysis of these parameters for the system gives evidence that HSA molecules are very likely to be attached to AuNPs surface predominantly as a flat monolayer to form a stable AuNPs-HSA conjugate with a core-shell structure, and the binding process takes place mainly through electrostatic and hydrogen-bond interactions between the positively amino acid residues of HSA and the negatively carboxyl group of citrate on AuNPs surface. The results obtained are verified by transmission electron microscopy, zeta potential, circular dichroism spectroscopy and Fourier transform infrared spectroscopy, showing the potential of UV-vis spectroscopy for study of evolution of protein nanoconjugate. In parallel, concentration evolutions of pure species resolved by MCR-ALS are used to construct a sensitive spectroscopic biosensor for HSA with a linear range from 1.8 nM to 28.1 nM and a detection limit of 0.8 nM.

  10. A novel quantification strategy of transferrin and albumin in human serum by species-unspecific isotope dilution laser ablation inductively coupled plasma mass spectrometry (ICP-MS).

    PubMed

    Feng, Liuxing; Zhang, Dan; Wang, Jun; Shen, Dairui; Li, Hongmei

    2015-07-16

    Species-specific (SS) isotope dilution analysis with gel electrophoresis (GE)-laser ablation (LA)-ICP-MS is a promising technique for the quantification of particular metal-binding proteins in biological samples. However, unavailable isotopically enriched spike and metal losses in GE separation are main limitations for SS-isotope dilution PAGE-LA-ICP-MS. In this study, we report for the first time the absolute quantification of transferrin (Tf) and albumin (Alb) in human serum by non-denaturing (native) GE combined with species-unspecific isotope dilution mass spectrometry (IDMS). In order to achieve a homogeneous distribution of both protein and isotope-enriched spike (simulated isotope equilibration), immersing the protein strips with (34)S spike solution after gel electrophoresis was demonstrated to be an effective way of spike addition. Furthermore, effects of immersion time and (34)S spike concentration were investigated to obtain optimal conditions of the post-electrophoresis isotope dilution method. The relative mass of spike and ablated sample (m(sp)/m(sam)) in IDMS equation was calculated by standard Tf and Alb proteins, which could be applied to the quantification of Tf and Alb in ERM-DA470k/IFCC for method confirmation. The results were in agreement with the certified value with good precision and small uncertainty (1.5-3%). In this method, species-specific spike protein is not necessary and the integrity of the heteroatom-protein could be maintained in sample preparation process. Moreover, the application of species-unspecific isotope dilution GE-LA-ICP-MS has the potential to offer reliable, direct and simultaneous quantification of proteins after conventional 1D and 2D gel electrophoretic separations.

  11. Effect of CdTe quantum dots size on the conformational changes of human serum albumin: results of spectroscopy and isothermal titration calorimetry.

    PubMed

    Yang, Bingjun; Liu, Rutao; Hao, Xiaopeng; Wu, Yongzhong; Du, Jie

    2013-10-01

    Quantum dots (QDs) are recognized as some of the most promising candidates for future applications in biomedicine. However, concerns about their safety have delayed their widespread application. Human serum albumin (HSA) is the main protein component of the circulatory system. It is important to explore the interaction of QDs with HSA for the potential in vivo application of QDs. Herein, using spectroscopy and isothermal titration calorimetry (ITC), the effect of glutathione-capped CdTe quantum dots of different sizes on the HSA was investigated. After correction for the inner filter effect, the fluorescence emission spectra and synchronous fluorescence spectra showed that the microenvironment of aromatic acid residues in the protein was slightly changed when the glutathione (GSH)-cadmium telluride (CdTe) QDs was added, and GSH-CdTe QDs with larger particle size exhibited a much higher effect on HSA than the small particles. Although a ground-state complex between HSA and GSH-CdTe QDs was formed, the UV-vis absorption and circular dichroism spectroscopic results did not find appreciable conformational changes of HSA. ITC has been used for the first time to characterize the binding of QDs with HSA. The ITC results revealed that the binding was a thermodynamically spontaneous process mainly driven by hydrophobic interactions, and the binding constant tended to increase as the GSH-CdTe QDs size increased. These findings are helpful in understanding the bioactivities of QDs in vivo and can be used to assist in the design of biocompatible and stable QDs.

  12. Preparation and characterization of Apo2L/TNF-related apoptosis-inducing ligand-loaded human serum albumin nanoparticles with improved stability and tumor distribution.

    PubMed

    Kim, Tae Hyung; Jiang, Hai-Hua; Youn, Yu Seok; Park, Chan Woong; Lim, Sung Mook; Jin, Cheng-Hao; Tak, Kyung Kook; Lee, Hye Suk; Lee, Kang Choon

    2011-02-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has received considerable attention as a potential anticancer agent. However, recombinant Apo2L/TRAIL has several limitations, which include a weak pharmacokinetic profile, namely, a short biological half-life and rapid renal clearance, and an inability to form a homotrimeric structure. In this research, we attempted to develop a sustained release nanoparticle (NP) formulation that stabilizes Apo2L/TRAIL and preserves its antitumor activity. Apo2L/TRAIL-loaded human serum albumin (HSA) NPs were prepared using a desolvation technique optimized by particle size, zeta-potential, and entrapment efficiency. Apo2L/TRAIL in HSA-NPs continuously released over 24 h at 37°C in phosphate buffered saline and rat plasma condition, and the biological activity of Apo2L/TRAIL-HSA-NPs was preserved (IC(50) = 67.2 ng/mL versus Apo2L/TRAIL IC(50) = 55.4 ng/mL) with negligible activity loss. Furthermore, in vivo pharmacokinetic profiles and tumor distribution demonstrated the superiority of Apo2L/TRAIL-HSA-NPs over Apo2L/TRAIL. The circulating half-life period was significantly prolonged from 9.8 to 90.7 min (9.2-fold enhancement), and drug bioavailability was clearly enhanced on the basis of area under the curve analysis (2.7-fold). And tumor distribution of Apo2L/TRAIL-HSA-NPs was also increased at 1 h after injection, which was about 14-fold (1-h point) over that of Apo2L/TRAIL. These results show that Apo2L/TRAIL-loaded HSA-NPs should be considered as potential long-acting cancer agents.

  13. Smart human serum albumin-indocyanine green nanoparticles generated by programmed assembly for dual-modal imaging-guided cancer synergistic phototherapy.

    PubMed

    Sheng, Zonghai; Hu, Dehong; Zheng, Mingbin; Zhao, Pengfei; Liu, Huilong; Gao, Duyang; Gong, Ping; Gao, Guanhui; Zhang, Pengfei; Ma, Yifan; Cai, Lintao

    2014-12-23

    Phototherapy, including photodynamic therapy (PDT) and photothermal therapy (PTT), is a light-activated local treatment modality that is under intensive preclinical and clinical investigations for cancer. To enhance the treatment efficiency of phototherapy and reduce the light-associated side effects, it is highly desirable to improve drug accumulation and precision guided phototherapy for efficient conversion of the absorbed light energy to reactive oxygen species (ROS) and local hyperthermia. In the present study, a programmed assembly strategy was developed for the preparation of human serum albumin (HSA)-indocyanine green (ICG) nanoparticles (HSA-ICG NPs) by intermolecular disulfide conjugations. This study indicated that HSA-ICG NPs had a high accumulation with tumor-to-normal tissue ratio of 36.12±5.12 at 24 h and a long-term retention with more than 7 days in 4T1 tumor-bearing mice, where the tumor and its margin, normal tissue were clearly identified via ICG-based in vivo near-infrared (NIR) fluorescence and photoacoustic dual-modal imaging and spectrum-resolved technology. Meanwhile, HSA-ICG NPs efficiently induced ROS and local hyperthermia simultaneously for synergetic PDT/PTT treatments under a single NIR laser irradiation. After an intravenous injection of HSA-ICG NPs followed by imaging-guided precision phototherapy (808 nm, 0.8 W/cm2 for 5 min), the tumor was completely suppressed, no tumor recurrence and treatments-induced toxicity were observed. The results suggest that HSA-ICG NPs generated by programmed assembly as smart theranostic nanoplatforms are highly potential for imaging-guided cancer phototherapy with PDT/PTT synergistic effects.

  14. Structure and properties of licochalcone A-human serum albumin complexes in solution: a spectroscopic, photophysical and computational approach to understand drug-protein interaction.

    PubMed

    Monti, Sandra; Manet, Ilse; Manoli, Francesco; Marconi, Giancarlo

    2008-11-28

    In the present contribution we address the study of the interaction of a flavonoid-derivative licochalcone A (LA) with human serum albumin (HSA). The application of circular dichroism, UV-Vis absorption, fluorescence and laser flash photolysis combined with molecular mechanics, molecular dynamics and quantum mechanical calculations of rotational strength afforded a clear picture of the modes of association of the LA neutral molecule to HSA, evidencing specific interactions with protein amino acids and their photophysical consequences. The drug is primarily associated in subdomain IIA where a strong interaction with Trp214 is established. At least two different positions of LA with respect to tryptophan are possible, one with the phenolic ring of the drug facing the aromatic ring of Trp214 and the other with the methoxyphenolic ring of LA in proximity to Trp214. In both cases LA is at ca. 4 angstroms from Trp214. This vicinity does not affect much the S1 singlet state deactivation of the bound drug, which exhibits a slightly higher fluorescence quantum yield and fluorescence lifetime on the order of that of the free molecule. The LA triplet lifetime appears to be somewhat shortened in this site. The secondary binding site is in subdomain IIIA. Here, the carbonyl group of LA experiences a strong H-bond with the OH-phenolic substituent of Tyr411. This interaction reduces substantially the LA molecular degrees of freedom, thereby determining a decrease of both radiative and nonradiative rate constants for decay of the singlet. The overall rigidity of the structure causes a lengthening of the triplet lifetime. PMID:18989470

  15. Analysis of multi-site drug-protein interactions by high-performance affinity chromatography: Binding by glimepiride to normal or glycated human serum albumin.

    PubMed

    Matsuda, Ryan; Li, Zhao; Zheng, Xiwei; Hage, David S

    2015-08-21

    High-performance affinity chromatography (HPAC) was used in a variety of formats to examine multi-site interactions between glimepiride, a third-generation sulfonylurea drug, and normal or in vitro glycated forms of the transport protein human serum albumin (HSA). Frontal analysis revealed that glimepiride interacts with normal HSA and glycated HSA at a group of high affinity sites (association equilibrium constant, or Ka, 9.2-11.8×10(5)M(-1) at pH 7.4 and 37°C) and a group of lower affinity regions (Ka, 5.9-16×10(3)M(-1)). Zonal elution competition studies were designed and carried out in both normal- and reversed-role formats to investigate the binding by this drug at specific sites. These experiments indicated that glimepiride was interacting at both Sudlow sites I and II. Allosteric effects were also noted with R-warfarin at Sudlow site I and with tamoxifen at the tamoxifen site on HSA. The binding at Sudlow site I had a 2.1- to 2.3-fold increase in affinity in going from normal HSA to the glycated samples of HSA. There was no significant change in the affinity for glimepiride at Sudlow site II in going from normal HSA to a moderately glycated sample of HSA, but a slight decrease in affinity was seen in going to a more highly glycated HSA sample. These results demonstrated how various HPAC-based methods can be used to profile and characterize multi-site binding by a drug such as glimepiride to a protein and its modified forms. The information obtained from this study should be useful in providing a better understanding of how drug-protein binding may be affected by glycation and of how separation and analysis methods based on HPAC can be employed to study systems with complex interactions or that involve modified proteins.

  16. Analysis of drug-protein binding using on-line immunoextraction and high-performance affinity microcolumns: Studies with normal and glycated human serum albumin.

    PubMed

    Matsuda, Ryan; Jobe, Donald; Beyersdorf, Jared; Hage, David S

    2015-10-16

    A method combining on-line immunoextraction microcolumns with high-performance affinity chromatography (HPAC) was developed and tested for use in examining drug-protein interactions with normal or modified proteins. Normal human serum albumin (HSA) and glycated HSA were used as model proteins for this work. High-performance immunoextraction microcolumns with sizes of 1.0-2.0 cm × 2.1mm i.d. and containing anti-HSA polyclonal antibodies were developed and tested for their ability to bind normal HSA or glycated HSA. These microcolumns were able to extract up to 82-93% for either type of protein at 0.05-0.10 mL/min and had a binding capacity of 0.34-0.42 nmol HSA for a 1.0 cm × 2.1mm i.d. microcolumn. The immunoextraction microcolumns and their adsorbed proteins were tested for use in various approaches for drug binding studies. Frontal analysis was used with the adsorbed HSA/glycated HSA to measure the overall affinities of these proteins for the drugs warfarin and gliclazide, giving comparable values to those obtained previously using similar protein preparations that had been covalently immobilized within HPAC columns. Zonal elution competition studies with gliclazide were next performed to examine the specific interactions of this drug at Sudlow sites I and II of the adsorbed proteins. These results were also comparable to those noted in prior work with covalently immobilized samples of normal HSA or glycated HSA. These experiments indicated that drug-protein binding studies can be carried out by using on-line immunoextraction microcolumns with HPAC. The same method could be used in the future with clinical samples and other drugs or proteins of interest in pharmaceutical studies or biomedical research.

  17. Analysis of multi-site drug-protein interactions by high-performance affinity chromatography: Binding by glimepiride to normal or glycated human serum albumin.

    PubMed

    Matsuda, Ryan; Li, Zhao; Zheng, Xiwei; Hage, David S

    2015-08-21

    High-performance affinity chromatography (HPAC) was used in a variety of formats to examine multi-site interactions between glimepiride, a third-generation sulfonylurea drug, and normal or in vitro glycated forms of the transport protein human serum albumin (HSA). Frontal analysis revealed that glimepiride interacts with normal HSA and glycated HSA at a group of high affinity sites (association equilibrium constant, or Ka, 9.2-11.8×10(5)M(-1) at pH 7.4 and 37°C) and a group of lower affinity regions (Ka, 5.9-16×10(3)M(-1)). Zonal elution competition studies were designed and carried out in both normal- and reversed-role formats to investigate the binding by this drug at specific sites. These experiments indicated that glimepiride was interacting at both Sudlow sites I and II. Allosteric effects were also noted with R-warfarin at Sudlow site I and with tamoxifen at the tamoxifen site on HSA. The binding at Sudlow site I had a 2.1- to 2.3-fold increase in affinity in going from normal HSA to the glycated samples of HSA. There was no significant change in the affinity for glimepiride at Sudlow site II in going from normal HSA to a moderately glycated sample of HSA, but a slight decrease in affinity was seen in going to a more highly glycated HSA sample. These results demonstrated how various HPAC-based methods can be used to profile and characterize multi-site binding by a drug such as glimepiride to a protein and its modified forms. The information obtained from this study should be useful in providing a better understanding of how drug-protein binding may be affected by glycation and of how separation and analysis methods based on HPAC can be employed to study systems with complex interactions or that involve modified proteins. PMID:26189669

  18. Use of entrapment and high-performance affinity chromatography to compare the binding of drugs and site-specific probes with normal and glycated human serum albumin

    PubMed Central

    Jackson, Abby J.; Anguizola, Jeanethe; Pfaunmiller, Erika L.; Hage, David S.

    2013-01-01

    Protein entrapment and high-performance affinity chromatography were used with zonal elution to examine the changes in binding that occurred for site-specific probes and various sulfonylurea drugs with normal and glycated forms of human serum albumin (HSA). Samples of this protein in a soluble form were physically entrapped within porous silica particles by using glycogen-capped hydrazide-activated silica; these supports were then placed into 1.0 cm × 2.1 mm inner diameter columns. Initial zonal elution studies were performed using (R)-warfarin and L-tryptophan as probes for Sudlow sites I and II (i.e., the major drug binding sites of HSA), giving quantitative measures of binding affinities in good agreement with literature values. It was also found for solutes with multisite binding to the same proteins, such as many sulfonylurea drugs, that this method could be used to estimate the global affinity of the solute for the entrapped protein. This entrapment and zonal approach provided retention information with precisions of ±0.1–3.3% (± one standard deviation) and elution within 0.50–3.00 min for solutes with binding affinities of 1 × 104–3 × 105 M−1. Each entrapped-protein column was used for many binding studies, which decreased the cost and amount of protein needed per injection (e.g., the equivalent of only 125–145 pmol of immobilized HSA or glycated HSA per injection over 60 sample application cycles). This method can be adapted for use with other proteins and solutes and should be valuable in high-throughput screening or quantitative studies of drug–protein binding or related biointeractions. PMID:23657448

  19. Analysis of Multi-Site Drug-Protein Interactions by High-Performance Affinity Chromatography: Binding by Glimepiride to Normal or Glycated Human Serum Albumin

    PubMed Central

    Matsuda, Ryan; Li, Zhao; Zheng, Xiwei; Hage, David S.

    2015-01-01

    High-performance affinity chromatography (HPAC) was used in a variety of formats to examine multi-site interactions between glimepiride, a third-generation sulfonylurea drug, and normal or in vitro glycated forms of the transport protein human serum albumin (HSA). Frontal analysis revealed that glimepiride interacts with normal HSA and glycated HSA at a group of high affinity sites (association equilibrium constant, or Ka, 9.2–11.8 × 105 M−1 at pH 7.4 and 37°C) and a group of lower affinity regions (Ka, 5.9–16.2 × 103 M−1). Zonal elution competition studies were designed and carried out in both normal- and reversed-role formats to investigate the binding by this drug at specific sites. These experiments indicated that glimepiride was interacting at both Sudlow sites I and II. Allosteric effects were also noted with R-warfarin at Sudlow site I and with tamoxifen at the tamoxifen site on HSA. The binding at Sudlow site I had a 2.1- to 2.3-fold increase in affinity in going from normal HSA to the glycated samples of HSA. There was no significant change in the affinity for glimepiride at Sudlow site II in going from normal HSA to a moderately glycated sample of HSA, but a slight decrease in affinity was seen in going to a more highly glycated HSA sample. These results demonstrated how various HPAC-based methods can be used to profile and characterize multi-site binding by a drug such as glimepiride to a protein and its modified forms. The information obtained from this study should be useful in providing a better understanding of how drug-protein binding may be affected by glycation and of how separation and analysis methods based on HPAC can be employed to study systems with complex interactions or that involve modified proteins. PMID:26189669

  20. HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY AND THE ANALYSIS OF DRUG INTERACTIONS WITH MODIFIED PROTEINS: BINDING OF GLICLAZIDE WITH GLYCATED HUMAN SERUM ALBUMIN

    PubMed Central

    Matsuda, Ryan; Anguizola, Jeanethe; Joseph, K.S.; Hage, David S.

    2011-01-01

    This study used high-performance affinity chromatography (HPAC) to examine the binding of gliclazide (i.e., a sulfonylurea drug used to treat diabetes) with the protein human serum albumin (HSA) at various stages of modification due to glycation. Frontal analysis conducted with small HPAC columns was first used to estimate the number of binding sites and association equilibrium constants (Ka) for gliclazide with normal HSA and glycated HSA. Both normal and glycated HSA interacted with gliclazide according to a two-site model, with a class of high affinity sites (average Ka, 7.1-10 × 104 M−1) and a group of lower affinity sites (average Ka, 5.7-8.9 × 103 M−1) at pH 7.4 and 37°C. Competition experiments indicated that Sudlow sites I and II of HSA were both involved in these interactions, with the Ka values for gliclazide at these sites being 1.9 × 104 M−1 and 6.0 × 104 M−1, respectively, for normal HSA. Two samples of glycated HSA had similar affinities to normal HSA for gliclazide at Sudlow site I, but one sample had a 1.9-fold increase in affinity at this site. All three glycated HSA samples differed from normal HSA in their affinity for gliclazide at Sudlow site II. This work illustrated how HPAC can be used to examine both the overall binding of a drug with normal or modified proteins and the site-specific changes that can occur in these interactions as a result of protein modification. PMID:21922305

  1. Revealing the ionization ability of binding site I of human serum albumin using 2-(2'-hydroxyphenyl)benzoxazole as a pH sensitive probe.

    PubMed

    Abou-Zied, Osama K

    2012-02-28

    The ability of site I of human serum albumin (HSA) to bind medium sized molecules is important for the distribution, metabolism, and efficacy of many drugs. Herein, we show that this binding site has the ionization ability that may alter the drug structure during the process of its delivery. We reveal this ability by employing 2-(2'-hydroxyphenyl)benzoxazole (HBO) as a pH sensitive probe. Binding of HBO in site I is studied here at physiological pH 7.2 using steady-state and lifetime spectroscopic measurements, molecular docking and molecular dynamics (MD) simulation methods. The complex photophysics of HBO and the unique fluorescence signature of its anionic form indicate that, upon binding with HSA, the molecule exists in equilibrium between the anionic and the syn-keto forms. The position of HBO inside the binding site was determined experimentally by measuring the fluorescence quenching of W214, the sole tryptophan residue in HSA. The ionization degree of HBO inside the binding site was estimated to be close to the ionization degree of HBO in an aqueous solution of pH 10. This was concluded by comparing the fluorescence behavior of bound HBO to that of HBO in different solvents and in aqueous solutions of different pH values. Molecular docking and MD simulations show that HBO binds in site I close to W214, confirming the experimental results, and pinpoint the dominant role of hydrophobic interactions in the binding site. The formation of the anionic form is proposed to be due to through-space interaction between the OH group of HBO and both R222 and I290 with a binding mode similar to that of warfarin in site I. Comparison of the results with those of HBO mixed with key amino acids in solution indicates the importance of through-space interaction in the formation of the anion, similar to enzymatic reactions.

  2. Preparation and Characterization of 125I Labeled Bovine Serum Albumin

    PubMed Central

    Ashwitha Rai, K. S.; Jyothi; Rasmi, R. R.; Sarnaik, Jayula; Kadwad, V. B.; Shenoy, K. B.; Somashekarappa, H. M.

    2015-01-01

    Bovine serum albumin is a model protein, which has been conventionally used as protein standard and in many areas of biochemistry, pharmacology and medicine. Radioiodination procedure for bovine serum albumin employing chloramine-T as an oxidant with slight modification was evaluated critically to establish the optimal conditions for the preparation of radiolabeled tracer (125I-BSA) with required specific activity without impairing the immune reactivity and biological activity. Optimized radioiodination procedure involving 10 µg of chloramine-T along with 20 µg of sodium metabisulphite with 60 seconds incubation at 2° yielded 125I-BSA with high integrity. PMID:25767326

  3. Study of the interaction between esculetin and human serum albumin by multi-spectroscopic method and molecular modeling

    NASA Astrophysics Data System (ADS)

    Yue, Yuanyuan; Zhang, Yaheng; Qin, Jin; Chen, Xingguo

    2008-10-01

    Esculetin derived from Cortex fraxin plays an important role as a traditional Chinese medicine because of its unique pharmacological properties. The interactions between esculetin and HSA were studied by fluorescence spectroscopic techniques under similar to human physiologic conditions. The binding parameters have been evaluated by fluorescence quenching methods. The results proved the mechanism of fluorescence quenching of HSA while interacting with esculetin is due to the formation of esculetin-HSA complex formation. The thermodynamic parameters like Δ H0 and Δ S0 were calculated to be -14.62 kJ/mol and 38.93 J/mol/K, respectively, which proves main interaction between esculetin and HSA is hydrophobic contact, but the electrostatic interaction cannot be excluded, which in agreement with the result of molecular docking study. The distance r between donor (HSA) and acceptor (esculetin) was obtained according to the Förster's theory of non-radiative energy transfer and found to be 2.89 nm. From the high value of fluorescence anisotropy ( r = 0.07) it was argued that the probe molecular was located in motionally restricted environment of the protein. The alterations of protein secondary structure in the presence of esculetin were confirmed by the evidences from UV, FT-IR and CD spectroscopes. In addition, the effects of common ions and amino acids on the constants of esculetin-HSA complex were also discussed.

  4. Spectroscopic characterization of effective components anthraquinones in Chinese medicinal herbs binding with serum albumins

    NASA Astrophysics Data System (ADS)

    Bi, Shuyun; Song, Daqian; Kan, Yuhe; Xu, Dong; Tian, Yuan; Zhou, Xin; Zhang, Hanqi

    2005-11-01

    The interactions of serum albumins such as human serum albumin (HSA) and bovine serum albumin (BSA) with emodin, rhein, aloe-emodin and aloin were assessed employing fluorescence quenching and absorption spectroscopic techniques. The results obtained revealed that there are relatively strong binding affinity for the four anthraquinones with HSA and BSA and the binding constants for the interactions of anthraquinones with HSA or BSA at 20 °C were obtained. Anthraquinone-albumin interactions were studied at different temperatures and in the presence of some metal ions. And the competition binding of anthraquinones with serum albumins was also discussed. The Stern-Volmer curves suggested that the quenching occurring in the reactions was the static quenching process. The binding distances and transfer efficiencies for each binding reactions were calculated according to the Föster theory of non-radiation energy transfer. Using thermodynamic equations, the main action forces of these reactions were also obtained. The reasons of the different binding affinities for different anthraquinone-albumin reactions were probed from the point of view of molecular structures.

  5. 21 CFR 640.80 - Albumin (Human).

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... a sterile solution of the albumin derived from human plasma. (b) Source material. The source material of Albumin (Human) shall be plasma recovered from Whole Blood prepared as prescribed in §§ 640.1 through 640.5, or Source Plasma prepared as prescribed in §§ 640.60 through 640.76. (c) Additives...

  6. 21 CFR 640.80 - Albumin (Human).

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... a sterile solution of the albumin derived from human plasma. (b) Source material. The source material of Albumin (Human) shall be plasma recovered from Whole Blood prepared as prescribed in §§ 640.1 through 640.5, or Source Plasma prepared as prescribed in §§ 640.60 through 640.76. (c) Additives...

  7. 21 CFR 640.80 - Albumin (Human).

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... a sterile solution of the albumin derived from human plasma. (b) Source material. The source material of Albumin (Human) shall be plasma recovered from Whole Blood prepared as prescribed in §§ 640.1 through 640.5, or Source Plasma prepared as prescribed in §§ 640.60 through 640.76. (c) Additives...

  8. 21 CFR 640.80 - Albumin (Human).

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... a sterile solution of the albumin derived from human plasma. (b) Source material. The source material of Albumin (Human) shall be plasma recovered from Whole Blood prepared as prescribed in §§ 640.1 through 640.5, or Source Plasma prepared as prescribed in §§ 640.60 through 640.76. (c) Additives...

  9. 21 CFR 640.80 - Albumin (Human).

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... a sterile solution of the albumin derived from human plasma. (b) Source material. The source material of Albumin (Human) shall be plasma recovered from Whole Blood prepared as prescribed in §§ 640.1 through 640.5, or Source Plasma prepared as prescribed in §§ 640.60 through 640.76. (c) Additives...

  10. Characterization and comparative analysis of 2,4-toluene diisocyanate and 1,6-hexamethylene diisocyanate haptenated human serum albumin and hemoglobin.

    PubMed

    Mhike, Morgen; Hettick, Justin M; Chipinda, Itai; Law, Brandon F; Bledsoe, Toni A; Lemons, Angela R; Nayak, Ajay P; Green, Brett J; Beezhold, Donald H; Simoyi, Reuben H; Siegel, Paul D

    2016-04-01

    Diisocyanates (dNCOs) are low molecular weight chemical sensitizers that react with autologous proteins to produce neoantigens. dNCO-haptenated proteins have been used as immunogens for generation of dNCO-specific antibodies and as antigens to screen for dNCO-specific antibodies in exposed individuals. Detection of dNCO-specific antibodies in exposed individuals for diagnosis of dNCO asthma has been hampered by poor sensitivities of the assay methods in that specific IgE can only be detected in approximately 25% of the dNCO asthmatics. Apart from characterization of the conjugates used for these immunoassays, the choice of the carrier protein and the dNCO used are important parameters that can influence the detection of dNCO-specific antibodies. Human serum albumin (HSA) is the most common carrier protein used for detection of dNCO specific-IgE and -IgG but the immunogenicity and/or antigenicity of other proteins that may be modified by dNCO in vivo is not well documented. In the current study, 2,4-toluene diisocyanate (TDI) and 1,6-hexamethylene diisocyanate (HDI) were reacted with HSA and human hemoglobin (Hb) and the resultant adducts were characterized by (i) HPLC quantification of the diamine produced from acid hydrolysis of the adducts, (ii) 2,4,6-trinitrobenzene sulfonic acid (TNBS) assay to assess extent of cross-linking, (iii) electrophoretic migration in polyacrylamide gels to analyze intra- and inter-molecular cross-linking, and (iv) evaluation of antigenicity using a monoclonal antibody developed previously to TDI conjugated to Keyhole limpet hemocyanin (KLH). Concentration-dependent increases in the amount of dNCO bound to HDI and TDI, cross-linking, migration in gels, and antibody-binding were observed. TDI reactivity with both HSA and Hb was significantly higher than HDI. Hb-TDI antigenicity was approximately 30% that of HSA-TDI. In conclusion, this data suggests that both, the extent of haptenation as well as the degree of cross-linking differs

  11. Characterizing the Interaction between tartrazine and two serum albumins by a hybrid spectroscopic approach.

    PubMed

    Pan, Xingren; Qin, Pengfei; Liu, Rutao; Wang, Jing

    2011-06-22

    Tartrazine is an artificial azo dye commonly used in food products. The present study evaluated the interaction of tartrazine with two serum albumins (SAs), human serum albumin (HSA) and bovine serum albumin (BSA), under physiological conditions by means of fluorescence, three-dimensional fluorescence, UV-vis absorption, and circular dichroism (CD) techniques. The fluorescence data showed that tartrazine could bind to the two SAs to form a complex. The binding process was a spontaneous molecular interaction procedure, in which van der Waals and hydrogen bond interactions played a major role. Additionally, as shown by the UV-vis absorption, three-dimensional fluorescence, and CD results, tartrazine could lead to conformational and some microenvironmental changes of both SAs, which may affect the physiological functions of SAs. The work provides important insight into the mechanism of toxicity of tartrazine in vivo. PMID:21591756

  12. NMR identification of endogenous metabolites interacting with fatted and non-fatted human serum albumin in blood plasma: Fatty acids influence the HSA-metabolite interaction.

    PubMed

    Jupin, Marc; Michiels, Paul J; Girard, Frederic C; Spraul, Manfred; Wijmenga, Sybren S

    2013-03-01

    Metabolites and their concentrations are direct reporters on body biochemistry. Thanks to technical developments metabolic profiling of body fluids, such as blood plasma, by for instance NMR has in the past decade become increasingly accurate enabling successful clinical diagnostics. Human Serum Albumin (HSA) is the main plasma protein (∼60% of all plasma protein) and responsible for the transport of endogenous (e.g. fatty acids) and exogenous metabolites, which it achieves thanks to its multiple binding sites and its flexibility. HSA has been extensively studied with regard to its binding of drugs (exogenous metabolites), but only to a lesser extent with regard to its binding of endogenous (non-fatty acid) metabolites. To obtain correct NMR measured metabolic profiles of blood plasma and/or potentially extract information on HSA and fatty acids content, it is necessary to characterize these endogenous metabolite/plasma protein interactions. Here, we investigate these metabolite-HSA interactions in blood plasma and blood plasma mimics. The latter contain the roughly twenty metabolites routinely detected by NMR (also most abundant) in normal relative concentrations with fatted or non-fatted HSA added or not. First, we find that chemical shift changes are small and seen only for a few of the metabolites. In contrast, a significant number of the metabolites display reduced resonance integrals and reduced free concentrations in the presence of HSA or fatted HSA. For slow-exchange (or strong) interactions, NMR resonance integrals report the free metabolite concentration, while for fast exchange (weak binding) the chemical shift reports on the binding. Hence, these metabolites bind strongly to HSA and/or fatted HSA, but to a limited degree because for most metabolites their concentration is smaller than the HSA concentration. Most interestingly, fatty acids decrease the metabolite-HSA binding quite significantly for most of the interacting metabolites. We further find

  13. Comparative solution equilibrium studies on pentamethylcyclopentadienyl rhodium complexes of 2,2'-bipyridine and ethylenediamine and their interaction with human serum albumin.

    PubMed

    Enyedy, Éva A; Mészáros, János P; Dömötör, Orsolya; Hackl, Carmen M; Roller, Alexander; Keppler, Bernhard K; Kandioller, Wolfgang

    2015-11-01

    Complex formation equilibrium processes of the (N,N) donor containing 2,2'-bipyridine (bpy) and ethylenediamine (en) with (η(5)-pentamethylcyclopentadienyl)rhodium(III) were investigated in aqueous solution via pH-potentiometry, (1)H NMR spectroscopy, and UV-vis spectrophotometry in the absence and presence of chloride ions. The structure of [RhCp*(en)Cl]ClO4 (Cp*, pentamethylcyclopentadienyl) was also studied by single-crystal X-ray diffraction. pKa values of 8.56 and 9.58 were determined for [RhCp*(bpy)(H2O)](2+) and [RhCp*(en)(H2O)](2+), respectively resulting in the formation of negligible amount of mixed hydroxido complexes at pH 7.4. Stability and the H2O/Cl(-) co-ligand exchange constants of bpy and en complexes considerably exceed those of the bidentate O-donor deferiprone. The strong affinity of the bpy and en complexes to chloride ions most probably contributes to their low antiproliferative effect. Interactions between human serum albumin (HSA) and [RhCp*(H2O)3](2+), its complexes formed with deferiprone, bpy and en were also monitored by (1)H NMR spectroscopy, ultrafiltration/UV-vis and spectrofluorometry. Numerous binding sites (≥ 8) are available for [RhCp*(H2O)3](2+); and the interaction takes place most probably via covalent bonds through the imidazole nitrogen of His. According to the various fluorescence studies [RhCp*(H2O)3](2+) binds on sites I and II, and coordination of surface side chain donor atoms of the protein is also feasible. The binding of the bpy and en complex is weaker and slower compared to that of [RhCp*(H2O)3](2+), and formation of ternary HSA-RhCp*-ligand adducts was proved. In the case of the deferiprone complex, the RhCp* fragment is cleaved off when HSA is loaded with low equivalents of the compound.

  14. Hybrid paclitaxel and gold nanorod-loaded human serum albumin nanoparticles for simultaneous chemotherapeutic and photothermal therapy on 4T1 breast cancer cells.

    PubMed

    Peralta, Donna V; Heidari, Zahra; Dash, Srikanta; Tarr, Matthew A

    2015-04-01

    The use of human serum albumin nanoparticles (HSAPs) as a drug carrier system for cancer treatment has proven successful through current marketable clinical formulations. Despite this success, there is a current lack of multifunctional HSAPs, which offer combinational therapies of more than one proven technique. Gold nanorods (AuNRs) have also shown medicinal promise due to their photothermal therapy capabilities. In this study, a desolvation and cross-linking approach was employed to successfully encapsulate gold nanorods into HSAPs simultaneously with the chemotherapeutic drug paclitaxel (PAC); forming PAC-AuNR-HSAPs with desirable overall particle sizes of 299 ± 6 nm. The loading efficiency of paclitaxel into PAC-AuNR-HSAPs reached up to 3 μg PAC/mg HSA. The PAC-AuNR-HSAPs experienced photothermal heating; with the bulk particle solution reaching up to 46 °C after 15 min of near-IR laser exposure. This heat increase marked the successful attainment of the temperature necessary to cause severe cellular hyperthermia and necrosis. The encasement strategy facilitated a colloidal hybrid treatment system capable of enhanced permeability and retention effects, photothermal ablation of cancer cells, and release of the active paclitaxel of up to 188 ng (from PAC-AuNR-HSAPs created with 30 mg HSA) in a single 15 min irradiation session. When treated with PAC-AuNR-HSAPs containing 20 μg PAC/mL particle solution, 4T1 mouse breast cancer cells experienced ∼82% cell death without irradiation and ∼94% cell death after just one irradiation session. The results for PAC-AuNR-HSAPs were better than that of free PAC, which only killed ∼77% of the cells without irradiation and ∼80% with irradiation. The hybrid particle system also lends itself to future customizable external functionalities via conjugated targeting ligands, such as antibodies. Internal entrapment of patient tailored medication combinations are also possible with this combination treatment platform, which

  15. Production of transgenic cattle highly expressing human serum albumin in milk by phiC31 integrase-mediated gene delivery.

    PubMed

    Luo, Yan; Wang, Yongsheng; Liu, Jun; Lan, Hui; Shao, Minghao; Yu, Yuan; Quan, Fusheng; Zhang, Yong

    2015-10-01

    Transgenic cattle expressing high levels of recombinant human serum albumin (HSA) in their milk may as an alternative source for commercial production. Our objective was to produce transgenic cattle highly expressing HSA in milk by using phiC31 integrase system and somatic cell nuclear transfer (SCNT). The mammary-specific expression plasmid pIACH(-), containing the attB recognition site for phiC31 integrase, were co-transfected with integrase expression plasmid pCMVInt into bovine fetal fibroblast cells (BFFs). PhiC31 integrase-mediated integrations in genome of BFFs were screened by nested inverse PCR. After analysis of sequence of the PCR products, 46.0% (23/50) of the both attB-genome junction sites (attL and attR) were confirmed, and four pseudo attP sites were identified. The integration rates in BF3, BF11, BF19 and BF4 sites were 4.0% (2/50), 6.0% (3/50), 16.0% (8/50) and 20.0% (10/50), respectively. BF3 is located in the bovine chromosome 3 collagen alpha-3 (VI) chain isomer 2 gene, while the other three sites are located in the non-coding region. The transgenic cell lines from BF11, BF19 and BF4 sites were used as donors for SCNT. Two calves from transgenic cells BF19 were born, one died within a few hours after birth, and another calf survived healthy. PCR and Southern blot analysis revealed integration of the transgene in the genome of cloned calves. The nested reverse PCR confirmed that the integration site in cloned calves was identical to the donor cells. The western blotting assessment indicated that recombinant HSA was expressed in the milk of transgenic cattle and the expression level was about 4-8 mg/mL. The present study demonstrated that phiC31 integrase system was an efficient and safety gene delivery tool for producing HSA transgenic cattle. The production of recombinant HSA in the milk of cattle may provide a large-scale and cost-effective resource.

  16. NMR identification of endogenous metabolites interacting with fatted and non-fatted human serum albumin in blood plasma: Fatty acids influence the HSA-metabolite interaction

    NASA Astrophysics Data System (ADS)

    Jupin, Marc; Michiels, Paul J.; Girard, Frederic C.; Spraul, Manfred; Wijmenga, Sybren S.

    2013-03-01

    Metabolites and their concentrations are direct reporters on body biochemistry. Thanks to technical developments metabolic profiling of body fluids, such as blood plasma, by for instance NMR has in the past decade become increasingly accurate enabling successful clinical diagnostics. Human Serum Albumin (HSA) is the main plasma protein (˜60% of all plasma protein) and responsible for the transport of endogenous (e.g. fatty acids) and exogenous metabolites, which it achieves thanks to its multiple binding sites and its flexibility. HSA has been extensively studied with regard to its binding of drugs (exogenous metabolites), but only to a lesser extent with regard to its binding of endogenous (non-fatty acid) metabolites. To obtain correct NMR measured metabolic profiles of blood plasma and/or potentially extract information on HSA and fatty acids content, it is necessary to characterize these endogenous metabolite/plasma protein interactions. Here, we investigate these metabolite-HSA interactions in blood plasma and blood plasma mimics. The latter contain the roughly twenty metabolites routinely detected by NMR (also most abundant) in normal relative concentrations with fatted or non-fatted HSA added or not. First, we find that chemical shift changes are small and seen only for a few of the metabolites. In contrast, a significant number of the metabolites display reduced resonance integrals and reduced free concentrations in the presence of HSA or fatted HSA. For slow-exchange (or strong) interactions, NMR resonance integrals report the free metabolite concentration, while for fast exchange (weak binding) the chemical shift reports on the binding. Hence, these metabolites bind strongly to HSA and/or fatted HSA, but to a limited degree because for most metabolites their concentration is smaller than the HSA concentration. Most interestingly, fatty acids decrease the metabolite-HSA binding quite significantly for most of the interacting metabolites. We further find

  17. Scaffold materials from glycosylated and PEGylated bovine serum albumin.

    PubMed

    Wang, Kai; David, Allan E; Choi, Young-Suk; Wu, Yonnie; Buschle-Diller, Gisela

    2015-09-01

    Bovine serum albumin has been PEGylated and glycosylated to create mimetic materials for the extracellular matrix (ECM) with potential tissue engineering applications. Different surfaces for cell adhesion were achieved by crosslinking the initial albumin product and forming either a coating or a sponge-like three-dimensional morphology to mimic the mesh structure of natural ECM. The biocompatibility of the albumin matrix with mammalian cells was evaluated using cell culture assays with NIH 3T3 cells. The results indicated that glycoprotein composition and specific morphology of the assembly can improve the cell growth environment. These ECM mimetic structures might eventually be considered to serve as alternatives for the more expensive collagen and elastin based ECM substances currently in use in tissue engineering. PMID:25691091

  18. Competitive binding of phenylbutazone and colchicine to serum albumin in multidrug therapy: A spectroscopic study

    NASA Astrophysics Data System (ADS)

    Sułkowska, A.; Maciążek-Jurczyk, M.; Bojko, B.; Równicka, J.; Zubik-Skupień, I.; Temba, E.; Pentak, D.; Sułkowski, W. W.

    2008-06-01

    The binding sites for phenylbutazone and colchicine were identified in tertiary structure of bovine and human serum albumin with the use of spectrofluorescence analysis. It was found that phenylbutazone has two binding sites in both sera albumins (HSA and BSA), while colchicine has one binding site in BSA as well as in HSA. The comparison of the quenching effect of BSA and HSA fluorescence by phenylbutazone and colchicine allows us to identify subdomain IIA in protein as the binding site for these two drugs. In this subdomain tryptophan 214 is located. The participation of tyrosyl and tryptophanyl residues of protein was also estimated in the drug-albumin complex. The comparison of quenching of fluorescence of HSA and BSA excited at 280 nm with that at 295 nm allowed us to state that the participation of tyrosyl residues of albumin in the phenylbutazone-serum albumin interaction is significant. The analysis of quenching of fluorescence of BSA in the binary and ternary systems showed that phenylbutazone does not affect the complex formed between colchicine and BSA. Similarly, colchicine has no effect on the Phe-BSA complex. However marked differences were observed for the complex with HSA. On the basis of Ka and KQ values it was concluded that colchicine may probably cause displacement of phenylbutazone from its complex with serum albumin (SA). Static and dynamic quenching for the binary and ternary systems is also discussed. The competition of phenylbutazone and colchicine in binding to serum albumin should be taken into account in the multi-drug therapy.

  19. A spectroscopic study of phenylbutazone and aspirin bound to serum albumin in rheumatoid diseases

    NASA Astrophysics Data System (ADS)

    Maciążek-Jurczyk, M.; Sułkowska, A.; Bojko, B.; Równicka-Zubik, J.; Sułkowski, W. W.

    2011-11-01

    Interaction of phenylbutazone (PBZ) and aspirin (ASA), two drugs recommended in rheumatoid diseases (RDs), when binding to human (HSA) and bovine (BSA) serum albumins, has been studied by quenching of fluorescence and proton nuclear magnetic resonance ( 1HNMR) techniques. On the basis of spectrofluorescence measurements high affinity binding sites of PBZ and ASA on albumin as well as their interaction within the binding sites were described. A low affinity binding site has been studied by proton nuclear magnetic resonance spectroscopy. Using fluorescence spectroscopy the location of binding site in serum albumin (SA) for PBZ and ASA was found. Association constants Ka were determined for binary (i.e. PBZ-SA and ASA-SA) and ternary complexes (i.e. PBZ-[ASA]-SA and ASA-[PBZ]-SA). PBZ and ASA change the affinity of each other to the binding site in serum albumin (SA). The presence of ASA causes the increase of association constants KaI of PBZ-SA complex. Similarly, PBZ influences KaI of ASA-SA complex. This phenomenon shows that the strength of binding and the stability of the complexes increase in the presence of the second drug. The decrease of KaII values suggests that the competition between PBZ and ASA in binding to serum albumin in the second class of binding sites occurs. The analysis of 1HNMR spectral parameters i.e. changes of chemical shifts and relaxation times of the drug indicate that the presence of ASA weakens the interaction of PBZ with albumin. Similarly PBZ weakens the interaction of ASA with albumin. This conclusion points to the necessity of using a monitoring therapy owning to the possible increase of uncontrolled toxic effects.

  20. Surface Plasmon Resonance Assay of Binding Properties of Antisense Oligonucleotides to Serum Albumins and Lipoproteins.

    PubMed

    Onishi, Reina; Watanabe, Ayahisa; Nakajima, Mado; Sekiguchi, Mitsuaki; Kugimiya, Akira; Kinouchi, Hiroki; Nihashi, Yoichiro; Kamimori, Hiroshi

    2015-01-01

    In the present study, we developed an assay to evaluate the kinetic binding properties of the unconjugated antisense oligonucleotide (ASO) and lipophilic and hydrophilic ligands conjugated ASOs to mouse and human serum albumin, and lipoproteins using surface plasmon resonance (SPR). The lipophilic ligands conjugated ASOs showed clear affinity to the albumins and lipoproteins, while the unconjugated and hydrophilic ligand conjugated ASOs showed no interaction. The SPR method showed reproducible immobilization of albumins and lipoproteins as ligands on the sensor chip, and reproducible affinity kinetic parameters of interaction of ASOs conjugated with the ligands could be obtained. The kinetic binding data of these ASOs to albumin and lipoproteins by SPR were related with the distributions in the whole liver in mice after administration of these conjugated ASOs. The results demonstrated that our SPR method could be a valuable tool for predicting the mechanism of the properties of delivery of conjugated ASOs to the organs.

  1. Binding of several benzodiazepines to bovine serum albumin: Fluorescence study

    NASA Astrophysics Data System (ADS)

    Machicote, Roberta G.; Pacheco, María E.; Bruzzone, Liliana

    2010-10-01

    The interactions of lorazepam, oxazepam and bromazepam with bovine serum albumin (BSA) were studied by fluorescence spectrometry. The Stern-Volmer quenching constants and corresponding thermodynamic parameters Δ H, Δ G and Δ S were calculated. The binding constants and the number of binding sites were also investigated. The distances between the donor (BSA) and the acceptors (benzodiazepines) were obtained according to fluorescence resonance energy transfer and conformational changes of BSA were observed from synchronous fluorescence spectra.

  2. Serum albumin-alginate coated beads: mechanical properties and stability.

    PubMed

    Edwards-Lévy, F; Lévy, M C

    1999-11-01

    According to a previously described method, alginate beads were prepared from a Na-alginate solution containing propylene glycol alginate (PGA) and human serum albumin (HSA). The solution was added dropwise to a CaCl2 solution. The beads were treated with NaOH, which started the formation of amide bonds between HSA and PGA at the periphery, giving a membrane. Batches of beads with increasingly thick membranes were prepared using growing concentrations of NaOH, and studied with a texture analyser. When raising NaOH concentration, the rupture strength progressively increased, and the resistance strength to a deformation of 50% of total height also increased before slightly decreasing for the highest NaOH concentration. Variations of bead elasticity were also observed. When the beads were prepared with saline reducing gelation time from 10 to 5 min, and reaction time from 15 to 5 min, mechanical properties varied more progressively with the NaOH concentration, while the results became more reproducible. A series of assays conducted with 0.01 M NaOH confirmed the importance of using a short gelation time, and saline rather than water. Stability assays were also performed. The results were compared to those of alginate-polylysine coated beads and showed the interest of the transacylation method. PMID:10535819

  3. Evaluation of the biointeraction of colorant flavazin with human serum albumin: insights from multiple spectroscopic studies, in silico docking and molecular dynamics simulation.

    PubMed

    Peng, Wei; Ding, Fei; Jiang, Yu-Ting; Sun, Ying; Peng, Yu-Kui

    2014-06-01

    Azo compounds are the largest chemical class of agents frequently used as colorants in a variety of consumer goods and farm produce; therefore, they may become a hazard to public health, because numerous azo compounds and their metabolites are proven to be carcinogens and mutagens. Herein several qualitative and quantitative analytical techniques, including steady state and time-resolved fluorescence, circular dichroism (CD), computer-aided molecular docking as well as molecular dynamics simulation, were employed to ascertain the molecular recognition between the principal vehicle of ligands in human plasma, albumin and a model azo compound, flavazin. The results show that the albumin spatial structure was changed in the presence of flavazin with a decrease of α-helix suggesting partial protein destabilization/self-regulation, as derived from steady state fluorescence, far-UV CD and detailed analyses of three-dimensional fluorescence spectra. Time-resolved fluorescence further evinced that the recognition mechanism is related to albumin-flavazin adduct formation with an association intensity of 10(4) M(-1), and the driving forces were found to be chiefly π-π interactions, hydrophobic interactions and hydrogen bonds. The specific binding domain of flavazin in protein was defined from molecular docking; subdomain IIA (Sudlow's site I) was found to retain high affinity for the ligand flavazin. This finding corroborates the results of competitive ligand displacement experiments, a hydrophobic 8-anilino-1-naphthalenesulfonic acid probe study and protein denaturation results, placing flavazin at the warfarin-azapropazone site. Based on molecular dynamics simulation, it can be said with certainty that the results of molecular docking are credible, and the key amino acid residues participating in the molecular recognition of flavazin by protein are clearly Trp-214, Arg-222 and Lys-436. The outcomes presented here will help to further comprehend the molecular recognition

  4. Evaluation of the biointeraction of colorant flavazin with human serum albumin: insights from multiple spectroscopic studies, in silico docking and molecular dynamics simulation.

    PubMed

    Peng, Wei; Ding, Fei; Jiang, Yu-Ting; Sun, Ying; Peng, Yu-Kui

    2014-06-01

    Azo compounds are the largest chemical class of agents frequently used as colorants in a variety of consumer goods and farm produce; therefore, they may become a hazard to public health, because numerous azo compounds and their metabolites are proven to be carcinogens and mutagens. Herein several qualitative and quantitative analytical techniques, including steady state and time-resolved fluorescence, circular dichroism (CD), computer-aided molecular docking as well as molecular dynamics simulation, were employed to ascertain the molecular recognition between the principal vehicle of ligands in human plasma, albumin and a model azo compound, flavazin. The results show that the albumin spatial structure was changed in the presence of flavazin with a decrease of α-helix suggesting partial protein destabilization/self-regulation, as derived from steady state fluorescence, far-UV CD and detailed analyses of three-dimensional fluorescence spectra. Time-resolved fluorescence further evinced that the recognition mechanism is related to albumin-flavazin adduct formation with an association intensity of 10(4) M(-1), and the driving forces were found to be chiefly π-π interactions, hydrophobic interactions and hydrogen bonds. The specific binding domain of flavazin in protein was defined from molecular docking; subdomain IIA (Sudlow's site I) was found to retain high affinity for the ligand flavazin. This finding corroborates the results of competitive ligand displacement experiments, a hydrophobic 8-anilino-1-naphthalenesulfonic acid probe study and protein denaturation results, placing flavazin at the warfarin-azapropazone site. Based on molecular dynamics simulation, it can be said with certainty that the results of molecular docking are credible, and the key amino acid residues participating in the molecular recognition of flavazin by protein are clearly Trp-214, Arg-222 and Lys-436. The outcomes presented here will help to further comprehend the molecular recognition

  5. Circular dichroism study of the interaction between mutagens and bilirubin bound to different binding sites of serum albumins.

    PubMed

    Orlov, Sergey; Goncharova, Iryna; Urbanová, Marie

    2014-05-21

    Although recent investigations have shown that bilirubin not only has a negative role in the organism but also exhibits significant antimutagenic properties, the mechanisms of interactions between bilirubin and mutagens are not clear. In this study, interaction between bilirubin bound to different binding sites of mammalian serum albumins with structural analogues of the mutagens 2-aminofluorene, 2,7-diaminofluorene and mutagen 2,4,7-trinitrofluorenone were investigated by circular dichroism and absorption spectroscopy. Homological human and bovine serum albumins were used as chiral matrices, which preferentially bind different conformers of bilirubin in the primary binding sites and make it observable by circular dichroism. These molecular systems approximated a real system for the study of mutagens in blood serum. Differences between the interaction of bilirubin bound to primary and to secondary binding sites of serum albumins with mutagens were shown. For bilirubin bound to secondary binding sites with low affinity, partial displacement and the formation of self-associates were observed in all studied mutagens. The associates of bilirubin bound to primary binding sites of serum albumins are formed with 2-aminofluorene and 2,4,7-trinitrofluorenone. It was proposed that 2,7-diaminofluorene does not interact with bilirubin bound to primary sites of human and bovine serum albumins due to the spatial hindrance of the albumins binding domains. The spatial arrangement of the bilirubin bound to serum albumin along with the studied mutagens was modelled using ligand docking, which revealed a possibility of an arrangement of the both bilirubin and 2-aminofluorene and 2,4,7-trinitrofluorenone in the primary binding site of human serum albumin.

  6. Non-covalent interaction between dietary stilbenoids and human serum albumin: Structure-affinity relationship, and its influence on the stability, free radical scavenging activity and cell uptake of stilbenoids.

    PubMed

    Cao, Hui; Jia, Xueping; Shi, Jian; Xiao, Jianbo; Chen, Xiaoqing

    2016-07-01

    Dietary stilbenoids are associated with many benefits for human health, which depend on their bioavailability and bioaccessibility. The stilbenoid-human serum albumin (HSA) interactions are investigated to explore the structure-affinity relationship and influence on the stability, free radical scavenging activity and cell uptake of stilbenoids. The structure-affinity relationship of the stilbenoids-HSA interaction was found as: (1) the methoxylation enhanced the affinity, (2) an additional hydroxyl group increases the affinity and (3) the glycosylation significantly weakened the affinity. HSA obviously masked the free radical scavenging potential of stilbenoids. The stabilities of stilbenoids in different medium were determined as: HSA solution>human plasma>Dulbecco's modified Eagle's medium. It appears that the milk enhanced the cell uptake of stilbenoids with multi-hydroxyl groups and weakened the cell uptake of stilbenoids with methoxyl group on EA.hy 926 endothelial cells. The stilbenoids are hardly absorbed by human umbilical vein endothelial cells in the presence of milk.

  7. Interaction of triprolidine hydrochloride with serum albumins: thermodynamic and binding characteristics, and influence of site probes.

    PubMed

    Sandhya, B; Hegde, Ashwini H; Kalanur, Shankara S; Katrahalli, Umesha; Seetharamappa, J

    2011-04-01

    The interaction between triprolidine hydrochloride (TRP) to serum albumins viz. bovine serum albumin (BSA) and human serum albumin (HSA) has been studied by spectroscopic methods. The experimental results revealed the static quenching mechanism in the interaction of TRP with protein. The number of binding sites close to unity for both TRP-BSA and TRP-HSA indicated the presence of single class of binding site for the drug in protein. The binding constant values of TRP-BSA and TRP-HSA were observed to be 4.75 ± 0.018 × 10(3) and 2.42 ± 0.024 × 10(4)M(-1) at 294 K, respectively. Thermodynamic parameters indicated that the hydrogen bond and van der Waals forces played the major role in the binding of TRP to proteins. The distance of separation between the serum albumin and TRP was obtained from the Förster's theory of non-radioactive energy transfer. The metal ions viz., K(+), Ca(2+), Co(2+), Cu(2+), Ni(2+), Mn(2+) and Zn(2+) were found to influence the binding of the drug to protein. Displacement experiments indicated the binding of TRP to Sudlow's site I on both BSA and HSA. The CD, 3D fluorescence spectra and FT-IR spectral results revealed the changes in the secondary structure of protein upon interaction with TRP.

  8. Fluorescence spectroscopic study of serum albumin-bromadiolone interaction: fluorimetric determination of bromadiolone.

    PubMed

    Deepa, Subbiah; Mishra, Ashok K

    2005-07-01

    Bromadiolone (BRD), a substituted 4-hydroxycoumarin derivative, is known to possess anti-coagulant activity with acute toxicity. In this paper, we report a study on the interaction of bromadiolone with the plasma proteins bovine serum albumin (BSA) and human serum albumin (HSA), using the intrinsic fluorescence emission properties of bromadiolone. Bromadiolone is weakly fluorescent in aqueous buffer medium, with an emission at 397 nm. Binding of bromadiolone with serum albumins (SA) leads to a marked enhancement in the fluorescence emission intensity and steady state fluorescence anisotropy (r(ss)), accompanied by a blueshift of 10 nm. In the serum albumin-bromadiolone complex, selective excitation of tryptophan (Trp) residue results in emission from bromadiolone, thereby indicating a Förster type energy transfer from Trp to BRD. This quenching of Trp fluorescence by BRD was used to estimate the binding constant of the SA-BRD complex. The binding constants for BRD with BSA and HSA were 7.5 x 10(4) and 3.7 x 10(5)L mol(-1), respectively. Based on this, a new method involving SA as fluorescence-enhancing reagent for estimation of BRD in aqueous samples has been suggested. The detection limits of bromadiolone under the optimum conditions were 0.77 and 0.19 microg mL(-1) in presence of BSA and HSA, respectively. PMID:15925260

  9. X-ray Structure Analysis of Indazolium trans-[Tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019) Bound to Human Serum Albumin Reveals Two Ruthenium Binding Sites and Provides Insights into the Drug Binding Mechanism

    PubMed Central

    2016-01-01

    Ruthenium(III) complexes are promising candidates for anticancer drugs, especially the clinically studied indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019) and its analogue sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (NKP-1339). Several studies have emphasized the likely role of human serum proteins in the transportation and accumulation of ruthenium(III) complexes in tumors. Therefore, the interaction between KP1019 and human serum albumin was investigated by means of X-ray crystallography and inductively coupled plasma mass spectrometry (ICP-MS). The structural data unambiguously reveal the binding of two ruthenium atoms to histidine residues 146 and 242, which are both located within well-known hydrophobic binding pockets of albumin. The ruthenium centers are octahedrally coordinated by solvent molecules revealing the dissociation of both indazole ligands from the ruthenium-based drug. However, a binding mechanism is proposed indicating the importance of the indazole ligands for binding site recognition and thus their indispensable role for the binding of KP1019. PMID:27196130

  10. Specific, sensitive and accurate quantification of albumin, retinol binding protein and transferrin in human urine and serum by zone immunoelectrophoresis assay (ZIA).

    PubMed

    Vesterberg, O

    1994-05-01

    For zone immunoelectrophoresis assay (ZIA) glass tubes, ID 2 mm and 90 mm high, are filled to 2/3 with buffer containing agarose and antibodies against the protein to be quantified, each sample being pipetted on top of separate agarose gel rods. On electrophoresis at 35-150 V for several hours, the sample proteins enter the gel with resultant immunoprecipitates, visualized by staining. The extension of each immunoprecipitation zone from the upper gel surface (measured with a ruler) is directly proportional to the amount of protein in each sample and can easily be quantitated by comparison with a linear calibration curve. ZIA can be used for quantification of several proteins in blood serum and plasma as well as in urine, as is illustrated for albumin, retinol-binding protein (RBP) and transferrin. The recovery of the pure proteins added to urine is often close to 100%. ZIA has many advantages: (i) simple apparatus and procedure (no gel punching nor cooling), (ii) minimal antiserum consumption (1 mL may allow > 1000 assays), (iii) electrophoresis can be performed within a few hours or overnight, (iv) low coefficient of variation (often < 4%), (v) linear calibration curves, (vi) low detection limit (< 20 ng/mL), (vii) wide concentration ranges, (viii) no kits nor unique antisera preparation are required, and (ix) good agreement with the results from other methods.

  11. Tamoxifen and curcumin binding to serum albumin. Spectroscopic study

    NASA Astrophysics Data System (ADS)

    Maciążek-Jurczyk, M.; Maliszewska, M.; Pożycka, J.; Równicka-Zubik, J.; Góra, A.; Sułkowska, A.

    2013-07-01

    Tamoxifen (TMX) is widely used for the breast cancer treatment and is known as chemopreventive agent. Curcumin (CUR) is natural phenolic compound with broad spectrum of biological activity e.g. anti-inflammatory, antimicrobial, antiviral, antifungal and chemopreventive. Combination of tamoxifen and curcumin could be more effective with lower toxicity than each agent alone in use for the treatment or chemoprevention of breast cancer. Binding of drugs to serum albumin is an important factor, which determines toxicity and therapeutic dosage of the drugs. When two drugs are administered together the competition between them for the binding site on albumin can result in a decrease in bound fraction and an increase in the concentration of free biologically active fraction of drug.

  12. Binding of oxprenolol and propranolol to serum, albumin and alpha 1-acid glycoprotein in man and other species.

    PubMed

    Belpaire, F M; Braeckman, R A; Bogaert, M G

    1984-07-01

    Species differences in binding of basic drugs have only occasionally been studied and we have therefore measured the binding of the beta-adrenergic blockers oxprenolol and propranolol in (1) serum of healthy humans, dogs, rats and rabbits and of rabbits with experimental arthritis, (2) a solution of albumin of these species and (3) a solution of human alpha 1-AGP. In humans, dogs, rats and arthritic rabbits, binding of oxprenolol and propranolol was much higher in serum than in albumin solution; in healthy rabbits serum binding was very low and not different from albumin binding. For both drugs, concentration-dependency was seen in serum of dogs, humans and rats and of arthritic rabbits; a similar concentration-dependency was found for human alpha 1-AGP solution, but not for human albumin and for serum of healthy rabbits. Tris (2-butoxyethyl)-phosphate (TBEP), a known displacer of drugs from alpha 1-AGP in humans, decreased binding in serum of all species except the rabbit. For both beta-blockers, species differences in capacity constants were found; species differences in affinity constants were present only for propranolol. These results suggest that in humans, dog and rat, but much less in rabbits, oxprenolol and propranolol bind mainly to alpha 1-AGP and that binding to alpha 1-AGP is more important for oxprenolol than for propranolol. PMID:6743355

  13. Molecular interactions between some non-steroidal anti-inflammatory drugs (NSAID's) and bovine (BSA) or human (HSA) serum albumin estimated by means of isothermal titration calorimetry (ITC) and frontal analysis capillary electrophoresis (FA/CE).

    PubMed

    Ràfols, Clara; Zarza, Sílvia; Bosch, Elisabeth

    2014-12-01

    The interactions between some non-steroidal anti-inflammatory drugs, NSAIDs, (naproxen, ibuprofen and flurbiprofen) and bovine (BSA) or human (HSA) serum albumin have been examined by means of two complementary techniques, isothermal titration calorimetry (ITC) and frontal analysis/capillary electrophoresis (FA/CE). It can be concluded that ITC is able to measure with high precision the strongest drug-albumin interactions but the higher order interactions can be better determined by means of FA/CE. Then, the combination of both techniques leads to a complete evaluation of the binding profiles between the selected NSAIDs and both kind of albumin proteins. When BSA is the binding protein, the NSAIDs show a strong primary interaction (binding constants: 1.5 × 10(7), 8 × 10(5) and 2 × 10(6) M(-1) for naproxen, ibuprofen and flurbiprofen, respectively), and also lower affinity interactions of the same order for the three anti-inflammatories (about 1.7 × 10(4) M(-1)). By contrast, when HSA is the binding protein two consecutive interactions can be observed by ITC for naproxen (9 × 10(5) and 7 × 10(4) M(-1)) and flurbiprofen (5 × 10(6) and 6 × 10(4) M(-1)) whereas only one is shown for ibuprofen (9 × 10(5) M(-1)). Measurements by FA/CE show a single interaction for each drug being the ones of naproxen and flurbiprofen the same that those evaluated by ITC as the second interaction events. Then, the ability of both techniques as suitable complementary tools to establish the whole interaction NSAIDs-albumin profile is experimentally demonstrated and allows foreseeing suitable strategies to establish the complete drug-protein binding profile. In addition, for the interactions analyzed by means of ITC, the thermodynamic signature is established and the relative contributions of the enthalpic and entropic terms discussed.

  14. Serum Glycated Albumin to Guide the Diagnosis of Diabetes Mellitus.

    PubMed

    Wu, Wan-Chen; Ma, Wen-Ya; Wei, Jung-Nan; Yu, Tse-Ya; Lin, Mao-Shin; Shih, Shyang-Rong; Hua, Cyue-Huei; Liao, Ying-Jhu; Chuang, Lee-Ming; Li, Hung-Yuan

    2016-01-01

    In the diagnosis of diabetes mellitus, hemoglobin A1c (HbA1c) is sometimes measured to determine the need of an oral glucose tolerance test (OGTT). However, HbA1c does not accurately reflect glycemic status in certain conditions. This study was performed to test the possibility that measurement of serum glycated albumin (GA) better assesses the need for OGTT. From 2006 to 2012, 1559 subjects not known to have diabetes or to use anti-diabetic medications were enrolled. Serum GA was measured, and a 75-g OGTT was then performed to diagnose diabetes. Serum GA correlated significantly to age (r = 0.27, p<0.001), serum albumin (r = -0.1179, age-adjusted p = 0.001), body mass index (r = -0.24, age-adjusted p<0.001), waist circumference (r = -0.16, age-adjusted p<0.001), and plasma GA (r = 0.999, p<0.001), but was unaffected by diet (p = 0.8). Using serum GA at 15% for diagnosis of diabetes, the sensitivity, specificity, and area under the receiver-operating characteristic curve were 74%, 85%, and 0.86, respectively. Applying a fasting plasma glucose (FPG) value of < 100 mg/dL to exclude diabetes and of ≥ 126 mg/dL to diagnose diabetes, 14.4% of the study population require an OGTT (OGTT%) with a sensitivity of 78.8% and a specificity of 100%. When serum GA value of 14% and 17% were used to exclude and diagnose diabetes, respectively, the sensitivity improved to 83.3%, with a slightly decrease in specificity (98.2%), but a significant increase in OGTT% (35%). Using combined FPG and serum GA cutoff values (FPG < 100 mg/dL plus serum GA < 15% to exclude diabetes and FPG ≥ 126 mg/dL or serum GA ≥ 17% to diagnose diabetes), the OGTT% was reduced to 22.5% and the sensitivity increased to 85.6% with no change in specificity (98.2%). In the diagnosis of diabetes, serum GA measurements can be used to determine the need of an OGTT.

  15. Crystal structure of equine serum albumin in complex with cetirizine reveals a novel drug binding site.

    PubMed

    Handing, Katarzyna B; Shabalin, Ivan G; Szlachta, Karol; Majorek, Karolina A; Minor, Wladek

    2016-03-01

    Serum albumin (SA) is the main transporter of drugs in mammalian blood plasma. Here, we report the first crystal structure of equine serum albumin (ESA) in complex with antihistamine drug cetirizine at a resolution of 2.1Å. Cetirizine is bound in two sites--a novel drug binding site (CBS1) and the fatty acid binding site 6 (CBS2). Both sites differ from those that have been proposed in multiple reports based on equilibrium dialysis and fluorescence studies for mammalian albumins as cetirizine binding sites. We show that the residues forming the binding pockets in ESA are highly conserved in human serum albumin (HSA), and suggest that binding of cetirizine to HSA will be similar. In support of that hypothesis, we show that the dissociation constants for cetirizine binding to CBS2 in ESA and HSA are identical using tryptophan fluorescence quenching. Presence of lysine and arginine residues that have been previously reported to undergo nonenzymatic glycosylation in CBS1 and CBS2 suggests that cetirizine transport in patients with diabetes could be altered. A review of all available SA structures from the PDB shows that in addition to the novel drug binding site we present here (CBS1), there are two pockets on SA capable of binding drugs that do not overlap with fatty acid binding sites and have not been discussed in published reviews. PMID:26896718

  16. Highly sensitive bovine serum albumin biosensor based on liquid crystal

    NASA Astrophysics Data System (ADS)

    Sharma, Vikash; Kumar, Ajay; Ganguly, Prasun; Biradar, A. M.

    2014-01-01

    A highly sensitive liquid crystal (LC) based bovine serum albumin (BSA) protein biosensor is designed. A uniform homeotropic alignment of nematic LC was observed in BSA free substrate which changed into homogeneous in presence of BSA. The change in the LC orientation is found to depend strongly on BSA concentration. This change in the LC alignment is attributed to the modification in the surface conditions which is verified by contact angle measurements. We have detected an ultra low concentration (0.5 μg/ml) of BSA. The present study demonstrates the utilization of LC in the realization of high sensitivity biosensors.

  17. Comparison of albumin receptors expressed on bovine and human group G streptococci.

    PubMed Central

    Raeder, R; Otten, R A; Boyle, M D

    1991-01-01

    The albumin receptor expressed by bovine group G streptococci was extracted and affinity purified. The protein was characterized for species reactivity, and monospecific antibodies were prepared to the purified receptor. The bovine group G albumin receptor was compared functionally, antigenically, and for DNA homology with the albumin-binding protein expressed by human group G streptococci. In agreement with previous reports, the albumin-binding activity of human strains was mediated by a unique domain of the type III immunoglobulin G-Fc-binding molecule, protein G. The albumin receptor expressed by bovine group G strains was found to lack any immunoglobulin G-binding potential but displayed a wider profile of species albumin reactivity than protein G. Both albumin receptors could inhibit the binding of the other to immobilized human serum albumin, and each displayed similar binding properties. Antigenic comparison of the two albumin receptors demonstrated a low level of cross-reactivity; however comparison at the DNA level, using an oligonucleotide probe specific for the albumin-binding region of protein G, demonstrated that the two albumin receptors expressed by human and bovine group G streptococcal strains do not display significant homology. Images PMID:1846128

  18. Comparative studies of the effects of copper sulfate and zinc sulfate on serum albumins

    NASA Astrophysics Data System (ADS)

    Plotnikova, O. A.; Melnikov, G. V.; Melnikov, A. G.; Kovalenko, A. V.

    2016-04-01

    The work is devoted to the study of the interaction of heavy metals with bovine serum albumin (BSA) and human serum albumin (HSA), by quenching of the intrinsic fluorescence of proteins and fluorescent probe pyrene by heavy metal ions. Sulfates of copper and zinc (CuSO4, ZnSO4) were taken as the metal salts. The value of the Stern-Volmer constants of quenching of intrinsic fluorescence of proteins and fluorescence probe pyrene reduced from Cu (II) to the Zn (II). It was experimentally found that the copper ions have a greater ability to fluorescence quenching, which is probably associated with the greater availability of protein chromophore groups to copper ions and with adsorbed fluorescent probe pyrene in the protein globule.

  19. Characterization and analytical application of Morin - Bovine serum albumin system by spectroscopic approaches

    NASA Astrophysics Data System (ADS)

    Wang, Feng; Huang, Wei; Miao, Xiaowen; Tang, Bo

    2012-12-01

    It is found that the fluorescence intensity of Morin can be strongly quenched by proteins. Based on this, a new fluorimetric method for the determination of protein was developed. Under optimum conditions, the quenchment of Morin fluorescence was in proportion to the concentration of proteins in the range 0.0001-0.1000 g · L-1 for bovine serum albumin (BSA) and 0.0005-0.1000 g · L-1 for human serum albumin (HSA). The reaction mechanism indicates that proteins can bind with Morin at the 3-hydroxyl and the 4-carbonyl and form a non-fluorescence complex 4:1 molar ratio of Morin/BSA, which results in the fluorescence of Morin and BSA are all quenched.

  20. The effect of structural alterations of three mammalian serum albumins on their binding properties

    NASA Astrophysics Data System (ADS)

    Równicka-Zubik, J.; Sułkowski, L.; Maciążek-Jurczyk, M.; Sułkowska, A.

    2013-07-01

    The binding of piroxicam (PIR) to human (HSA), bovine (BSA) and sheep (SSA) serum albumin in native and destabilized/denaturated state was studied by the fluorescence quenching technique. Quenching of the intrinsic fluorescence of three analyzed serum albumins was observed due to selective exciting of tryptophanyl and tyrosil residues at 295 nm and 280 nm. Based on fluorescence emission spectra the quenching (KQ) and binding constants (Ka) were determined. The results showed that PIR is bound mainly in IIA subdomain of HSA and is additionally able to interact with tyrosil groups located in subdomains IB, IIB or IIIA. PIR interacts only with tryptophanyl residues of BSA and SSA [Trp-214, Trp-237 (IIA) and Trp-135, Trp-158 (IB)]. The presence of denaturating factors modified the mechanism of fluorescence quenching of SSA by PIR. Linear Scatchard plots suggest that HSA, BSA and SSA bind PIR in one class of binding sites.

  1. Characterization of the binding of chrysoidine, an illegal food additive to bovine serum albumin.

    PubMed

    Yang, Bingjun; Hao, Fang; Li, Jiarong; Wei, Kai; Wang, Wenyu; Liu, Rutao

    2014-03-01

    Chrysoidine is an industrial azo dye and the presence of chrysoidine in water and food has become an environmental concern due to its negative effects on human beings. Binding of dyes to serum albumins significantly influence their absorption, distribution, metabolism, and excretion properties. In this work, the interactions of chrysoidine with bovine serum albumin (BSA) were explored. Isothermal titration calorimetry results reveal the binding stoichiometry of chrysoidine to BSA is 1:15.5, and van der Waals and hydrogen bonding interactions are the major driving force in the binding of chrysoidine to BSA. Molecular docking simulations show that chrysoidine binds to BSA at a cavity close to Sudlow site I in domain IIA. However, no detectable conformational change of BSA occurs in the presence of chrysoidine as revealed by UV-vis absorption, circular dichroism and fluorescence spectroscopy studies.

  2. Synthesis, characterization and interaction studies of 1,3,4-oxadiazole derivatives of fatty acid with human serum albumin (HSA): A combined multi-spectroscopic and molecular docking study.

    PubMed

    Laskar, Khairujjaman; Alam, Parvez; Khan, Rizwan Hasan; Rauf, Abdul

    2016-10-21

    An efficient synthesis of fatty acid derivatives of 1,3,4-oxadiazole has been reported and comparative study between conventional heating to that of microwave irradiation also described. The newly synthesized compounds (2A-F) were characterized by FT-IR, (1)H NMR, (13)C NMR and mass spectral analysis. The binding interaction of (Z)-2-(heptadec-8'-enyl)-5-methyl-1,3,4-oxadiazole (2C) with human serum albumin (HSA) has been evaluated by UV, fluorescence, circular dichroism (CD) and molecular docking studies. Fluorescence results showed that compound 2C interacts with HSA through static quenching mechanism with binding affinity of 2 × 10(3) M(-1) and Gibbs free energy change (ΔG) was found to be -16.83 kJ mol(-1). Molecular docking studies have been performed to evaluate possible mode of interaction of compound 2C with HSA.

  3. Detection and analysis of tupaia hepatocytes via mAbs against tupaia serum albumin

    PubMed Central

    Liu, Xuan; Yuan, Lunzhi; Yuan, Quan; Zhang, Yali; Wu, Kun; Zhang, Tianying; Wu, Yong; Hou, Wangheng; Wang, Tengyun; Liu, Pingguo; Shih, James Wai Kuo; Cheng, Tong; Xia, Ningshao

    2015-01-01

    On the basis of its close phylogenetic relationship with primates, the development of Tupaia belangeri as an infection animal model and drug metabolism model could provide a new option for preclinical studies, especially in hepatitis virus research. As a replacement for primary human hepatocytes (PHHs), primary tupaia hepatocytes (PTHs) have been widely used. Similar to human serum albumin, tupaia serum albumin (TSA) is the most common liver synthesis protein and is an important biomarker for PTHs and liver function. However, no detection or quantitative method for TSA has been reported. In this study, mouse monoclonal antibodies (mAbs) 4G5 and 9H3 against TSA were developed to recognize PTHs, and they did not show cross-reactivity with serum albumin from common experimental animals, such as the mouse, rat, cow, rabbit, goat, monkey, and chicken. The two mAbs also exhibited good performance in fluorescence activated cell sorting (FACS) analysis and immunofluorescence (IF) detection of PTHs. A chemiluminescent enzyme immune assay method using the two mAbs, with a linear range from 96.89 pg/ml to 49,609.38 pg/ml, was developed for the quantitative detection of TSA. The mAbs and the CLEIA method provide useful tools for research on TSA and PTHs. PMID:26597317

  4. Biologically active protein fragments containing specific binding regions of serum albumin or related proteins

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C. (Inventor)

    1998-01-01

    In accordance with the present invention, biologically active protein fragments can be constructed which contain only those specific portions of the serum albumin family of proteins such as regions known as subdomains IIA and IIIA which are primarily responsible for the binding properties of the serum albumins. The artificial serums that can be prepared from these biologically active protein fragments are advantageous in that they can be produced much more easily than serums containing the whole albumin, yet still retain all or most of the original binding potential of the full albumin proteins. In addition, since the protein fragment serums of the present invention can be made from non-natural sources using conventional recombinant DNA techniques, they are far safer than serums containing natural albumin because they do not carry the potentially harmful viruses and other contaminants that will be found in the natural substances.

  5. Synthesis of fluorine-18 radio-labeled serum albumins for PET blood pool imaging.