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Sample records for albumin hsa binding

  1. 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

  2. Spectroscopic and molecular modeling study on the separate and simultaneous bindings of alprazolam and fluoxetine hydrochloride to human serum albumin (HSA): with the aim of the drug interactions probing.

    PubMed

    Dangkoob, Faeze; Housaindokht, Mohmmad Reza; Asoodeh, Ahmad; Rajabi, Omid; Rouhbakhsh Zaeri, Zeinab; Verdian Doghaei, Asma

    2015-02-25

    The objective of the present research is to study the interaction of separate and simultaneous of alprazolam (ALP) and fluoxetine hydrochloride (FLX) with human serum albumin (HSA) in phosphate buffer (pH 7.4) using different kinds of spectroscopic, cyclic voltammetry and molecular modeling techniques. The absorbance spectra of protein, drugs and protein-drug showed complex formation between the drugs and HSA. Fluorescence analysis demonstrated that ALP and FLX could quench the fluorescence spectrum of HSA and demonstrated the conformational change of HSA in the presence of both drugs. Also, fluorescence quenching mechanism of HSA-drug complexes both separately and simultaneously was suggested as static quenching. The analysis of UV absorption data and the fluorescence quenching of HSA in the binary and ternary systems showed that FLX decreased the binding affinity between ALP and HSA. On the contrary, ALP increased the binding affinity of FLX and HSA. The results of synchronous fluorescence and three-dimensional fluorescence spectra indicated that the binding of drugs to HSA would modify the microenvironment around the Trp and Tyr residues and the conformation of HSA. The distances between Trp residue and the binding sites of the drugs were estimated according to the Förster theory, and it was demonstrated that non-radiative energy transfer from HSA to the drugs occurred with a high probability. Moreover, according to CV measurements, the decrease of peak current in the cyclic voltammogram of the both drugs in the presence of HSA revealed that they interacted with albumin and binding constants were calculated for binary systems which were in agreement with the binding constants obtained from UV absorption and fluorescence spectroscopy. The prediction of the best binding sites of ALP and FLX in binary and ternary systems in molecular modeling approach was done using of Gibbs free energy.

  3. Spectroscopic and molecular modeling study on the separate and simultaneous bindings of alprazolam and fluoxetine hydrochloride to human serum albumin (HSA): With the aim of the drug interactions probing

    NASA Astrophysics Data System (ADS)

    Dangkoob, Faeze; Housaindokht, Mohmmad Reza; Asoodeh, Ahmad; Rajabi, Omid; Rouhbakhsh Zaeri, Zeinab; Verdian Doghaei, Asma

    2015-02-01

    The objective of the present research is to study the interaction of separate and simultaneous of alprazolam (ALP) and fluoxetine hydrochloride (FLX) with human serum albumin (HSA) in phosphate buffer (pH 7.4) using different kinds of spectroscopic, cyclic voltammetry and molecular modeling techniques. The absorbance spectra of protein, drugs and protein-drug showed complex formation between the drugs and HSA. Fluorescence analysis demonstrated that ALP and FLX could quench the fluorescence spectrum of HSA and demonstrated the conformational change of HSA in the presence of both drugs. Also, fluorescence quenching mechanism of HSA-drug complexes both separately and simultaneously was suggested as static quenching. The analysis of UV absorption data and the fluorescence quenching of HSA in the binary and ternary systems showed that FLX decreased the binding affinity between ALP and HSA. On the contrary, ALP increased the binding affinity of FLX and HSA. The results of synchronous fluorescence and three-dimensional fluorescence spectra indicated that the binding of drugs to HSA would modify the microenvironment around the Trp and Tyr residues and the conformation of HSA. The distances between Trp residue and the binding sites of the drugs were estimated according to the Förster theory, and it was demonstrated that non-radiative energy transfer from HSA to the drugs occurred with a high probability. Moreover, according to CV measurements, the decrease of peak current in the cyclic voltammogram of the both drugs in the presence of HSA revealed that they interacted with albumin and binding constants were calculated for binary systems which were in agreement with the binding constants obtained from UV absorption and fluorescence spectroscopy. The prediction of the best binding sites of ALP and FLX in binary and ternary systems in molecular modeling approach was done using of Gibbs free energy.

  4. 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.

  5. 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.

  6. Investigation of human serum albumin (HSA) binding specificity of certain photosensitizers related to pyropheophorbide-a and bacteriopurpurinimide by circular dichroism spectroscopy and its correlation with in vivo photosensitizing efficacy.

    PubMed

    Chen, Yihui; Miclea, Razvan; Srikrishnan, Thamarapu; Balasubramanian, Sathyamangalam; Dougherty, Thomas J; Pandey, Ravindra K

    2005-07-01

    A series of pyropheophorbide-a and bacteriopurpurinimides were investigated to understand the correlation between HSA (site II) binding affinity and in vivo photosensitizing activity. In our study, photosensitizers that bound to site II of HSA produced a significant difference in the circular dichroism spectra of the corresponding complexes, especially at Soret band region of the photosensitizers. Our results suggest that CD spectroscopy of the photosensitizer-HSA complexes could be a valuable tool in screening new photosensitizers before evaluating them for in vivo efficacy.

  7. Interaction of an antiepileptic drug, lamotrigine with human serum albumin (HSA): Application of spectroscopic techniques and molecular modeling methods.

    PubMed

    Poureshghi, Fatemeh; Ghandforoushan, Parisa; Safarnejad, Azam; Soltani, Somaieh

    2017-01-01

    Lamotrigine (an epileptic drug) interaction with human serum albumin (HSA) was investigated by fluorescence, UV-Vis, FTIR, CD spectroscopic techniques, and molecular modeling methods. Binding constant (Kb) of 5.74×10(3) and number of binding site of 0.97 showed that there is a slight interaction between lamotrigine and HSA. Thermodynamic studies was constructed using the flourimetric titrations in three different temperatures and the resulted data used to calculate the parameters using Vant Hoff equation. Decreased Stern Volmer quenching constant by enhanced temperature revealed the static quenching mechanism. Negative standard enthalpy (ΔH) and standard entropy (ΔS) changes indicated that van der Waals interactions and hydrogen bonds were dominant forces which facilitate the binding of Lamotrigine to HSA, the results were confirmed by molecular docking studies which showed no hydrogen binding. The FRET studies showed that there is a possibility of energy transfer between Trp214 and lamotrigine. Also the binding of lamotrigine to HSA in the studied concentrations was not as much as many other drugs, but the secondary structure of the HSA was significantly changed following the interaction in a way that α-helix percentage was reduced from 67% to 57% after the addition of lamotrigine in the molar ratio of 4:1 to HSA. According to the docking studies, lamotrigine binds to IB site preferably.

  8. Spectroscopic, structural and thermodynamic properties of chlorpyrifos bound to serum albumin: A comparative study between BSA and HSA.

    PubMed

    Han, Xiao-Le; Tian, Fang-Fang; Ge, Yu-Shu; Jiang, Feng-Lei; Lai, Lu; Li, Dong-Wei; Yu, Qiu-Liyang; Wang, Jia; Lin, Chen; Liu, Yi

    2012-04-02

    Chlorpyrifos (CPF) is a widely used organophosphate insecticide which could bind with human serum albumin (HSA) and bovine serum albumin (BSA). The binding behavior was studied employing fluorescence, three-dimensional fluorescence, Circular dichroism (CD) spectroscopy, UV-vis absorption spectroscopy, electrochemistry and molecular modeling methods. The fluorescence spectra revealed that CPF causes the quenching of the fluorescence emission of serum albumin. Stern-Volmer plots were made and quenching constants were thus obtained. The results suggested the formation of the complexes of CPF with serum albumins, which were in good agreement with the results from electrochemical experiments. Association constants at 25°C were 3.039 × 10(5) mol L(-1) for HSA, and 0.3307 × 10(5) mol L(-1) for BSA, which could affect the distribution, metabolism, and excretion of pesticide. The alterations of protein secondary structure in the presence of CPF were confirmed by the evidences from UV and CD spectra. Site competitive experiments also suggested that the primary binding site for CPF on serum albumin is close to tryptophan residues 214 of HSA and 212 of BSA, which was further confirmed by molecular modeling.

  9. Binding of Sulpiride to Seric Albumins.

    PubMed

    da Silva Fragoso, Viviane Muniz; de Morais Coura, Carla Patrícia; Hoppe, Luanda Yanaan; Soares, Marília Amável Gomes; Silva, Dilson; Cortez, Celia Martins

    2016-01-04

    The aim of this work was to study the interaction of sulpiride with human serum albumin (HSA) and bovine serum albumin (BSA) through the fluorescence quenching technique. As sulpiride molecules emit fluorescence, we have developed a simple mathematical model to discriminate the quencher fluorescence from the albumin fluorescence in the solution where they interact. Sulpiride is an antipsychotic used in the treatment of several psychiatric disorders. We selectively excited the fluorescence of tryptophan residues with 290 nm wavelength and observed the quenching by titrating HSA and BSA solutions with sulpiride. Stern-Volmer graphs were plotted and quenching constants were estimated. Results showed that sulpiride form complexes with both albumins. Estimated association constants for the interaction sulpiride-HSA were 2.20 (±0.08) × 10⁴ M(-1), at 37 °C, and 5.46 (±0.20) × 10⁴ M(-1), at 25 °C. Those for the interaction sulpiride-BSA are 0.44 (±0.01) × 10⁴ M(-1), at 37 °C and 2.17 (±0.04) × 10⁴ M(-1), at 25 °C. The quenching intensity of BSA, which contains two tryptophan residues in the peptide chain, was found to be higher than that of HSA, what suggests that the primary binding site for sulpiride in albumin should be located next to the sub domain IB of the protein structure.

  10. Binding of Sulpiride to Seric Albumins

    PubMed Central

    da Silva Fragoso, Viviane Muniz; de Morais Coura, Carla Patrícia; Hoppe, Luanda Yanaan; Soares, Marília Amável Gomes; Silva, Dilson; Cortez, Celia Martins

    2016-01-01

    The aim of this work was to study the interaction of sulpiride with human serum albumin (HSA) and bovine serum albumin (BSA) through the fluorescence quenching technique. As sulpiride molecules emit fluorescence, we have developed a simple mathematical model to discriminate the quencher fluorescence from the albumin fluorescence in the solution where they interact. Sulpiride is an antipsychotic used in the treatment of several psychiatric disorders. We selectively excited the fluorescence of tryptophan residues with 290 nm wavelength and observed the quenching by titrating HSA and BSA solutions with sulpiride. Stern-Volmer graphs were plotted and quenching constants were estimated. Results showed that sulpiride form complexes with both albumins. Estimated association constants for the interaction sulpiride–HSA were 2.20 (±0.08) × 104 M−1, at 37 °C, and 5.46 (±0.20) × 104 M−1, at 25 °C. Those for the interaction sulpiride-BSA are 0.44 (±0.01) × 104 M−1, at 37 °C and 2.17 (±0.04) × 104 M−1, at 25 °C. The quenching intensity of BSA, which contains two tryptophan residues in the peptide chain, was found to be higher than that of HSA, what suggests that the primary binding site for sulpiride in albumin should be located next to the sub domain IB of the protein structure. PMID:26742031

  11. 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

  12. Antioxidant flavonoids bind human serum albumin

    NASA Astrophysics Data System (ADS)

    Kanakis, C. D.; Tarantilis, P. A.; Polissiou, M. G.; Diamantoglou, S.; Tajmir-Riahi, H. A.

    2006-10-01

    Human serum albumin (HSA) is a principal extracellular protein with a high concentration in blood plasma and carrier for many drugs to different molecular targets. Flavonoids are powerful antioxidants and prevent DNA damage. The antioxidative protections are related to their binding modes to DNA duplex and complexation with free radicals in vivo. However, flavonoids are known to inhibit the activities of several enzymes such as calcium phospholipid-dependent protein kinase, tyrosine protein kinase from rat lung, phosphorylase kinase, phosphatidylinositol 3-kinase and DNA topoisomerases that exhibit the importance of flavonoid-protein interaction. This study was designed to examine the interaction of human serum albumin (HSA) with quercetin (que), kaempferol (kae) and delphinidin (del) in aqueous solution at physiological conditions, using constant protein concentration of 0.25 mM (final) and various drug contents of 1 μM-1 mM. FTIR and UV-vis spectroscopic methods were used to determine the polyphenolic binding mode, the binding constant and the effects of flavonoid complexation on protein secondary structure. The spectroscopic results showed that flavonoids are located along the polypeptide chains through H-bonding interactions with overall affinity constant of Kque = 1.4 × 10 4 M -1, Kkae = 2.6 × 10 5 M -1 and Kdel = 4.71 × 10 5 M -1. The protein secondary structure showed no alterations at low pigment concentration (1 μM), whereas at high flavonoid content (1 mM), major reduction of α-helix from 55% (free HSA) to 42-46% and increase of β-sheet from 15% (free HSA) to 17-19% and β-anti from 7% (free HSA) to 10-20% occurred in the flavonoid-HSA adducts. The major reduction of HSA α-helix is indicative of a partial protein unfolding upon flavonoid interaction.

  13. Evaluation of albumin structural modifications through cobalt-albumin binding (CAB) assay.

    PubMed

    Lee, Eunyoung; Eom, Ji-Eun; Jeon, Kyung-Hwa; Kim, Tae Hee; Kim, Eunnam; Jhon, Gil-Ja; Kwon, Youngjoo

    2014-03-01

    Human serum albumin (HSA) is the most abundant protein in the human body. HSA injections prepared by fractionating human blood have mainly covered the demand for albumin to treat hypoalbuminemia, the state of low concentration of albumin in blood. HSA in solution may exist in various forms such as monomers, oligomers, polymers, or as mixtures, and its conformational change and/or aggregation may occur easily. Considering these characteristics, there is a great chance of modification and polymer formation during the preparation processes of albumin products, especially injections. The albumin cobalt binding (ACB) test reported by Bar-Or et al. was originally designed to detect ischemia modified albumin (IMA), which contains the modified HSA N-terminal sequence by cleavage of the last two amino acids. In this study, we developed a cobalt albumin binding (CAB) assay to correct the flaws of the ACB test with improving the sensitivity and precision. The newly developed CAB assay easily detects albumin configuration alterations and may be able to be used in developing a quality control method for albumin and its pharmaceutical formulations including albumin injections.

  14. Binding and Irradition Study of the Porphyrin-Protein Complex TSPP-HSA

    NASA Astrophysics Data System (ADS)

    Palos-Chavez, Jorge

    2013-04-01

    Porphyrins are a class of organic molecules that have found numerous applications in biological physics, such as, for example, photodynamic therapy in the treatment of malignant tumors and serving as fluorescent tags for proteins. In this study, we focus on the porphyryn TSPP and its role as a photoactive ligand to the protein HSA (Human Serum Albumin), capable of mediating conformational changes to the structure of HSA via irradiation. The effect of irradiation on the conformation and binding behavior of HSA in buffer solution at physiological pH will be deduced from a combination of spectroscopy tools including absorption, fluorescence, circular dichroism, and fluorescence lifetime decay spectroscopy. Additionally, computational modeling will be employed to complement experimental data. This work was supported through the grant TWD MARC GM07717.

  15. Resveratrol binding to human serum albumin.

    PubMed

    N' soukpoe-Kossi, C N; St-Louis, C; Beauregard, M; Subirade, M; Carpentier, R; Hotchandani, S; Tajmir-Riahi, H A

    2006-12-01

    Resveratrol (Res), a polyphenolic compound found largely in the skin of red grape and wine, exhibits a wide range of pharmaceutical properties and plays a role in prevention of human cardiovascular diseases [Pendurthi et al., Arterioscler. Thromb. Vasc. Biol. 19, 419-426 (1999)]. It shows a strong affinity towards protein binding and used as inhibitor for cyclooxygenase and ribonuclease reductase. The aim of this study was to examine the interaction of resveratrol with human serum albumin (HSA) in aqueous solution at physiological conditions, using a constant protein concentration (0.3 mM) and various pigment contents (microM to mM). FTIR, UV-Visible, CD, and fluorescence spectroscopic methods were used to determine the resveratrol binding mode, the binding constant and the effects of pigment complexation on protein secondary structure. Structural analysis showed that resveratrol bind non-specifically (H-bonding) via polypeptide polar groups with overall binding constant of K(Res) = 2.56 x 10(5) M(-1). The protein secondary structure, analysed by CD spectroscopy, showed no major alterations at low resveratrol concentrations (0.125 mM), whereas at high pigment content (1 mM), major increase of alpha-helix from 57% (free HSA) to 62% and a decrease of beta-sheet from 10% (free HSA) to 7% occurred in the resveratrol-HSA complexes. The results indicate a partial stabilization of protein secondary structure at high resveratrol content.

  16. Binding of coumarins to site I of human serum albumin. Effect of the fatty acids.

    PubMed

    Zatón, A M; Ferrer, J M; Ruiz de Gordoa, J C; Marquínez, M A

    1995-07-14

    It is known that binding site I on human serum albumin (HSA) consists of a zone of two overlapping regions: the specific binding region represented by warfarin binding and the specific binding region represented by azapropazone and phenylbutazone binding. In this paper binding parameters to defatted HSA and to HSA with fatty acids (molar ratio of fatty acid/HSA = 4) were compared. High-affinity binding sites for warfarin, 4-chromanol, 4-hydroxycoumarin, coumarin, 3-acetylcoumarin and phenylbutazone (759,549 M-1 > Ka > 67,024 M-1) constitute binding site I on HSA. In this binding area defatted HSA can bind two molecules of warfarin, but the presence of fatty acids diminish the binding capacity of warfarin to HSA (2 > n > 1).

  17. Interactive association of drugs binding to human serum albumin.

    PubMed

    Yang, Feng; Zhang, Yao; Liang, Hong

    2014-02-27

    Human serum albumin (HSA) is an abundant plasma protein, which attracts great interest in the pharmaceutical industry since it can bind a remarkable variety of drugs impacting their delivery and efficacy and ultimately altering the drug's pharmacokinetic and pharmacodynamic properties. Additionally, HSA is widely used in clinical settings as a drug delivery system due to its potential for improving targeting while decreasing the side effects of drugs. It is thus of great importance from the viewpoint of pharmaceutical sciences to clarify the structure, function, and properties of HSA-drug complexes. This review will succinctly outline the properties of binding site of drugs in IIA subdomain within the structure of HSA. We will also give an overview on the binding characterization of interactive association of drugs to human serum albumin that may potentially lead to significant clinical applications.

  18. 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

  19. 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

  20. Effects of glycation on meloxicam binding to human serum albumin

    NASA Astrophysics Data System (ADS)

    Trynda-Lemiesz, Lilianna; Wiglusz, Katarzyna

    2011-05-01

    The current study reports a binding of meloxicam a pharmacologically important new generation, non-steroidal anti-inflammatory drug to glycated form of the human serum albumin (HSA). The interaction of the meloxicam with nonglycated and glycated albumin has been studied at pH 7.4 in 0.05 M sodium phosphate buffer with 0.1 M NaCl, using fluorescence quenching technique and circular dichroism spectroscopy. Results of the present study have shown that the meloxicam could bind both forms of albumin glycated and nonglycated at a site, which was close to the tryptophan residues. Similarly, how for native albumin glycated form has had one high affinity site for the drug with association constants of the order of 10 5 M -1. The glycation process of the HSA significantly has affected the impact of the meloxicam on the binding of other ligands such as warfarin and bilirubin. The affinity of the glycated albumin for bilirubin as for native albumin has been reduced by meloxicam but observed effect was weaker by half (about 20%) compared with nonglycated albumin. In contrast to the native albumin meloxicam binding to glycated form of the protein only slightly affected the binding of warfarin. It seemed possible that the effects on warfarin binding might be entirely attributable to the Lys 199 modification which was in site I.

  1. Spectroscopic study on binding of rutin to human serum albumin

    NASA Astrophysics Data System (ADS)

    Pastukhov, Alexander V.; Levchenko, Lidiya A.; Sadkov, Anatoli P.

    2007-10-01

    Steady-state and time-resolved fluorescence spectroscopy techniques were used to study the interaction of the flavonoid rutin with human serum albumin (HSA) as well as spectral properties of the protein-bound flavonoid. Both quenching of the intrinsic fluorescence of the protein (Trp214) and the ligand fluorescence, appearing upon complexation with HSA, were used to determine binding parameters. The binding constant determined from the quenching of the Trp214 fluorescence by rutin is equal to 6.87 ± 0.22 × 10 4 M -1 and that obtained from the fluorescence of HSA-bound rutin is 3.8 ± 0.4 × 10 4 M -1. Based on the Job plot analysis, the 1:1 binding stoichiometry for the HSA-rutin complex was determined. The efficient quenching of the Trp214 fluorescence by rutin, fluorescence resonance energy transfer from excited Trp214 to rutin, and competitive binding of warfarin indicate that the binding site for the flavonoid is situated within subdomain IIA of HSA. The presence of the sugar moiety in the flavonoid molecule reduces affinity of rutin for binding to HSA but does not affect the binding stoichiometry and location of the binding site compared with aglycone analogues.

  2. Investigation of the behavior of HSA upon binding to amlodipine and propranolol: Spectroscopic and molecular modeling approaches

    NASA Astrophysics Data System (ADS)

    Housaindokht, Mohammad Reza; Rouhbakhsh Zaeri, Zeinab; Bahrololoom, Mahmood; Chamani, Jamshid; Bozorgmehr, Mohammad Reza

    2012-01-01

    The interaction between human serum albumin (HSA) and two drugs - amlodipine and propranolol - was investigated using fluorescence, UV absorption and circular dichroism (CD) spectroscopy. In addition, the binding site was established by applying molecular modeling technique. Fluorescence data suggest that amlodipine will quench the intrinsic fluorescence of HSA; whereas propranolol enhances the fluorescence of HSA. The binding constants for the interaction of amlodipine and propranolol with HSA were found to be 3.63 × 10 5 M -1 and 2.29 × 10 4 M -1, respectively. The percentage of secondary structure feature of each one of the HSA-bound drugs, i.e. the α-helix content, was estimated empirically by circular dichroism. The results indicated that amlodipine causes an increase, and that propranolol leads to a decrease in α-helix content of HSA. The spectroscopic analysis indicates that the binding mechanisms of the two drugs are different from each other. The data obtained by the molecular modeling study indicated that these drugs bind, with different affinity, to different sites located in subdomain IIA and IIIA.

  3. Species-dependent stereoselective drug binding to albumin: a circular dichroism study.

    PubMed

    Pistolozzi, Marco; Bertucci, Carlo

    2008-03-01

    Drug binding to albumins from different mammalian species was investigated to disclose evidence of species-dependent stereoselectivity in drug-binding processes and affinities. This aspect is important for evaluating the reliability of extrapolating distribution data among species. The circular dichroism (CD) signal induced by drug binding to the albumins [human serum albumin (HSA), bovine serum albumin (BSA), rat serum albumin (RSA), and dog serum albumin (DSA)] were measured and analyzed. The binding of selected drugs and metabolites to HSA significantly differed from the binding to the other albumins in terms of affinity and conformation of the bound ligands. In particular, phenylbutazone, a marker of site one on HSA, showed a higher affinity for binding to BSA with respect to RSA, HSA, and DSA, respectively. In the case of diazepam, a marker of site two on HSA, the affinity decreased in order from HSA to DSA, RSA, and BSA. The induced CD spectra were similar in terms of energy and band signs, suggesting almost the same conformation for the bound drug to the different albumins. Stereoselectivity was high for the binding of ketoprofen to HSA and RSA. A different sign was observed for the CD spectra induced by the drug to the two albumins because of the prevalence of a different conformation of the bound drug. Interestingly, the same induced CD spectra were obtained using either the racemic form or the (S)-enantiomer. Finally, significant differences were observed in the affinity of bilirubin, being highest for BSA, then decreasing for RSA, HSA, and DSA. A more complex conformational equilibrium was observed for bound bilirubin.

  4. 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.

  5. Comparison of the binding behavior of FCCP with HSA and HTF as determined by spectroscopic and molecular modeling techniques.

    PubMed

    Moghaddam, Maryam Mahmoodian; Pirouzi, Malihe; Saberi, Mohammad Reza; Chamani, Jamshidkhan

    2014-06-01

    The interaction of carbonylcyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) with human serum albumin (HSA) and human transferrin (HTF) was investigated using multiple spectroscopy, molecular modeling, zeta-potential and conductometry measurements of aqueous solutions at pH 7.4. The fluorescence, UV/vis and polarization fluorescence spectroscopy data disclosed that the drug-protein complex formation occurred through a remarkable static quenching. Based on the fluorescence quenching, two sets of binding sites with distinct affinities for FCCP existed in the two proteins. Steady-state and polarization fluorescence analysis showed that there were more affinities between FCCP and HSA than HTF. Far UV-CD and synchronous fluorescence studies indicated that FCCP induced more structural changes on HSA. The resonance light scattering (RLS) and zeta-potential measurements suggested that HTF had a greater resistance to drug aggregation, whereas conductometry measurements expressed the presence of free ions improving the resistance of HSA to aggregation. Thermodynamic measurements implied that a combination of electrostatic and hydrophobic forces was involved in the interaction between FCCP with both proteins. The phase diagram plots indicated that the presence of second binding site on HSA and HTF was due to the existence of intermediate structures. Site marker competitive experiments demonstrated that FCCP had two distinct binding sites in HSA which were located in sub-domains IIA and IIIA and one binding site in the C-lobe of HTF as confirmed by molecular modeling. The obtained results suggested that both proteins could act as drug carriers, but that the HSA potentially had a higher capacity for delivering FCCP to cancerous tissues.

  6. 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.

  7. Production of Recombinant Polypeptides Containing One GA-Module and Analysis of Their Ability to Bind to Human Albumin.

    PubMed

    Bormotova, E A; Gupalova, T V

    2016-11-01

    Surface proteins of many bacterial species interact with human serum albumin (HSA) via a special region of amino acid sequence termed GA module. For instance, surface peptostreptococcal albumin-binding protein of anaerobic bacteria Peptostreptococcus magnus contains one HSA-binding GA-module. Protein G from group G and C Streptococcus strains isolated from humans has HSA-binding region consisting of three GA-modules. HSA-binding protein containing two GA-modules was found in strains of group G Streptococcus of animal origin. We obtained two recombinant polypeptides GA1 and GA2 congaing one GA-module each. Recombinant polypeptide with two GA-modules binds HSA with a much higher affinity than polypeptides GA1 and GA2 containing one GA-module. Polypeptide with the second GAmodule more effectively binds HSA than polypeptides with the GA-module.

  8. A comparison investigation of DNP-binding effects to HSA and HTF by spectroscopic and molecular modeling techniques.

    PubMed

    Zolfagharzadeh, Mahboobeh; Pirouzi, Maliheh; Asoodeh, Ahmad; Saberi, Mohammad Reza; Chamani, Jamshidkhan

    2014-12-01

    This paper describes the interaction between 2,4-dinitrophenol (DNP) with the two drug carrier proteins - human serum albumin (HSA) and human holo transferrin (HTF). Hence, binding characteristics of DNP to HSA and HTF were analyzed by spectroscopic and molecular modeling techniques. Based on results obtained from fluorescence spectroscopy, DNP had a strong ability to quench the intrinsic fluorescence of HSA and HTF through a static quenching procedure. The binding constant and the number of binding sites were calculated as 2.3 × 10(11) M(-1) and .98 for HSA, and 1.7 × 10(11) M(-1) and 1.06 for HTF, respectively. In addition, synchronous fluorescence results showed that the microenvironment of Trp had a slight tendency of increasing its hydrophobicity, whereas the microenvironment of the Tyr residues of HSA did not change and that of HTF showed a significant trend (red shift of about 4 nm) of an increase in polarity. The distance between donor and acceptor was obtained by the Förster energy according to fluorescence resonance energy transfer, and was found to be 3.99 and 3.72 nm for HSA and HTF, respectively. The critical induced aggregation concentration (CCIAC) of the drug on both proteins was determined and confirmed by an inflection point of the zeta potential behavior. Circular dichroism data revealed that the presence of DNP caused a decrease of the α-helical content of HSA and HTF, and induced a remarkable mild denaturation of both proteins. The molecular modeling data confirmed our experimental results. This study is deemed useful for determining drug dosage.

  9. Camptothecin-binding site in human serum albumin and protein transformations induced by drug binding.

    PubMed

    Fleury, F; Ianoul, A; Berjot, M; Feofanov, A; Alix, A J; Nabiev, I

    1997-07-14

    Circular dichroism (CD) and Raman spectroscopy were employed in order to locate a camptothecin (CPT)-binding site within human serum albumin (HSA) and to identify protein structural transformations induced by CPT binding. A competitive binding of CPT and 3'-azido-3'-deoxythymidine (a ligand occupying IIIA structural sub-domain of the protein) to HSA does not show any competition and demonstrates that the ligands are located in the different binding sites, whereas a HSA-bound CPT may be replaced by warfarin, occupying IIA structural sub-domain of the protein. Raman and CD spectra of HSA and HSA/CPT complexes show that the CPT-binding does not induce changes of the global protein secondary structure. On the other hand, Raman spectra reveal pronounced CPT-induced local structural modifications of the HSA molecule, involving changes in configuration of the two disulfide bonds and transfer of a single Trp-residue to hydrophilic environment. These data suggest that CPT is bound in the region of interdomain connections within the IIA structural domain of HSA and it induces relative movement of the protein structural domains.

  10. Biosensor analysis of the interaction between immobilized human serum albumin and drug compounds for prediction of human serum albumin binding levels.

    PubMed

    Frostell-Karlsson, A; Remaeus, A; Roos, H; Andersson, K; Borg, P; Hämäläinen, M; Karlsson, R

    2000-05-18

    The interactions between a set of drugs, selected on the basis of reported human serum albumin (HSA) binding levels, and immobilized HSA were investigated using surface plasmon resonance technology. Major HSA binding sites were available after immobilization. The intensity of the signal obtained from the interaction of the drug with the HSA surface was correlated with the reported HSA binding level. Drugs were classified into groups corresponding to high, medium, or low HSA binding based on the injection of the drug at 80 microM concentration. A set of 10 drugs binding to alpha(1)-acid glycoprotein (AGP) was also investigated and correlated with reported AGP binding data. The throughput of the presented assay is 100 compounds/24 h, and the sample consumption is less than 100 microL (8 nmol).

  11. Binding site identification of metformin to human serum albumin and glycated human serum albumin by spectroscopic and molecular modeling techniques: a comparison study.

    PubMed

    Rahnama, Elaheh; Mahmoodian-Moghaddam, Maryam; Khorsand-Ahmadi, Sabra; Saberi, Mohammad Reza; Chamani, Jamshidkhan

    2015-01-01

    The interaction between metformin and human serum albumin (HSA), as well as its glycated form (gHSA) was investigated by multiple spectroscopic techniques, zeta potential, and molecular modeling under physiological conditions. The steady state and time-resolved fluorescence data displayed the quenching mechanism of HSA-metformin and gHSA-metformin was static. The binding information, including the binding constants, number of binding sites, effective quenching constant showed that the binding affinity of metformin to HSA was greater than to gHSA which also confirmed by anisotropy measurements. It was determined that metformin had two and one set of binding sites on HSA and gHSA, respectively. Far-UV CD spectra of proteins demonstrated that the α-helical content decreased with increasing metformin concentration. The zeta potential and resonance light scattering (RLS) diagrams provided that lower drug concentration induced metformin aggregation on gHSA surface as compare to HSA. The increase in polarizability was one of the important factors for the enhancement of RLS and the formation of drug/protein complexes. The zeta potential results suggested that both hydrophobic and electrostatic interactions played important roles in the protein-metformin complex formation. Site marker experiments and molecular modeling showed that the metformin bound to subdomain IIIA (Sudlow's site II) on HSA and gHSA.

  12. Immunochemical studies on HNE-modified HSA: Anti-HNE-HSA antibodies as a probe for HNE damaged albumin in SLE.

    PubMed

    Khan, Farzana; Moinuddin; Mir, Abdul Rouf; Islam, Sidra; Alam, Khursheed; Ali, Asif

    2016-05-01

    Non-enzymatic lipid peroxidation of cellular membranes occurs during periods of sustained oxidative stress. 4-Hydroxynonenal (HNE), the most reactive lipid peroxidation product, is capable of modifying and/or cross-linking proteins leading to impaired physiological functions. The formation of protein adducts produce structural modifications which generate neo-antigens and induce auto-antibodies. Enhanced oxidative stress and accumulation of HNE-modified proteins are associated with systemic lupus erythematosus (SLE) and other autoimmune diseases. This study has probed the role of lipid peroxidation derived aldehydes in SLE. We report the structural perturbations in human serum albumin (HSA) upon modification with HNE and the consequential enhanced immunogenicity. The induced antibodies were found to be highly specific for the immunogen and exhibited cross-reactivity with HNE-modified epitopes on proteins, amino acids and nucleic acid. The experimentally induced anti-HNE-HSA antibodies appreciably recognized HNE modified epitopes on the HSA obtained from SLE patients. These antibodies, therefore, form a good immunochemical probe to detect such damages in lupus patients. Possible role of anti-HNE-HSA antibodies as a marker for detection/progression of SLE has been discussed.

  13. Modulation of linoleic acid-binding properties of human serum albumin by divalent metal cations.

    PubMed

    Nemashkalova, Ekaterina L; Permyakov, Eugene A; Permyakov, Sergei E; Litus, Ekaterina A

    2017-03-16

    Human serum albumin (HSA) is an abundant multiligand carrier protein, linked to progression of Alzheimer's disease (AD). Blood HSA serves as a depot of amyloid β (Aβ) peptide. Aβ peptide-buffering properties of HSA depend on interaction with its ligands. Some of the ligands, namely, linoleic acid (LA), zinc and copper ions are involved into AD progression. To clarify the interplay between LA and metal ion binding to HSA, the dependence of LA binding to HSA on Zn(2+), Cu(2+), Mg(2+) and Ca(2+) levels and structural consequences of these interactions have been explored. Seven LA molecules are bound per HSA molecule in the absence of the metal ions. Zn(2+) binding to HSA causes a loss of one bound LA molecule, while the other metals studied exert an opposite effect (1-2 extra LA molecules are bound). In most cases, the observed effects are not related to the metal-induced changes in HSA quaternary structure. However, the Zn(2+)-induced decline in LA capacity of HSA could be due to accumulation of multimeric HSA forms. Opposite to Ca(2+)/Mg(2+)-binding, Zn(2+) or Cu(2+) association with HSA induces marked changes in its hydrophobic surface. Overall, the divalent metal ions modulate LA capacity and affinity of HSA to a different extent. LA- and Ca(2+)-binding to HSA synergistically support each other. Zn(2+) and Cu(2+) induce more pronounced changes in hydrophobic surface and quaternary structure of HSA and its LA capacity. A misbalanced metabolism of these ions in AD could modify interactions of HSA with LA, other fatty acids and hydrophobic substances, associated with AD.

  14. Imidazolium ionic liquids as solvents of pharmaceuticals: influence on HSA binding and partition coefficient of nimesulide.

    PubMed

    Azevedo, Ana M O; Ribeiro, Diogo M G; Pinto, Paula C A G; Lúcio, Marlene; Reis, Salette; Saraiva, M Lúcia M F S

    2013-02-25

    In this work, the influence of imidazolium ionic liquids (ILs) on bio-chemical parameters that influence the in vivo behavior of nimesulide was evaluated. In this context, the binding of nimesulide to human serum albumin (HSA), in IL media, was studied. In parallel, the evaluation of the interaction of drug-IL systems, with micelles of hexadecylphosphocholine (HDPC), enabled the calculation of partition coefficients (K(p)). Both assays were performed in buffered media in the absence and in the presence of emim [BF(4)], emim [Ms] and emim [TfMs] 1%. Even though there was an increase of the dissociation constant (K(d)) in IL media, nimesulide still binds to HSA by means of strong interactions. The thermodynamic analysis indicates that the interaction is spontaneous for all the tested systems. Moreover, the studied systems exhibited properties that are favorable to the interaction of the drug with biological membranes, with K(p) values 2.5-3.5 higher than in aqueous environment. The studied nimesulide-IL systems presented promising characteristics regarding the absorption and distribution of the drug in vivo, so that the studied solvents seem to be good options for drug delivery.

  15. Reversible binding of ethacrynic acid to human serum albumin: difference circular dichroism study.

    PubMed

    Bertucci, C; Nanni, B; Salvadori, P

    1999-01-01

    The reversible binding of ethacrynic acid was characterized by a difference circular dichroism method. A 2/1 stoichiometry was determined for the [drug]/[HSA] (human serum albumin) complex. The reversible binding of ethacrynic acid to HSA determines direct competition with ligands that selectivity bind to site II and to the fatty acid site. Furthermore, indirect competition was shown for ligands for site I (anti-cooperative) and to site III (cooperative).

  16. 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.

  17. Bromophenol blue binding to mammalian albumins and displacement of albumin-bound bilirubin.

    PubMed

    Kim, B Boon; Abdul Kadir, H; Tayyab, S

    2008-10-15

    Interaction of bromophenol blue (BPB) with serum albumins from different mammalian species, namely, human (HSA), bovine (BSA), goat (GSA), sheep (SSA), rabbit (RbSA), porcine (PSA) and dog (DSA) was studied using absorption and absorption difference spectroscopy. BPB-albumin complexes showed significant differences in the spectral characteristics, i.e., extent of bathochromic shift and hypochromism relative to the spectral features of free BPB. Absorption difference spectra of these complexes also showed variations in the position of maxima and absorption difference (deltaAbs.) values. Absorption difference spectra of different bilirubin (BR)-albumin complexes showed a significant blue shift accompanied by decrease in deltaAbs. values in presence of BPB which were indicative of the displacement of bound BR from its binding site in BR-albumin complexes. These changes in the difference spectral characteristics of BR-albumin complexes were more marked at higher BPB concentration. However, the extent of these changes was different for different BR-albumin complexes. Taken together, all these results suggest that BPB partially shares BR binding site on albumin and different mammalian albumins show differences in the microenvironment of the BR/BPB binding site.

  18. 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).

  19. Protein-protein binding before and after photo-modification of albumin

    NASA Astrophysics Data System (ADS)

    Rozinek, Sarah C.; Glickman, Randolph D.; Thomas, Robert J.; Brancaleon, Lorenzo

    2016-03-01

    Bioeffects of directed-optical-energy encompass a wide range of applications. One aspect of photochemical interactions involves irradiating a photosensitizer with visible light in order to induce protein unfolding and consequent changes in function. In the past, irradiation of several dye-protein combinations has revealed effects on protein structure. Beta lactoglobulin, human serum albumin (HSA) and tubulin have all been photo-modified with meso-tetrakis(4- sulfonatophenyl)porphyrin (TSPP) bound, but only in the case of tubulin has binding caused a verified loss of biological function (loss of ability to form microtubules) as a result of this light-induced structural change. The current work questions if the photo-induced structural changes that occur to HSA, are sufficient to disable its biological function of binding to osteonectin. The albumin-binding protein, osteonectin, is about half the molecular weight of HSA, so the two proteins and their bound product can be separated and quantified by size exclusion high performance liquid chromatography. TSPP was first bound to HSA and irradiated, photo-modifying the structure of HSA. Then native HSA or photo-modified HSA (both with TSPP bound) were compared, to assess loss in HSA's innate binding ability as a result of light-induced structure modification.

  20. Polyamine analogues bind human serum albumin.

    PubMed

    Beauchemin, R; N'soukpoé-Kossi, C N; Thomas, T J; Thomas, T; Carpentier, R; Tajmir-Riahi, H A

    2007-10-01

    Polyamine analogues show antitumor activity in experimental models, and their ability to alter activity of cytotoxic chemotherapeutic agents in breast cancer is well documented. Association of polyamines with nucleic acids and protein is included in their mechanism of action. The aim of this study was to examine the interaction of human serum albumin (HSA) with several polyamine analogues, such as 1,11-diamino-4,8-diazaundecane (333), 3,7,11,15-tetrazaheptadecane.4HCl (BE-333), and 3,7,11,15,19-pentazahenicosane.5HCl (BE-3333), in aqueous solution at physiological conditions using a constant protein concentration and various polyamine contents (microM to mM). FTIR, UV-visible, and CD spectroscopic methods were used to determine the polyamine binding mode and the effects of polyamine complexation on protein stability and secondary structure. Structural analysis showed that polyamines bind nonspecifically (H-bonding) via polypeptide polar groups with binding constants of K333 = 9.30 x 10(3) M(-1), KBE-333 = 5.63 x 10(2) M(-1), and KBE-3333 = 3.66 x 10(2) M(-1). The protein secondary structure showed major alterations with a reduction of alpha-helix from 55% (free protein) to 43-50% and an increase of beta-sheet from 17% (free protein) to 29-36% in the 333, BE-333, and BE-3333 complexes, indicating partial protein unfolding upon polyamine interaction. HSA structure was less perturbed by polyamine analogues compared to those of the biogenic polyamines.

  1. Binding of suprofen to human serum albumin. Role of the suprofen carboxyl group.

    PubMed

    Maruyama, T; Lin, C C; Yamasaki, K; Miyoshi, T; Imai, T; Yamasaki, M; Otagiri, M

    1993-03-09

    The binding of suprofen (SP), a non-steroidal anti-inflammatory drug of the arylpropionic acid class, and its methyl ester derivative (SPM) to human serum albumin (HSA) was studied by dialysis and spectroscopic techniques. In spite of the remarkable differences in the physicochemical properties of SP and SPM, the binding of each molecule to HSA was quantitatively very similar. Thermodynamic analysis suggests that the interaction of SP with HSA may be caused by electrostatic as well as hydrophobic forces, whereas the interactions with SPM may be explained by hydrophobic and van der Waals forces. Similarities in the difference UV absorption spectra between ligand-detergent micelle and -HSA systems indicate that the SP and SPM molecules are inserted into a hydrophobic crevice on HSA. The same studies suggest that the carboxyl group of SP interacts with a cationic sub-site which is closely associated with the SP binding site. Proton relaxation rate measurements indicate that the thiophen ring and propanoate portion of the SP molecule is the major binding site for HSA. The locations of SP and SPM binding sites were identified by using fluorescence probes which bind to a known site on HSA. The displacement data implied that SP primarily binds to Site II, while the high affinity site of SPM as well as low affinity site of SP are at the warfarin binding site in the Site I area. From binding data with chemically modified HSA derivatives, it is likely that highly reactive tyrosine (Tyr) and lysine (Lys) residues, which may be Tyr-411 and Lys-195, are specifically involved in SP binding. In contrast, these two residues are clearly separated from the SPM binding site. The binding of SP and SPM is independent of conformational changes on HSA that accompany N-B transition. There is evidence that the carboxyl group may play a crucial role in the high affinity binding processes of SP to HSA.

  2. 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.

  3. Alteration of methotrexate binding to human serum albumin induced by oxidative stress. Spectroscopic comparative study

    NASA Astrophysics Data System (ADS)

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

    2016-01-01

    Changes of oxidative modified albumin conformation by comparison of non-modified (HSA) and modified (oHSA) human serum albumin absorption spectra, Red Edge Excitation Shift (REES) effect and fluorescence synchronous spectra were investigated. Studies of absorption spectra indicated that changes in the value of absorbance associated with spectral changes in the region from 200 to 250 nm involve structural alterations related to variations in peptide backbone conformation. Analysis of the REES effect allowed for the observation of changes caused by oxidation in the region of the hydrophobic pocket containing the tryptophanyl residue. Synchronous fluorescence spectroscopy confirmed changes of the position of the tryptophanyl and tyrosil residues fluorescent band. Effect of oxidative stress on binding of methotrexate (MTX) was investigated by spectrofluorescence, UV-VIS and 1HNMR spectroscopy. MTX caused the fluorescence quenching of non-modified (HSA) and modified (oHSA) human serum albumin molecule. The values of binding constants, Hill's coefficients and a number of binding sites in the protein molecule in the high affinity binding site were calculated for the binary MTX-HSA and MTX-oHSA systems. For these systems, qualitative analysis in the low affinity binding sites was performed with the use of the 1HNMR technique.

  4. 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.

  5. Development of an albumin copper binding (ACuB) assay to detect ischemia modified albumin.

    PubMed

    Eom, Ji-Eun; Lee, Eunyoung; Jeon, Kyung-Hwa; Sim, Jeongeun; Suh, Minah; Jhon, Gil-Ja; Kwon, Youngjoo

    2014-01-01

    Myocardial ischemia (MI) induces many changes in the body, including pH decrease and electrolyte imbalance. No obvious symptoms of MI appear until irreversible cellular injuries occur. Since early treatment is critical for recovery from ischemia, the development of reliable diagnostic tool is demanded to detect the early ischemic status. Ischemia modified albumin (IMA), formed by cleavage of the last two amino acids of the human serum albumin (HSA) N-terminus, has been considered so far as the most trustworthy and accurate marker for the investigation of ischemia. IMA levels are elevated in plasma within a few minutes of ischemic onset, and may last for up to 6 h. In the present study, we developed a novel assay for the examination of IMA levels to ameliorate the known albumin cobalt binding (ACB) test established previously. We observed a stronger copper ion bound to the HSA N-terminal peptide than cobalt ion by HPLC and ESI-TOF mass spectrometric analyses. The copper ion was employed with lucifer yellow (LY), a copper-specific reagent to develop a new albumin copper binding (ACuB) assay. The parameters capable of affecting the assay results were optimized, and the finally-optimized ACuB assay was validated. The result of the IMA level measurement in normal versus stroke rat serum suggests that the ACuB assay is likely to be a reliable and sensitive method for the detection of ischemic states.

  6. Adherence of platelets to in situ albumin-binding surfaces under flow conditions: role of surface-adsorbed albumin.

    PubMed

    Guha Thakurta, Sanjukta; Miller, Robert; Subramanian, Anuradha

    2012-08-01

    Surfaces that preferentially bind human serum albumin (HSA) were generated by grafting albumin-binding linear peptide (LP1) onto silicon surfaces. The research aim was to evaluate the adsorption pattern of proteins and the adhesion of platelets from platelet-poor plasma and platelet-rich plasma, respectively, by albumin-binding surfaces under physiological shear rate (96 and 319 s(-1)) conditions. Bound proteins were quantified using enzyme-linked immunosorbent assays (ELISAs) and two-dimensional gel electrophoresis. A ratio of ∼1000:100:1 of adsorbed HSA, human immunoglobulin (HIgG) and human fibrinogen (HFib) was noted, respectively, on LP1-functionalized surfaces, and a ratio of ∼5:2:1 of the same was noted on control surfaces, as confirmed by ELISAs. The surface-adsorbed von Willebrand factor was undetectable by sensitive ELISAs. The amount of adhered platelets correlated with the ratio of adsorbed HSA/HFib. Platelet morphology was more rounded on LP1-functionalized surfaces when compared to control surfaces. The platelet adhesion response on albumin-binding surfaces can be explained by the reduction in the co-adsorption of other plasma proteins in a surface environment where there is an excess of albumin molecules, coupled with restrictions in the conformational transitions of other surface-adsorbed proteins into hemostatically active forms.

  7. Regulation of amantadine hydrochloride binding with IIA subdomain of human serum albumin by fatty acid chains.

    PubMed

    Yang, Feng; Lee, Philbert; Ma, Zhiyuan; Ma, Li; Yang, Guoping; Wu, Xiaoyang; Liang, Hong

    2013-01-01

    Human serum albumin (HSA) is a major protein component of blood plasma that has been exploited to bind and transport a wide variety of endogenous and exogenous organic compounds. Although anionic drugs readily associate with the IIA subdomain of HSA, most cationic drugs poorly associate with HSA at this subdomain. In this study, we propose to improve the association between cationic drugs and HSA by modifying HSA with fatty acid chains. For our experiments, we tested amantadine hydrochloride, a cationic drug with antiviral and antiparkinsonian effects. Our results suggest that extensive myristoylation of HSA can help stabilize the interaction between amantadine and HSA in vitro. Our X-ray crystallography data further elucidate the structural basis of this regulation. Additionally, our crystallography data suggest that anionic drugs, with a functional carboxylate group, may enhance the association between amantadine and HSA by a mechanism similar to myristoylation. Ultimately, our results provide critical structural insight into this novel association between cationic drugs and the HSA IIA subdomain, raising the tempting possibility to fully exploit the unique binding capacity of HSA's IIA subdomain to achieve simultaneous delivery of anionic and cationic drugs.

  8. Optical spectroscopic exploration of binding of Cochineal Red A with two homologous serum albumins.

    PubMed

    Bolel, Priyanka; Mahapatra, Niharendu; Halder, Mintu

    2012-04-11

    Cochineal Red A is a negatively charged synthetic azo food colorant and a potential carcinogen. We present here the study of binding of Cochineal Red A with two homologous serum albumins, human (HSA) and bovine (BSA), in aqueous pH 7.4 buffer by optical spectroscopic techniques. Protein intrinsic fluorescence quenching by Cochineal Red A occurs through ground-state static interaction and its binding with BSA is stronger than with HSA. The magnitudes of thermodynamic parameters suggest that dye binding occurs principally via electrostatic complexation. Site-marker competitive binding shows that Cochineal Red A binds primarily to site I of serum albumins. Circular dichroic spectra indicate that dye binding results in some conformational modification of serum albumins. Increased ionic strength of the medium results in lowering of binding. This study provides an important insight into possible means of removal of dye toxicity.

  9. Studies on binding interactions between clenbuterol hydrochloride and two serum albumins by multispectroscopic approaches in vitro.

    PubMed

    Wang, Qin; Zhang, Shengrui

    2014-08-01

    In this study, binding properties of clenbuterol hydrochloride (CL) with human serum albumin (HSA) and bovine serum albumin (BSA) were examined using constant protein concentrations and various CL contents under physiological conditions. The binding parameters were confirmed using fluorescence quenching spectroscopy at various temperatures. The experimental results confirmed that the quenching mechanisms of CL and HSA/BSA were both static quenching processes. The thermodynamic parameters, namely, enthalpy change (ΔH) and entropy change (ΔS), were calculated according to the van't Hoff equation, which suggested that the electrostatic interactions were the predominant intermolecular forces in stabilizing the CL-HSA complex, and hydrogen bonds and van der Waals force were the predominant intermolecular forces in stabilizing the CL-BSA complex. Furthermore, the conformational changes of HSA/BSA in the presence of CL were determined using the data obtained from three-dimensional fluorescence spectroscopy, ultraviolet-visible absorption spectroscopy and circular dichroism spectroscopy.

  10. 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.

  11. 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.

  12. 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.

  13. Photo-isomerization and oxidation of bilirubin in mammals is dependent on albumin binding.

    PubMed

    Goncharova, Iryna; Jašprová, Jana; Vítek, Libor; Urbanová, Marie

    2015-12-01

    The bilirubin (BR) photo-conversion in the human body is a protein-dependent process; an effective photo-isomerization of the potentially neurotoxic Z,Z-BR as well as its oxidation to biliverdin in the antioxidant redox cycle is possible only when BR is bound on serum albumin. We present a novel analytical concept in the study of linear tetrapyrroles metabolic processes based on an in-depth mapping of binding sites in the structure of human serum albumin (HSA). A combination of fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular modeling methods was used for recognition of the binding site for BR, its derivatives (mesobilirubin and bilirubin ditaurate), and the products of the photo-isomerization and oxidation (lumirubin, biliverdin, and xanthobilirubic acid) on HSA. The CD spectra and fluorescent quenching of the Trp-HSA were used to calculate the binding constants. The results of the CD displacement experiments performed with hemin were interpreted together with the findings of molecular docking performed on the pigment-HSA complexes. We estimated that Z,Z-BR and its metabolic products bind on two independent binding sites. Our findings support the existence of a reversible antioxidant redox cycle for BR and explain an additional pathway of the photo-isomerization process (increase of HSA binding capacity; the excess free [unbound] BR can be converted and also bound to HSA).

  14. Binding interactions of pefloxacin mesylate with bovine lactoferrin and human serum albumin*

    PubMed Central

    Fan, Ji-cai; Chen, Xiang; Wang, Yun; Fan, Cheng-ping; Shang, Zhi-cai

    2006-01-01

    The binding of pefloxacin mesylate (PFLX) to bovine lactoferrin (BLf) and human serum albumin (HSA) in dilute aqueous solution was studied using fluorescence spectra and absorbance spectra. The binding constant K and the binding sites n were obtained by fluorescence quenching method. The binding distance r and energy-transfer efficiency E between pefloxacin mesylate and bovine lactoferrin as well as human serum albumin were also obtained according to the mechanism of Förster-type dipole-dipole nonradiative energy-transfer. The effects of pefloxacin mesylate on the conformations of bovine lactoferrin and human serum albumin were also analyzed using synchronous fluorescence spectroscopy. PMID:16691639

  15. Probing the binding of fluoxetine hydrochloride to human serum albumin by multispectroscopic techniques

    NASA Astrophysics Data System (ADS)

    Katrahalli, Umesha; Jaldappagari, Seetharamappa; Kalanur, Shankara S.

    2010-01-01

    The interaction between human serum albumin (HSA) and fluoxetine hydrochloride (FLX) have been studied by using different spectroscopic techniques viz., fluorescence, UV-vis absorption, circular dichroism and FTIR under simulated physiological conditions. Fluorescence results revealed the presence of static type of quenching mechanism in the binding of FLX to HSA. The values of binding constant, K of FLX-HSA were evaluated at 289, 300 and 310 K and were found to be 1.90 × 10 3, 1.68 × 10 3 and 1.45 × 10 3 M -1, respectively. The number of binding sites, n was noticed to be almost equal to unity thereby indicating the presence of a single class of binding site for FLX on HSA. Based on the thermodynamic parameters, Δ H0 and Δ S0 nature of binding forces operating between HSA and FLX were proposed. Spectral results revealed the conformational changes in protein upon interaction. Displacement studies indicated the site I as the main binding site for FLX on HSA. The effect of common ions on the binding of FLX to HSA was also investigated.

  16. 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.

  17. 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.

  18. 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

    2011-04-05

    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.

  19. 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.

  20. An artificially evolved albumin binding module facilitates chemical shift epitope mapping of GA domain interactions with phylogenetically diverse albumins.

    PubMed

    He, Yanan; Chen, Yihong; Rozak, David A; Bryan, Philip N; Orban, John

    2007-07-01

    Protein G-related albumin-binding (GA) modules occur on the surface of numerous Gram-positive bacterial pathogens and their presence may promote bacterial growth and virulence in mammalian hosts. We recently used phage display selection to evolve a GA domain, PSD-1 (phage selected domain-1), which tightly bound phylogenetically diverse albumins. With respect to PSD-1's broad albumin binding specificity, it remained unclear how the evolved binding epitope compared to those of naturally occurring GA domains and whether PSD-1's binding mode was the same for different albumins. We investigate these questions here using chemical shift perturbation measurements of PSD-1 with rabbit serum albumin (RSA) and human serum albumin (HSA) and put the results in the context of previous work on structure and dynamics of GA domains. Combined, these data provide insights into the requirements for broad binding specificity in GA-albumin interactions. Moreover, we note that using the phage-optimized PSD-1 protein significantly diminishes the effects of exchange broadening at the binding interface between GA modules and albumin, presumably through stabilization of a ligand-bound conformation. The employment of artificially evolved domains may be generally useful in NMR structural studies of other protein-protein complexes.

  1. Influence of Molecular Structure on O2-Binding Properties and Blood Circulation of Hemoglobin‒Albumin Clusters

    PubMed Central

    Yamada, Kana; Yokomaku, Kyoko; Haruki, Risa; Taguchi, Kazuaki; Nagao, Saori; Maruyama, Toru; Otagiri, Masaki; Komatsu, Teruyuki

    2016-01-01

    A hemoglobin wrapped covalently by three human serum albumins, a Hb-HSA3 cluster, is an artificial O2-carrier with the potential to function as a red blood cell substitute. This paper describes the synthesis and O2-binding properties of new hemoglobin‒albumin clusters (i) bearing four HSA units at the periphery (Hb-HSA4, large-size variant) and (ii) containing an intramolecularly crosslinked Hb in the center (XLHb-HSA3, high O2-affinity variant). Dynamic light scattering measurements revealed that the Hb-HSA4 diameter is greater than that of either Hb-HSA3 or XLHb-HSA3. The XLHb-HSA3 showed moderately high O2-affinity compared to the others because of the chemical linkage between the Cys-93(β) residues in Hb. Furthermore, the blood circulation behavior of 125I-labeled clusters was investigated by assay of blood retention and tissue distribution after intravenous administration into anesthetized rats. The XLHb-HSA3 was metabolized faster than Hb-HSA3 and Hb-HSA4. Results suggest that the molecular structure of the protein cluster is a factor that can influence in vivo circulation behavior. PMID:26895315

  2. Screening major binding sites on human serum albumin by affinity capillary electrophoresis.

    PubMed

    Kim, Hee Seung; Austin, John; Hage, David S

    2004-01-01

    A screening method is described for determining whether a drug or small solute has significant interactions at the two major binding sites on human serum albumin (HSA). This method uses affinity capillary electrophoresis (ACE) to perform a mobility shift assay, where the solute of interest is injected in both the presence of pH 7.4, 0.067 M phosphate buffer, and the same buffer containing a known concentration of HSA. Dextran is also used in the running buffer to adjust the mobility of HSA. Two types of modified HSA are used in this assay. The first is modified with 2-hydroxy-5-nitrobenzyl bromide (HNB), which selectively blocks HSA's warfarin-azapropazone site. The second type of HSA is modified with tetranitromethane (TNM), which decreases binding at the indole-benzodiazepine site. By comparing the mobility of a solute in the presence of these two modified forms of HSA vs normal HSA, it is possible to detect solute interactions at these binding sites. This approach is illustrated using warfarin and ibuprofen as examples of test solutes.

  3. Evaluation of the enantioselective binding of imazalil to human serum albumin by capillary electrophoresis.

    PubMed

    Asensi-Bernardi, Lucía; Martín-Biosca, Yolanda; Escuder-Gilabert, Laura; Sagrado, Salvador; Medina-Hernández, María José

    2015-11-01

    In this work, a methodology for the evaluation of enantioselective binding of imazalil (IMA) enantiomers to human serum albumin (HSA) that does not require the separation of free and bound to HSA fractions is developed. This methodology comprises the incubation of IMA-HSA designed mixtures for 30 min directly in the capillary electrophoresis system and the subsequent direct injection and chiral separation of IMA employing highly sulfated β-cyclodextrin as chiral selector and the complete filling technique. Two mathematical approaches were used to estimate apparent affinity constants (K1), protein binding and enantioselectivity (ES) for both enantiomers of IMA. Moderate enantioselective binding of IMA enantiomers to HSA (ES = 2.0) was shown by the 1:1 stoichiometry and log K1 values of 3.4 ± 0.4 and 3.1 ± 0.3 for the first and second eluted enantiomers, respectively.

  4. 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.

  5. 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.

  6. Species-dependent binding of new synthesized bicalutamide analogues to albumin by optical biosensor analysis.

    PubMed

    Fortugno, Cecilia; van der Gronde, Toon; Varchi, Greta; Guerrini, Andrea; Bertucci, Carlo

    2015-01-01

    The binding of some novel bicalutamide analogues to human serum albumin (HSA) and rat serum albumin (RSA) was investigated by surface plasmon resonance (SPR) based optical biosensor technique. The serum protein binding of the bicalutamide analogues was determined and compared to that of the parent compound. Furthermore, HSA and RSA were used as target plasma proteins, in order to highlight possible differences among species when performing pharmacokinetic studies. HSA and RSA were covalently immobilized on carboxymethyl dextran matrixes, using an amine coupling procedure. The anchoring method was validated by determining the dissociation constant (KD) of a standard analyte to confirm that the binding properties of the proteins were maintained. The ranking of the bicalutamide analogues for their HSA and RSA bound fractions was used to compare the behaviour of the two albumins. Most of the bicalutamide analogues showed higher binding levels with respect to the lead compound, (R)-bicalutamide. Further, meaningful differences in the binding level to the two serum proteins were obtained. The dissociation constants (KD) of the interaction between the lead compound, (R)-bicalutamide, and the two proteins were calculated. As a result, the KD obtained with HSA was one order of magnitude higher than that obtained with RSA. The observed differences in the HSA and RSA bonding of the bicalutamide analogues increase the knowledge on the possible low reliability in extrapolating the distribution data obtained on animals to humans. This work demonstrates that SPR based optical biosensor technique is well suited for the medium-high throughput screening of compounds' ligand binding to serum albumins.

  7. 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.

  8. Binding of hydroxylated polybrominated diphenyl ethers with human serum albumin: Spectroscopic characterization and molecular modeling.

    PubMed

    Yang, Lulu; Yang, Wu; Wu, Zhiwei; Yi, Zhongsheng

    2017-02-21

    Three hydroxylated polybrominated diphenyl ethers (OH-PBDEs), 3-OH-BDE-47, 5-OH-BDE-47, and 6-OH-BDE-47, were selected to investigate the interactions between OH-PBDEs with human serum albumin (HSA) under physiological conditions. The observed fluorescence quenching can be attributed to the formation of complexes between HSA and OH-PBDEs. The thermodynamic parameters at different temperatures indicate that the binding was caused by hydrophobic forces and hydrogen bonds. Molecular modeling and three-dimensional fluorescence spectrum showed conformational and microenvironmental changes in HSA. Circular dichroism analysis showed that the addition of OH-PBDEs changed the conformation of HSA with a minor reduction in α-helix content and increase in β-sheet content. Furthermore, binding distance r between the donor (HSA) and acceptor (three OH-PBDEs) calculated using Förster's nonradiative energy transfer theory was <7 nm; therefore, the quenching mechanisms for the binding between HSA and OH-PBDEs involve static quenching and energy transfer. Combined with molecular dynamics simulations, the binding free energies (ΔGbind ) were calculated using molecular mechanics/Poisson - Boltzmann surface area method, and the crucial residues in HSA were identified.

  9. Fluorometric Investigation on the Binding of Letrozole and Resveratrol with Serum Albumin.

    PubMed

    Maliszewska, Monika; Maciążek-Jurczyk, Małgorzata; Pożycka, Jadwiga; Szkudlarek, Agnieszka; Chudzik, Mariola; Sułkowska, Anna

    2016-01-01

    Breast cancer is the most common cancer in women worldwide. Combination of drugs during long-therm cancer therapy can increase free, biological active form of the drug and cause dangerous side effects. The 21st century is a period of searching for a progress in cancer chemotherapy. The simultaneous dosage of drugs and natural agents isolated from fruits and vegetables used in breast cancer treatment could be more effective and less toxic. The aim of the study was to determine the binding sites of both letrozole (LET) and polyphenol product, resveratrol (RES) in tertiary structure of human serum albumin (HSA) based on the fluorescence spectroscopy. The binding of LET and RES to HSA was studied by monitoring the changes in emission fluorescence spectra of albumin in the presence of ligands at 280 nm and 295 nm excitation wavelengths. To identify the binding sites of LET and RES on HSA, warfarin (WAR) and 5-(Dimethylamino)-1-naphthalenesulfonamide (DNSA) were used as site probes for binding site I, while dansyl-L-proline (DP) was studied as a marker for binding site II. The binding sites for LET and RES in HSA were found to be located in subdomain IIIA. Based on the Stern - Volmer and binding isotherm using non-linear regression methods, the formation of complexes of LET and RES with HSA and association constants were obtained. The binding analysis showed that the association constants indicated a stronger interaction of HSA with RES than LET and the presence of RES in the tertiary system alters the stability of LET-albumin complex. This conclusion points to the necessity of precaution and monitoring therapy when resveratrol as a natural compound and letrozole are used together.

  10. α-Mangostin Extraction from the Native Mangosteen (Garcinia mangostana L.) and the Binding Mechanisms of α-Mangostin to HSA or TRF

    PubMed Central

    Guo, Ming; Wang, Xiaomeng; Lu, Xiaowang; Wang, Hongzheng; Brodelius, Peter E.

    2016-01-01

    In order to obtain the biological active compound, α-mangostin, from the traditional native mangosteen (Garcinia mangostana L.), an extraction method for industrial application was explored. A high yield of α-mangostin (5.2%) was obtained by extraction from dried mangosteen pericarps with subsequent purification on macroporous resin HPD-400. The chemical structure of α-mangostin was verified mass spectrometry (MS), nuclear magnetic resonance (1H NMR and 13C NMR), infrared spectroscopy (IR) and UV-Vis spectroscopy. The purity of the obtained α-mangostin was 95.6% as determined by HPLC analysis. The binding of native α-mangostin to human serum albumin (HSA) or transferrin (TRF) was explored by combining spectral experiments with molecular modeling. The results showed that α-mangostin binds to HSA or TRF as static complexes but the binding affinities were different in different systems. The binding constants and thermodynamic parameters were measured by fluorescence spectroscopy and absorbance spectra. The association constant of HSA or TRF binding to α-mangostin is 6.4832×105 L/mol and 1.4652×105 L/mol at 298 K and 7.8619×105 L/mol and 1.1582×105 L/mol at 310 K, respectively. The binding distance, the energy transfer efficiency between α-mangostin and HSA or TRF were also obtained by virtue of the Förster theory of non-radiation energy transfer. The effect of α-mangostin on the HSA or TRF conformation was analyzed by synchronous spectrometry and fluorescence polarization studies. Molecular docking results reveal that the main interaction between α-mangostin and HSA is hydrophobic interactions, while the main interaction between α-mangostin and TRF is hydrogen bonding and Van der Waals forces. These results are consistent with spectral results. PMID:27584012

  11. Determination of energies and sites of binding of PFOA and PFOS to human serum albumin.

    PubMed

    Salvalaglio, Matteo; Muscionico, Isabella; Cavallotti, Carlo

    2010-11-25

    Structure and energies of the binding sites of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) to human serum albumin (HSA) were determined through molecular modeling. The calculations consisted of a compound approach based on docking, followed by molecular dynamics simulations and by the estimation of the free binding energies adopting WHAM-umbrella sampling and semiempirical methodologies. The binding sites so determined are common either to known HSA fatty acids sites or to other HSA sites known to bind to pharmaceutical compounds such as warfarin, thyroxine, indole, and benzodiazepin. Among the PFOA binding sites, five have interaction energies in excess of -6 kcal/mol, which become nine for PFOS. The calculated binding free energy of PFOA to the Trp 214 binding site is the highest among the PFOA complexes, -8.0 kcal/mol, in good agreement with literature experimental data. The PFOS binding site with the highest energy, -8.8 kcal/mol, is located near the Trp 214 binding site, thus partially affecting its activity. The maximum number of ligands that can be bound to HSA is 9 for PFOA and 11 for PFOS. The calculated data were adopted to predict the level of complexation of HSA as a function of the concentration of PFOA and PFOS found in human blood for different levels of exposition. The analysis of the factors contributing to the complex binding energy permitted to outline a set of guidelines for the rational design of alternative fluorinated surfactants with a lower bioaccumulation potential.

  12. The adsorption of human serum albumin (HSA) on CO2 laser modified magnesia partially stabilised zirconia (MgO-PSZ).

    PubMed

    Hao, L; Lawrence, J

    2004-03-15

    Magnesia partially stabilised zirconia (MgO-PSZ), a bioinert ceramic, exhibits high mechanical strength, excellent corrosion resistance and good biocompatibility, but it does not naturally form a direct bond with bone resulting in a lack of osteointegration. The surface properties and structure of a biomaterial play an essential role in protein adsorption. As such, changes in the surface properties and structure of biomaterials may in turn alter their bioactivity. So, the fundamental reactions at the interface of biomaterials and tissue should influence their integration and bone-bonding properties. To this end, CO2 laser radiation was used to modify the surface roughness, crystal size, phase and surface energy of the MgO-PSZ. The basic mechanisms active in improving the surface energy were analysed and found to be the phase change and augmented surface area. The adsorption of human serum albumin (HSA), which is a non-cell adhesive protein, was compared on the untreated and CO2 laser modified MgO-PSZ. It was observed that the thickness of the adsorbed HSA decreased as the polar surface energy of the MgO-PSZ increased, indicating that HSA adsorbed more effectively on the hydrophobic MgO-PSZ surface than the hydrophilic surface. The current study provided important information regarding protein-biomaterial interactions and possible mechanisms behind the cell interaction and in vivo behaviour.

  13. Locating the binding sites of Pb(II) ion with human and bovine serum albumins.

    PubMed

    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 K(Pb-HSA) = 8.2 (±0.8)×10(4) M(-1) and K(Pb-BSA) = 7.5 (±0.7)×10(4) 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.

  14. Probing the binding sites of antibiotic drugs doxorubicin and N-(trifluoroacetyl) doxorubicin with human and bovine serum albumins.

    PubMed

    Agudelo, Daniel; Bourassa, Philippe; Bruneau, Julie; Bérubé, Gervais; Asselin, Eric; Tajmir-Riahi, Heidar-Ali

    2012-01-01

    We located the binding sites of doxorubicin (DOX) and N-(trifluoroacetyl) doxorubicin (FDOX) with bovine serum albumin (BSA) and human serum albumins (HSA) at physiological conditions, using constant protein concentration and various drug contents. FTIR, CD and fluorescence spectroscopic methods as well as molecular modeling were used to analyse drug binding sites, the binding constant and the effect of drug complexation on BSA and HSA stability and conformations. Structural analysis showed that doxorubicin and N-(trifluoroacetyl) doxorubicin bind strongly to BSA and HSA via hydrophilic and hydrophobic contacts with overall binding constants of K(DOX-BSA) = 7.8 (± 0.7) × 10(3) M(-1), K(FDOX-BSA) = 4.8 (± 0.5)× 10(3) M(-1) and K(DOX-HSA) = 1.1 (± 0.3)× 10(4) M(-1), K(FDOX-HSA) = 8.3 (± 0.6)× 10(3) M(-1). The number of bound drug molecules per protein is 1.5 (DOX-BSA), 1.3 (FDOX-BSA) 1.5 (DOX-HSA), 0.9 (FDOX-HSA) in these drug-protein complexes. Docking studies showed the participation of several amino acids in drug-protein complexation, which stabilized by H-bonding systems. The order of drug-protein binding is DOX-HSA > FDOX-HSA > DOX-BSA > FDOX>BSA. Drug complexation alters protein conformation by a major reduction of α-helix from 63% (free BSA) to 47-44% (drug-complex) and 57% (free HSA) to 51-40% (drug-complex) inducing a partial protein destabilization. Doxorubicin and its derivative can be transported by BSA and HSA in vitro.

  15. 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.

  16. Binding of plant alkaloids berberine and palmatine to serum albumins: a thermodynamic investigation.

    PubMed

    Khan, Asma Yasmeen; Hossain, Maidul; Kumar, Gopinatha Suresh

    2013-01-01

    The thermodynamics of the interaction of two pharmaceutically important isoquinoline alkaloids berberine and palmatine with bovine and human serum albumin was investigated using calorimetric techniques, and the data was supplemented with fluorescence and circular dichroism studies. Thermodynamic results revealed that there was only one class of binding sites for both alkaloids on BSA and HSA. The equilibrium constant was of the order of 10(4) M(-1) for both the alkaloids to serum albumins but the magnitude was slightly higher with HSA. Berberine showed higher affinity over palmatine to both proteins. The binding was enthalpy dominated and entropy favoured for both the alkaloids to BSA and HSA. Salt dependent studies suggested that electrostatic interaction had a significant role in the binding process, the binding affinity reduced as the salt concentration increased. Temperature dependent calorimetric data yielded heat capacity values that suggested the involvement of different molecular forces in the complexation of the two alkaloids with BSA and HSA. 3D fluorescence, synchronous fluorescence and circular dichroism data suggested that the binding of the alkaloids changed the conformation of proteins by reducing their helicity. Destabilization of the protein conformation was also revealed from differential scanning calorimetry studies. Overall, the alkaloids bound strongly to serum albumins, but berberine was a better binder to both serum proteins compared to palmatine.

  17. Synthetic human serum albumin Sudlow I binding site mimics.

    PubMed

    Karlsson, Björn C G; Rosengren, Annika M; Näslund, Inga; Andersson, Per Ola; Nicholls, Ian A

    2010-11-25

    Here, we report the design, synthesis, and characterization of molecularly imprinted polymer (MIP) derived mimics of the human serum albumin (HSA) Sudlow I site-the binding site for the anticoagulant warfarin. MIP design was based upon a combination of experimental ((1)H NMR) and computational (molecular dynamics) methods. Two MIPs and corresponding nonimprinted reference polymers were synthesized and characterized (scanning electron microscopy; nitrogen sorption; and Fourier transform infrared spectroscopy). MIP-ligand recognition was examined using radioligand binding studies, where the largest number of selective sites was found in a warfarin-imprinted methacrylic acid-ethylene dimethacrylate copolymer (MAA-MIP). The warfarin selectivity of this MIP was confirmed using radioligand displacement and zonal chromatographic studies. A direct comparison of MIP-warfarin binding characteristics with those of the HSA Sudlow I binding site was made, and similarities in site population (per gram polymer or protein) and affinities were observed. The warfarin selectivity of the MIP suggests its potential for use as a recognition element in a MIP-based warfarin sensor and even as a model to aid in understanding and steering blood-plasma protein-regulated transport processes or even for the development of warfarin sensors.

  18. Potential toxicity of amphenicol antibiotic: binding of chloramphenicol to human serum albumin.

    PubMed

    Chen, Huilun; Rao, Honghao; He, Pengzhen; Qiao, Yongxiang; Wang, Fei; Liu, Haijun; Cai, Minmin; Yao, Jun

    2014-10-01

    Antibiotics are widely used in daily life but their abuse has posed a potential threat to human health. To evaluate the toxicity of chloramphenicol (CAP) at the protein level, the interaction between CAP and human serum albumin (HSA) was investigated by fluorescence, Ultraviolet-visible (UV-Vis) absorption, Fourier transform infrared (FT-IR) spectroscopy and molecular docking methods. Fluorescence data revealed that the fluorescence quenching of HSA by CAP was the result of the formation of CAP-HSA complex, and the binding constant was determined to be 3.196 × 10(4) L mol(-1) at 310 K. The thermodynamic determination indicated that the interaction was driven by enthalpy change and entropy change together, where the multiple hydrogen bonds (CAP and the residues Arg 222 and His 242 of HSA) and van der Waals forces were the dominant binding force. The site marker competition revealed that CAP bound into sub-domain IIA of HSA. The binding of CAP induced the drastic reduction in α-helix conformation and the significant enhancement in β-sheet conformation of HSA. Molecular docking study further confirmed the binding mode obtained by experimental study. This work provides a new quantitative evaluation method for antibiotics to cause the protein damage.

  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. 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.

  1. Exploring the binding mechanism of ondansetron hydrochloride to serum albumins: Spectroscopic approach

    NASA Astrophysics Data System (ADS)

    Sandhya, B.; Hegde, Ashwini H.; K. C., Ramesh; Seetharamappa, J.

    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.

  2. Cu(II) Bis(thiosemicarbazone) Radiopharmaceutical Binding to Serum Albumin: Further Definition of Species-Dependence and Associated Substituent Effects

    PubMed Central

    Basken, Nathan E.; Green, Mark A.

    2009-01-01

    Introduction The Cu-PTSM (pyruvaldehyde bis(N4-methylthiosemicarbazonato)copper(II)) and Cu-ATSM (diacetyl bis(N4-methylthiosemicarbazonato)copper(II)) radiopharmaceuticals exhibit strong, species-dependent binding to the IIA site of human serum albumin (HSA), while the related Cu-ETS (ethylglyoxal bis(thiosemicarbazonato)copper(II)) radiopharmaceutical appears to only exhibit non-specific binding to human and animal serum albumins. Methods To further probe the structural basis for the species-dependence of this albumin binding interaction, protein binding of these three radiopharmaceuticals was examined in solutions of albumin and/or serum from a broader array of mammalian species (rat, sheep, donkey, rabbit, cow, pig, dog, baboon, mouse, cat, elephant). We also evaluated the albumin binding of several copper(II) bis(thiosemicarbazone) chelates offering more diverse substitution of the ligand backbone. Results Cu-PTSM and Cu-ATSM exhibit a strong interaction with HSA that is not apparent with the albumins of other species, while the binding of Cu-ETS to albumin is much less species-dependent. The strong interaction of Cu-PTSM with HSA does not appear to simply correlate with variation, relative to the animal albumins, of a single amino acid lining HSA's IIA site. Those agents that selectively interact with HSA share the common feature of only methyl or hydrogen substitution at the carbon atoms of the diimine fragment of the ligand backbone. Conclusions The interspecies variations in albumin binding of Cu-PTSM and Cu-ATSM are not simply explained by unique amino acid substitutions in the IIA binding pocket of the serum albumins. However, the specific affinity for this region of HSA is disrupted when substituents bulkier than a methyl group appear on the imine carbons of the copper bis(thiosemicarbazone) chelate. PMID:19520290

  3. 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.

  4. Estimating the relative position of risperidone primary binding site in Sera Albumins. Modeling from spectrofluorimetric data

    NASA Astrophysics Data System (ADS)

    Cortez, Celia Martins; Fragoso, Viviane Muniz S.; Silva, Dilson

    2014-10-01

    In this work, we used a mathematical model to study the interaction of risperidone with human and bovine serum albumins estimating the relative position of the primary binding site, based on the fluorescence quenching theory. Results have shown that the model was able to demonstrate that primary binding site for risperidone in HSA and BSA is very close to the position where is tryptophan 134 of BSA, possibly in domain 1B.

  5. Binding and conformational changes of human serum albumin upon interaction with 4-aminoantipyrine studied by spectroscopic methods and cyclic voltammetry.

    PubMed

    Gowda, Jayant I; Nandibewoor, Sharanappa T

    2014-04-24

    The interactions of 4-aminoantipyrine (AAP) with human serum albumin (HSA) have been studied by UV-visible spectroscopy, fluorescence spectroscopy and cyclic voltammetry. The binding of 4-aminoantipyrine quenches the HSA fluorescence, revealing a 1:1 interaction with a binding constant of about 10(5) M(-1). The experimental results showed that AAP effectively quenched the intrinsic fluorescence of HSA via dynamic type of quenching. In addition, according to the synchronous fluorescence spectra of HSA in presence of 4-aminoantipyrine, the tryptophan residue of the proteins are most perturbed by the binding process. The number of binding sites, the binding constant, site probe study, some common metal ions effect and the thermodynamic parameters were calculated.

  6. Interactions between quercetin and warfarin for albumin binding: A new eye on food/drug interference.

    PubMed

    Di Bari, Lorenzo; Ripoli, Silvia; Pradhan, Sanghamitra; Salvadori, Piero

    2010-06-01

    The interaction between quercetin, a popular antioxidant flavonoid, and human serum albumin (HSA) is investigated and characterized by means of induced circular dichroism and saturation transfer difference NMR. These techiques demonstrate the reversible binding of quercetin to the carrier protein, which is responsible for its dissolution in aqueous medium. Competition experiments with two classical probes for HSA binding sites, namely Ibuprofen and Warfarin (a common anticoagulant coumarin), demonstrate that quercetin has a primary binding site located in the subdomain IIA, where coumarins are hosted. The affinity for this site is large and we found that quercetin may effectively displace warfarin from HSA. This may have relevant consequences in rationalizing the interferences of common dietary compounds and food supplements to anticoagulant treatments.

  7. 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.

  8. 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.

  9. Stabilization of Human Serum Albumin by the Binding of Phycocyanobilin, a Bioactive Chromophore of Blue-Green Alga Spirulina: Molecular Dynamics and Experimental Study.

    PubMed

    Radibratovic, Milica; Minic, Simeon; Stanic-Vucinic, Dragana; Nikolic, Milan; Milcic, Milos; Cirkovic Velickovic, Tanja

    2016-01-01

    Phycocyanobilin (PCB) binds with high affinity (2.2 x 106 M-1 at 25°C) to human serum albumin (HSA) at sites located in IB and IIA subdomains. The aim of this study was to examine effects of PCB binding on protein conformation and stability. Using 300 ns molecular dynamics (MD) simulations, UV-VIS spectrophotometry, CD, FT-IR, spectrofluorimetry, thermal denaturation and susceptibility to trypsin digestion, we studied the effects of PCB binding on the stability and rigidity of HSA, as well as the conformational changes in PCB itself upon binding to the protein. MD simulation results demonstrated that HSA with PCB bound at any of the two sites showed greater rigidity and lower overall and individual domain flexibility compared to free HSA. Experimental data demonstrated an increase in the α-helical content of the protein and thermal and proteolytic stability upon ligand binding. PCB bound to HSA undergoes a conformational change to a more elongated conformation in the binding pockets of HSA. PCB binding to HSA stabilizes the structure of this flexible transport protein, making it more thermostable and resistant to proteolysis. The results from this work explain at molecular level, conformational changes and stabilization of HSA structure upon ligand binding. The resultant increased thermal and proteolytic stability of HSA may provide greater longevity to HSA in plasma.

  10. Stabilization of Human Serum Albumin by the Binding of Phycocyanobilin, a Bioactive Chromophore of Blue-Green Alga Spirulina: Molecular Dynamics and Experimental Study

    PubMed Central

    Stanic-Vucinic, Dragana; Nikolic, Milan; Milcic, Milos; Cirkovic Velickovic, Tanja

    2016-01-01

    Phycocyanobilin (PCB) binds with high affinity (2.2 x 106 M-1 at 25°C) to human serum albumin (HSA) at sites located in IB and IIA subdomains. The aim of this study was to examine effects of PCB binding on protein conformation and stability. Using 300 ns molecular dynamics (MD) simulations, UV-VIS spectrophotometry, CD, FT-IR, spectrofluorimetry, thermal denaturation and susceptibility to trypsin digestion, we studied the effects of PCB binding on the stability and rigidity of HSA, as well as the conformational changes in PCB itself upon binding to the protein. MD simulation results demonstrated that HSA with PCB bound at any of the two sites showed greater rigidity and lower overall and individual domain flexibility compared to free HSA. Experimental data demonstrated an increase in the α-helical content of the protein and thermal and proteolytic stability upon ligand binding. PCB bound to HSA undergoes a conformational change to a more elongated conformation in the binding pockets of HSA. PCB binding to HSA stabilizes the structure of this flexible transport protein, making it more thermostable and resistant to proteolysis. The results from this work explain at molecular level, conformational changes and stabilization of HSA structure upon ligand binding. The resultant increased thermal and proteolytic stability of HSA may provide greater longevity to HSA in plasma. PMID:27959940

  11. New cobalt(II) and nickel(II) complexes of benzyl carbazate Schiff bases: Syntheses, crystal structures, in vitro DNA and HSA binding studies.

    PubMed

    Nithya, Palanivelu; Helena, Sannasi; Simpson, Jim; Ilanchelian, Malaichamy; Muthusankar, Aathi; Govindarajan, Subbiah

    2016-12-01

    In the present study, new Schiff base complexes with the composition [M(NCS)2(L1)2]·nH2O, where M=Co (n=0) (1) and Ni (n=2) (2); [M(NCS)2(L2)2], M=Co (3) and Ni (4) as well as [M(NCS)2(L3)2], M=Co (5) and Ni (6); (L1=benzyl 2-(propan-2-ylidene)hydrazinecarboxylate, L2=benzyl 2-(butan-2-ylidene)hydrazinecarboxylate and L3=benzyl 2-(pentan-3-ylidene)hydrazinecarboxylate) have been synthesized by a template method. The complexes were characterised by analytical methods, spectroscopic studies, thermal and X-ray diffraction techniques. The structures of all the complexes explore that the metal(II) cation has a trans-planar coordination environment, the monomeric units containing a six-coordinated metal center in octahedral geometry with N-bound isothiocyanate anions coordinated as terminal ligands. Furthermore, the binding of the two Schiff base ligands to the metal centers involves the azomethine nitrogen and the carbonyl oxygen in mutually trans configuration. The binding interactions of all the complexes with Calf thymus-deoxyribonucleic acid (CT-DNA) and human serum albumin (HSA) have been investigated using absorption and emission spectral techniques. The CT-DNA binding properties of these complexes reveal that they bind to CT-DNA through a partial intercalation mode and the binding constant values were calculated using the absorption and emission spectral data. The binding constant values (~10×10(6)moldm(-3)) indicate strong binding of metal complexes with CT-DNA. HSA binding interaction studies showed that the cobalt and nickel complexes can quench the intrinsic fluorescence of HSA through static quenching process. Also, molecular docking studies were supported out to apprehend the binding interactions of these complexes with DNA and HSA which offer new understandings into the experimental model observations.

  12. A review of albumin binding in CKD.

    PubMed

    Meijers, Björn K I; Bammens, Bert; Verbeke, Kristin; Evenepoel, Pieter

    2008-05-01

    Hypoalbuminemia is associated with excess mortality in patients with kidney disease. Albumin is an important oxidant scavenger and an abundant carrier protein for numerous endogenous and exogenous compounds. Several specific binding sites for anionic, neutral, and cationic ligands were described. Overall, the extent of binding depends on the ligand and albumin concentration, albumin-binding affinity, and presence of competing ligands. Chronic kidney disease affects all these determinants. This may result in altered pharmacokinetics and increased risk of toxicity. Renal clearance of albumin-bound solutes mainly depends on tubular clearance. Dialytic clearance by means of conventional hemodialysis/hemofiltration and peritoneal dialysis is limited. Other epuration techniques combining hemodialysis with adsorption have been developed. However, the benefit of these techniques remains to be proved.

  13. Synthesis, characterization, crystal structure and HSA binding of two new N,O,O-donor Schiff-base ligands derived from dihydroxybenzaldehyde and tert-butylamine

    NASA Astrophysics Data System (ADS)

    Khosravi, Iman; Hosseini, Farnaz; Khorshidifard, Mahsa; Sahihi, Mehdi; Rudbari, Hadi Amiri

    2016-09-01

    Two new o-hydroxy Schiff-bases compounds, L1 and L2, were derived from the 1:1 M condensation of 2,3-dihydroxybenzaldehyde and 2,4-dihydroxybenzaldehyde with tert-butylamine and were characterized by elemental analysis, FT-IR, 1H and 13C NMR spectroscopies. The crystal structure of L2 was also determined by single crystal X-ray analysis. The crystal structure of L2 showed that the compound exists as a zwitterionic form in the solid state, with the H atom of the phenol group being transferred to the imine N atom. It adopts an E configuration about the central Cdbnd N double bond. Furthermore, binding of these Schiff base ligands to Human Serum Albumin (HSA) was investigated by fluorescence quenching, absorption spectroscopy, molecular docking and molecular dynamics (MD) simulation methods. The fluorescence emission of HSA was quenched by ligands. Also, suitable models were used to analyze the UV-vis absorption spectroscopy data for titration of HSA solution by various amounts of Schiff bases. The spectroscopic studies revealed that these Schiff bases formed 1:1 complex with HSA. Energy transfer mechanism of quenching was discussed and the values of 3.35 and 1.57 nm as the mean distances between the bound ligands and the HSA were calculated for L1 and L2, respectively. Molecular docking results indicated that the main active binding site for these Schiff bases ligands is in subdomain IB. Moreover, MD simulation results suggested that this Schiff base complex can interact with HSA, with a slight modification of its tertiary structure.

  14. Interaction between curcumin and human serum albumin in the presence of excipients and the effect of binding on curcumin photostability.

    PubMed

    Vukićević, Milica; Tønnesen, Hanne Hjorth

    2016-01-01

    Curcumin (Cur) is known to bind to human serum albumin (HSA) which may lead to a reduced phototoxic effect of the compound in the presence of serum or saliva. The influence of excipients on the Cur-HSA binding was studied by HSA florescence quenching and Cur absorption and emission spectroscopy in the presence and absence of the selected excipients. Photostabilty of Cur in the presence of HSA was evaluated, as well as the effect of excipients on HSA bound Cur photodegradation. Cyclodextrins (CDs) (2-hydroxypropyl-β-cyclodextrin and 2-hydroxypropyl-γ-cyclodextrin) and polymers (polyethylene glycol 400, PEG 400 and Pluronic F-127, PF-127) were selected for the study. CDs and PF-127 seem to decrease Cur binding to HSA, probably through competitive binding. Cur was still bound to HSA in polyethylene glycol (PEG) solutions at the highest investigated concentration (5% w/v). However, high PEG concentration appears to have effect on the protein conformation, as shown by the fluorescence quenching study. Low Cur photostability in the presence of HSA could be improved by the addition of hydroxylpropyl-γ-cyclodextrin (HPγCD) to the samples, whereas PEG and PF-127 showed no effect.

  15. Anti-coagulant rodenticide binding properties of human serum albumin: a biochromatographic approach.

    PubMed

    André, Claire; Guillaume, Yves Claude

    2004-03-05

    In this paper, the anti-coagulant rodenticide-human serum albumin (HSA) binding was investigated using a perturbation method to calculate the solute distribution isotherms. It was shown that rodenticide can bound either on the benzodiazepine HSA site with low affinity (site I) or on the warfarin HSA site with high affinity (site II). The thermodynamic parameters of this association were calculated for the two HSA binding sites. For the site II, the rodenticide-HSA association was governed enthalpically whereas for the site I, this one was driven entropically. Moreover, the role of the magnesium (Mg(2+)) and calcium (Ca(2+)) on this association was carried out. It was clearly demonstrated that the rodenticide affinity for the site I was not affected by modifying the bulk solvent surface tension whereas for the site II the association constant increased strongly with the Mg(2+) or the Ca(2+) concentration in the bulk solvent. These results showed that the rodenticide-HSA affinity and thus the rodenticide toxicological effect depends on the Mg(2+) or Ca(2+) concentration.

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

    PubMed

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

    2014-01-03

    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 10(4) 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.

  17. Mn(II) binding to human serum albumin: a ¹H-NMR relaxometric study.

    PubMed

    Fanali, Gabriella; Cao, Yu; Ascenzi, Paolo; Fasano, Mauro

    2012-12-01

    Human serum albumin (HSA) displays several metal binding sites, participating to essential and toxic metal ions disposal and transport. The major Zn(II) binding site, called Site A, is located at the I/II domain interface, with residues His67, Asn99, His247, and Asp249 contributing with five donor atoms to the metal ion coordination. Additionally, one water molecule takes part of the octahedral coordination geometry. The occurrence of the metal-coordinated water molecule allows the investigation of the metal complex geometry by water (1)H-NMR relaxation, provided that the diamagnetic Zn(II) is replaced by the paramagnetic Mn(II). Here, the (1)H-NMR relaxometric study of Mn(II) binding to HSA is reported. Mn(II) binding to HSA is modulated by Zn(II), pH, and myristate through competitive inhibition and allosteric mechanisms. The body of results indicates that the primary binding site of Zn(II) corresponds to the secondary binding site of Mn(II), i.e. the multimetal binding site A. Excess Zn(II) completely displaces Mn(II) from its primary site suggesting that the primary Mn(II) site corresponds to the secondary Zn(II) site. This uncharacterized site is functionally-linked to FA1; moreover, metal ion binding is modulated by myristate and pH. Noteworthy, water (1)H-NMR relaxometry allowed a detailed analysis of thermodynamic properties of HSA-metal ion complexes.

  18. Binding studies of L-3,4-dihydroxyphenylalanine with human serum albumin.

    PubMed

    Yeggoni, Daniel Pushparaju; Subramanyam, Rajagopal

    2014-12-01

    L-Dopa has been used to increase dopamine concentrations in the treatment of Parkinson's disease and dopamine-responsive dystonia. The binding interaction between L-dopa (phytochemical) and human serum albumin (HSA) under simulated physiological conditions was investigated by spectroscopic and molecular modeling methods. The results revealed that L-dopa caused fluorescence emission quenching of HSA through a static quenching procedure and the binding constant obtained was 2.3 ± 0.01 × 10(4) M(-1), which is corresponding to -5.9 kcal M(-1) of free energy at 25 °C. Interestingly, L-dopa is not binding to the α-1-acidglycoprotein, which is also a plasma protein and an acute phase protein. Furthermore, circular dichroism results confirm that in the presence of L-dopa the secondary structure of HSA is altered due to partial unfolding of the protein. Importantly, the displacement experiment with site specific probes, phenylbutazone (site I) and ibuprofen (site II), depicts that L-dopa binds particularly to site II of HSA. In addition, the molecular modeling results also confirmed that L-dopa is binding to the subdomain IIIA of HSA and is stabilized by hydrogen bonds and hydrophilic forces. Additionally, the molecular dynamic simulation studies showed that the HSA-L-dopa complex reaches an equilibration state at around 2 ns, which indicates that the HSA-L-dopa complex is very stable. These results provided valuable information of pharmacological mechanisms of L-dopa under in vivo conditions and play a pivotal role in the development of L-dopa-inspired drugs.

  19. Ligand binding strategies of human serum albumin: how can the cargo be utilized?

    PubMed

    Varshney, Ankita; Sen, Priyankar; Ahmad, Ejaz; Rehan, Mohd; Subbarao, Naidu; Khan, Rizwan Hasan

    2010-01-01

    Human serum albumin (HSA), being the most abundant carrier protein in blood and a modern day clinical tool for drug delivery, attracts high attention among biologists. Hence, its unfolding/refolding strategies and exogenous/endogenous ligand binding preference are of immense use in therapeutics and clinical biochemistry. Among its fellow proteins albumin is known to carry almost every small molecule. Thus, it is a potential contender for being a molecular cargo/or nanovehicle for clinical, biophysical and industrial purposes. Nonetheless, its structure and function are largely regulated by various chemical and physical factors to accommodate HSA to its functional purpose. This multifunctional protein also possesses enzymatic properties which may be used to convert prodrugs to active therapeutics. This review aims to highlight current overview on the binding strategies of protein to various ligands that may be expected to lead to significant clinical applications.

  20. Seeking to shed some light on the binding of fluoroquinolones to albumins.

    PubMed

    Bosca, Francisco

    2012-03-22

    Interactions between serum albumins (HSA, human, and BSA, bovine) and fluoroquinolones (FQs), such as enoxacin, norfloxacin, ciprofloxacin, and ofloxacin, have been studied using the laser flash photolysis technique. Lifetimes and quantum yields of FQs triplet excited states ((3)FQs) are not affected by the presence of albumins, however, the quenching of (3)FQs by tryptophan and tyrosine and the subsequent generation of FQs radical anions and tyrosyl or tryptophanyl radicals were detected. These results, besides agreeing with association constants (K(a)) for FQs binding to albumins lower than 6 × 10(2) M(-1), are highly relevant to understanding the process of photohapten formation, the first event in the onset of photoallergy. The emission of tryptophan within albumin is not affected by the presence of FQs when the inner filter effects (IFE) of these drugs are taken into account, which explains the discrepancies reported in the literature about K(a) of FQs with albumins.

  1. Binding of reactive brilliant red to human serum albumin: insights into the molecular toxicity of sulfonic azo dyes.

    PubMed

    Li, W Y; Chen, F F; Wang, Shi-Long

    2010-05-01

    The non-covalent interaction of reactive brilliant red (RBR) as a representative of sulfonic azo compounds with human serum albumin (HSA) was investigated by a combination of UV-VIS spectrometry, fluorophotometry, circular dichroism (CD) and isothermal titration calorimetry (ITC) technique. The thermodynamic characterization of the interaction was performed. The saturation binding numbers of RBR on peptide chains were determined and the effects of electrolytes and temperature were investigated. The ionic interaction induced a combination of multiple non-covalent bonds including hydrogen bonds, hydrophobic interactions and van der Waals force. A three-step binding model of RBR was revealed. The binding of RBR molecules might occur on the external surface of HSA via electric interaction when the mole ratio of RBR to HSA was less than 40 and RBR molecules entered the hydrophobic intracavity of HSA when ratio was more than 40. Moreover, RBR binding resulted in a conformational change in the structure of HSA or even the precipitation of HSA and inhibited its function accordingly. The possible binding site and the conformational transition of HSA were hypothesized and illustrated. This work provides a new insight into non-covalent interactions between sulfonic azo compounds and proteins, which may be further used to investigate the potential toxicity mechanism of azo dyes.

  2. Connecting simulated, bioanalytical, and molecular docking data on the stereoselective binding of (±)-catechin to human serum albumin.

    PubMed

    Sabela, Myalowenkosy I; Gumede, Njabulo J; Escuder-Gilabert, Laura; Martín-Biosca, Yolanda; Bisetty, Khirsna; Medina-Hernández, María-Jose; Sagrado, Salvador

    2012-02-01

    The stereoselective binding of the frequently ingested nutraceutical (±)-catechin, with demonstrated differential biological activity between enantiomers, to human serum albumin (HSA), with the largest complexation and enantioselectivity potential among the plasmatic proteins, is studied by combining simulations to optimize the experimental design, robust in vitro electrokinetic chromatographic data, and molecular docking-chiral recognition estimates. Methodological and mathematical drawbacks in previous reports on (±)-catechin-HSA are detected and eliminated. Recent and novel direct equations extracted from the classical interaction model allows advantageous univariate mathematical data treatment, providing the first evidence of quantitative (±)-catechin-HSA enantioselectivity. Also, the binding site in HSA of the enantiomers is approached, and both the experimental enantioselectivity and the main binding site information are contrasted with a molecular docking approach.

  3. Structure-affinity relationship of flavones on binding to serum albumins: effect of hydroxyl groups on ring A.

    PubMed

    Xiao, Jianbo; Cao, Hui; Wang, Yuanfeng; Yamamoto, Koichiro; Wei, Xinlin

    2010-07-01

    Four flavones (flavone, 7-hydroxyflavone, chrysin, and baicalein) sharing the same B- and C-ring structure but a different numbers of hydroxyl groups on the A-ring were studied for their affinities for BSA and HSA. The hydroxylation on ring A of flavones increased the binding constants (K(a)) and the number of binding sites (n) between flavones and serum albumins. The affinities of 7-hydroxyflavone for BSA and HSA were about 800 times and 40 times higher than that of flavone, respectively. It appears that the optimal number of hydroxyl groups introduced to the ring A of flavones is one. As more hydroxyl groups were introduced to positions at C-5, C-6, and/or C-7 of flavones, the affinities for serum albumins decrease. The critical energy transfer distances (R(0)) between the hydroxylated flavones (1-3 OH on the ring A) and serum albumins decreased with the increasing affinities for serum albumins.

  4. 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.

  5. 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.

  6. Spectroscopic and molecular modeling evidence of clozapine binding to human serum albumin at subdomain IIA

    NASA Astrophysics Data System (ADS)

    Wu, Xinhu; Liu, Jianjun; Wang, Qiang; Xue, Weiwei; Yao, Xiaojun; Zhang, Yan; Jin, Jing

    2011-09-01

    Various spectroscopy and molecular docking methods were used to examine the binding of Clozapine (CLZ) to human serum albumin (HSA) in this paper. By monitoring the intrinsic fluorescence of single Trp214 residue and performing Dansylamide (DNSA) displacement measurement, the specific binding of CLZ in the vicinity of Sudlow's Site I of HSA has been clarified. An apparent distance of 27.3 Å between the Trp214 and CLZ was obtained via fluorescence resonance energy transfer (FRET) method. In addition, the changes in the secondary structure of HSA after its complexation with CLZ ligand were studied with CD spectroscopy, which indicate that CLZ does not has remarkable effect on the structure of the protein. Moreover, thermal denaturation experiment shows that the HSA-CLZ complexes are conformationally more stable. Finally, the binding details between CLZ and HSA were further confirmed by molecular docking studies, which revealed that CLZ was bound at subdomain IIA through multiple interactions, such as hydrophobic effect, van der Waals forces and hydrogen bonding.

  7. Spectroscopic and molecular modeling evidence of clozapine binding to human serum albumin at subdomain IIA.

    PubMed

    Wu, Xinhu; Liu, Jianjun; Wang, Qiang; Xue, Weiwei; Yao, Xiaojun; Zhang, Yan; Jin, Jing

    2011-09-01

    Various spectroscopy and molecular docking methods were used to examine the binding of Clozapine (CLZ) to human serum albumin (HSA) in this paper. By monitoring the intrinsic fluorescence of single Trp214 residue and performing Dansylamide (DNSA) displacement measurement, the specific binding of CLZ in the vicinity of Sudlow's Site I of HSA has been clarified. An apparent distance of 27.3 Å between the Trp214 and CLZ was obtained via fluorescence resonance energy transfer (FRET) method. In addition, the changes in the secondary structure of HSA after its complexation with CLZ ligand were studied with CD spectroscopy, which indicate that CLZ does not has remarkable effect on the structure of the protein. Moreover, thermal denaturation experiment shows that the HSA-CLZ complexes are conformationally more stable. Finally, the binding details between CLZ and HSA were further confirmed by molecular docking studies, which revealed that CLZ was bound at subdomain IIA through multiple interactions, such as hydrophobic effect, van der Waals forces and hydrogen bonding.

  8. 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.

  9. A spectroscopic and molecular docking approach on the binding of tinzaparin sodium with human serum albumin

    NASA Astrophysics Data System (ADS)

    Abdullah, Saleh M. S.; Fatma, Sana; Rabbani, Gulam; Ashraf, Jalaluddin M.

    2017-01-01

    Protein bound toxins are poorly removed by conventional extracorporeal therapies. Venous thromboembolism (VTE) is a major cause of morbidity and mortality in patients with cancer. The interaction between tinzaparin, an inhibitor of angiotensin converting enzyme and human serum albumin, a principal plasma protein in the liver has been investigated in vitro under a simulated physiological condition by UV-vis spectrophotometry and fluorescence spectrometry. The intrinsic fluorescence intensity of human serum albumin was strongly quenched by tinzaparin (TP). The binding constants and binding stoichiometry can be calculated from the data obtained from fluorescence quenching experiments. The negative value of ΔG° reveals that the binding process is a spontaneous process. Thermodynamic analysis shows that the HSA-TP complex formation occurs via hydrogen bonds, hydrophobic interactions and undergoes slight structural changes as evident by far-UV CD. It indicated that the hydrophobic interactions play a main role in the binding of TP to human serum albumin. In addition, the distance between TP (acceptor) and tryptophan residues of human serum albumin (donor) was estimated to be 2.21 nm according to the Förster's resonance energy transfer theory. For the deeper understanding of the interaction, thermodynamic, and molecular docking studies were performed as well. Our docking results suggest that TP forms stable complex with HSA (Kb ∼ 104) and its primary binding site is located in subdomain IIA (Sudlow Site I). The results obtained herein will be of biological significance in pharmacology and clinical medicine.

  10. 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.

  11. 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.

  12. Surface plasmon resonance and circular dichroism characterization of cucurbitacins binding to serum albumins for early pharmacokinetic profiling.

    PubMed

    Fabini, Edoardo; Fiori, Giovana Maria Lanchoti; Tedesco, Daniele; Lopes, Norberto Peporine; Bertucci, Carlo

    2016-04-15

    Cucurbitacins are a group of tetracyclic triterpenoids, known for centuries for their anti-cancer and anti-inflammatory properties, which are being actively investigated over the past decades in order to elucidate their mechanism of action. In perspective of being used as therapeutic molecules, a pharmacokinetic characterization is crucial to assess the affinity toward blood carrier proteins and extrapolate distribution volumes. Usually, pharmacokinetic data are first collected on animal models and later translated to humans; therefore, an early characterization of the interaction with carrier proteins from different species is highly desirable. In the present study, the interactions of cucurbitacins E and I with human and rat serum albumins (HSA and RSA) were investigated by means of surface plasmon resonance (SPR)-based optical biosensing and circular dichroism (CD) spectroscopy. Active HSA and RSA sensor chip surfaces were prepared through an amine coupling reaction protocol, and the equilibrium dissociation constants (Kd) for the different cucurbitacins-serum albumins complexes were then determined by SPR analysis. Further information on the binding of cucurbitacins to serum albumins was obtained by CD competition experiments with biliverdin, a specific marker binding to subdomain IB of HSA. SPR data unveiled a previously unreported binding event between CucI and HSA; the determined binding affinities of both compounds were slightly higher for RSA with respect to HSA, even though all the compounds can be ranked as high-affinity binders for both carriers. CD analysis showed that the two cucurbitacins modify the binding of biliverdin to serum albumins through opposite allosteric modulation (positive for HSA, negative for RSA), confirming the need for caution in the translation of pharmacokinetic data across species.

  13. 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.

  14. 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.

  15. [Microcalorimetric study of the parameters of dicloxacillin binding with human serum albumin at different temperatures].

    PubMed

    Markovich, M N; Isakovich, L G; Klinichev, V F

    1986-08-01

    The thermodynamic parameters of human serum albumin (HSA) binding with dicloxacillin, an antibiotic widely used in clinical practice, were determined with the method of differential flow microcalorimetry at 18, 25, 30, 37 and 45 degrees C. The experiments were performed at two ionic strengths: 0.02 and 0.15. Two hypothetic models of interaction in the HSA-drug system were considered in processing the data for the curves of calorimetric titration. The first model implies the presence of independent homogeneous active sites on the protein. In accordance with the second model there are one primary and secondary independent homogeneous active sites on the biopolymer molecule. It is shown that dicloxacillin association with HSA proceeds according to the mechanism suggesting the presence of one primary and one secondary active sites on the protein molecule. The binding process in the system studied is exothermic, the enthalpy increasing at the temperature change from 18 to 45 degrees C. At the same time the binding constant and enthropy of the system decrease. The influence of the solution ionic strength on the binding process was practically lacking. On the basis of the analysis of the thermodynamic data it is concluded that the character of the binding in the HSA-dicloxacillin system at 18-30 degrees C is hydrophobic. With an increase in the temperature the hydrophoby level decreases.

  16. Thiophilic interaction chromatography of serum albumins.

    PubMed

    Bourhim, Mustapha; Rajendran, Anita; Ramos, Yanira; Srikrishnan, Thamarapu; Sulkowski, Eugene

    2008-07-01

    An investigation of the binding of native and recombinant human serum albumin and bovine serum albumin on three thiophilic gels, PyS, 2S, and 3S was performed. In addition to these proteins, we studied serum albumins from several species such as goat, rabbit, guinea pig, rat, hamster, baboon, and pig. Our results reveal that recombinant human serum albumin (rHSA) binds completely to PyS whereas native human serum albumin and bovine serum albumin bind only partially to PyS. The binding affinities of rHSA, human serum albumin and bovine serum albumin to 2S and 3S gels are less than their binding to PyS. Serum albumins from goat, rabbit, guinea pig, rat, hamster, baboon, and pig bind much stronger to 3S gel than human and bovine serum albumins. The binding of pig and hamster serum albumins is stronger than that of rat, goat, baboon, and rabbit.

  17. A new ternary copper(II) complex derived from 2-(2";-pyridyl)benzimidazole and glycylglycine: Synthesis, characterization, DNA binding and cleavage, antioxidation and HSA interaction

    NASA Astrophysics Data System (ADS)

    Fu, Xia-Bing; Lin, Zi-Hua; Liu, Hai-Feng; Le, Xue-Yi

    2014-03-01

    A new ternary copper(II)-dipeptide complex [Cu(glygly)(HPB)(Cl)]ṡ2H2O (glygly = glycylglycine anion, HPB = 2-(2";-pyridyl)benzimidazole) has been synthesized and characterized. The DNA interaction of the complex was studied by spectroscopic methods, viscosity, and electrophoresis measurements. The antioxidant activity was also investigated using the pyrogallol autoxidation assay. Besides, the interaction of the complex with human serum albumin (HSA) in vitro was examined by multispectroscopic techniques. The complex partially intercalated to CT-DNA with a high binding constant (Kb = 7.28 × 105 M-1), and cleaved pBR322 DNA efficiently via an oxidative mechanism in the presence of Vc, with the HO· and O2-rad as the active species, and the SOD as a promoter. Furthermore, the complex shows a considerable SOD-like activity with the IC50 value of 3.8386 μM. The complex exhibits desired binding affinity to HSA, in which hydrogen bond or vander Waals force played a major role. The alterations of HSA secondary structure induced by the complex were confirmed by UV-visible, CD, synchronous fluorescence and 3D fluorescence spectroscopy.

  18. Kaempferol-human serum albumin interaction: Characterization of the induced chirality upon binding by experimental circular dichroism and TDDFT calculations

    NASA Astrophysics Data System (ADS)

    Matei, Iulia; Ionescu, Sorana; Hillebrand, Mihaela

    2012-10-01

    The experimental induced circular dichroism (ICD) and absorption spectra of the achiral flavonoid kaempferol upon binding to human serum albumin (HSA) were correlated to electronic CD and UV-vis spectra theoretically predicted by time-dependent density functional theory (TDDFT). The neutral and four anionic species of kaempferol in various conformations were considered in the calculations. The appearance of the experimental ICD signal was rationalized in terms of kaempferol binding to HSA in a distorted, chiral, rigid conformation. The comparison between the experimental and simulated spectra allowed for the identification of the kaempferol species that binds to HSA, namely the anion generated by deprotonation of the hydroxyl group in position 7. This approach constitutes a convenient method for evidencing the binding species and for determining its conformation in the binding pocket of the protein. Its main advantage over the UV-vis absorption method lays in the fact that only the bound ligand species gives an ICD signal.

  19. Investigation of the binding of Salvianolic acid B to human serum albumin and the effect of metal ions on the binding

    NASA Astrophysics Data System (ADS)

    Chen, Tingting; Cao, Hui; Zhu, Shajun; Lu, Yapeng; Shang, Yanfang; Wang, Miao; Tang, Yanfeng; Zhu, Li

    2011-10-01

    The studies on the interaction between HSA and drugs have been an interesting research field in life science, chemistry and clinical medicine. There are also many metal ions present in blood plasma, thus the research about the effect of metal ions on the interaction between drugs and plasma proteins is crucial. In this study, the interaction of Salvianolic acid B (Sal B) with human serum albumin (HSA) was investigated by the steady-state, synchronous fluorescence and circular dichroism (CD) spectroscopies. The results showed that Sal B had a strong ability to quench the intrinsic fluorescence of HSA through a static quenching mechanism. Binding parameters calculated showed that Sal B was bound to HSA with the binding affinities of 10 5 L mol -1. The thermodynamic parameters studies revealed that the binding was characterized by positive enthalpy and positive entropy changes, and hydrophobic interactions were the predominant intermolecular forces to stabilize the complex. The specific binding distance r (2.93 nm) between donor (HSA) and acceptor (Sal B) was obtained according to Förster non-radiative resonance energy transfer theory. The synchronous fluorescence experiment revealed that Sal B cannot lead to the microenvironmental changes around the Tyr and Trp residues of HSA, and the binding site of Sal B on HSA is located in hydrophobic cavity of subdomain IIA. The CD spectroscopy indicated the secondary structure of HSA is not changed in the presence of Sal B. Furthermore, The effect of metal ions (e.g. Zn 2+, Cu 2+, Co 2+, Ni 2+, Fe 3+) on the binding constant of Sal B-HSA complex was also discussed.

  20. 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.

  1. 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.

  2. Study of the binding interaction between fluorinated matrix metalloproteinase inhibitors and Human Serum Albumin.

    PubMed

    Digilio, Giuseppe; Tuccinardi, Tiziano; Casalini, Francesca; Cassino, Claudio; Dias, David M; Geraldes, Carlos F G C; Catanzaro, Valeria; Maiocchi, Alessandro; Rossello, Armando

    2014-05-22

    Fluorinated, arylsulfone-based inhibitors of Matrix Metalloproteinases (MMP) have been used, in the [(18)F]-radiolabelled version, as radiotracers targeted to MMP-2/9 for Positron Emission Tomography (PET). Although they showed acceptable tumour uptake, specificity was rather low. To get further insights into the reason of low specificity, the binding interaction of these compounds with Human Serum Albumin (HSA) has been investigated. (19)F NMR spectroscopy showed that all compounds considered partition between multiple HSA binding sites, being characterized by either slow-exchange kinetics (with Ka in the order of 10(5) M(-1)) and fast-exchange kinetics (with Ka in the order of 10(4) M(-1)). For 2-(2-(4'-(2-fluoroethoxy)biphenyl-4-ylsulfonyl)phenyl)acetic acid (1a) and 2-(2-(4'-(2-fluoroacetamido)biphenyl-4-ylsulfonyl)phenyl)acetic acid (1c), these slow and fast-exchanging binding sites could be mapped to Sudlow's site I and II, respectively. It is shown that high affinity albumin binding constitutes a theoretical limitation for the specificity achievable by MMP-inhibitors as MMP-targeted PET tracers in cancer imaging, because albumin accumulating aspecifically in tumours lowers the binding potential of radiotracers.

  3. Binding of human serum albumin to PEGylated liposomes: insights into binding numbers and dynamics by fluorescence correlation spectroscopy.

    PubMed

    Kristensen, Kasper; Urquhart, Andrew J; Thormann, Esben; Andresen, Thomas L

    2016-12-01

    Liposomes for medical applications are often administered by intravenous injection. Once in the bloodstream, the liposomes are covered with a "protein corona", which impacts the behavior and eventual fate of the liposomes. Currently, many aspects of the liposomal protein corona are not well understood. For example, there is generally a lack of knowledge about the liposome binding affinities and dynamics of common types of blood plasma proteins. Fluorescence correlation spectroscopy (FCS) is a powerful experimental technique that potentially can provide such knowledge. In this study, we have used FCS to investigate the binding of human serum albumin (HSA) to standard types of PEGylated fluid-phase liposomes (consisting of DOPC and DOPE-PEG2k) and PEGylated gel-phase liposomes (consisting of DSPC and DSPE-PEG2k) with various PEG chain surface densities. We detected no significant binding of HSA to the PEGylated fluid-phase liposomes. In contrast, we found that HSA bound tightly to the PEGylated gel-phase liposomes, although only a low number of HSA molecules could be accommodated per liposome. Overall, we believe that our data provides a useful benchmark for other researchers interested in studying the liposomal protein corona.

  4. Elucidation of the binding mechanism of coumarin derivatives with human serum albumin.

    PubMed

    Garg, Archit; Manidhar, Darla Mark; Gokara, Mahesh; Malleda, Chandramouli; Suresh Reddy, Cirandur; Subramanyam, Rajagopal

    2013-01-01

    Coumarin is a benzopyrone which is widely used as an anti-coagulant, anti-oxidant, anti-cancer and also to cure arthritis, herpes, asthma and inflammation. Here, we studied the binding of synthesized coumarin derivatives with human serum albumin (HSA) at physiological pH 7.2 by using fluorescence spectroscopy, circular dichroism spectroscopy, molecular docking and molecular dynamics simulation studies. By addition of coumarin derivatives to HSA the maximum fluorescence intensity was reduced due to quenching of intrinsic fluorescence upon binding of coumarin derivatives to HSA. The binding constant and free energy were found to be 1.957±0.01×10(5) M(-1), -7.175 Kcal M(-1) for coumarin derivative (CD) enamide; 0.837±0.01×10(5) M(-1), -6.685 Kcal M(-1) for coumarin derivative (CD) enoate, and 0.606±0.01×10(5) M(-1), -6.49 Kcal M(-1) for coumarin derivative methylprop (CDM) enamide. The CD spectroscopy showed that the protein secondary structure was partially unfolded upon binding of coumarin derivatives. Further, the molecular docking studies showed that coumarin derivatives were binding to HSA at sub-domain IB with the hydrophobic interactions and also with hydrogen bond interactions. Additionally, the molecular dynamics simulations studies contributed in understanding the stability of protein-drug complex system in the aqueous solution and the conformational changes in HSA upon binding of coumarin derivatives. This study will provide insights into designing of the new inspired coumarin derivatives as therapeutic agents against many life threatening diseases.

  5. Elucidation of the Binding Mechanism of Coumarin Derivatives with Human Serum Albumin

    PubMed Central

    Gokara, Mahesh; Malleda, Chandramouli; Suresh Reddy, Cirandur; Subramanyam, Rajagopal

    2013-01-01

    Coumarin is a benzopyrone which is widely used as an anti-coagulant, anti-oxidant, anti-cancer and also to cure arthritis, herpes, asthma and inflammation. Here, we studied the binding of synthesized coumarin derivatives with human serum albumin (HSA) at physiological pH 7.2 by using fluorescence spectroscopy, circular dichroism spectroscopy, molecular docking and molecular dynamics simulation studies. By addition of coumarin derivatives to HSA the maximum fluorescence intensity was reduced due to quenching of intrinsic fluorescence upon binding of coumarin derivatives to HSA. The binding constant and free energy were found to be 1.957±0.01×105 M−1, −7.175 Kcal M−1 for coumarin derivative (CD) enamide; 0.837±0.01×105 M−1, −6.685 Kcal M−1 for coumarin derivative (CD) enoate, and 0.606±0.01×105 M−1, −6.49 Kcal M−1 for coumarin derivative methylprop (CDM) enamide. The CD spectroscopy showed that the protein secondary structure was partially unfolded upon binding of coumarin derivatives. Further, the molecular docking studies showed that coumarin derivatives were binding to HSA at sub-domain IB with the hydrophobic interactions and also with hydrogen bond interactions. Additionally, the molecular dynamics simulations studies contributed in understanding the stability of protein-drug complex system in the aqueous solution and the conformational changes in HSA upon binding of coumarin derivatives. This study will provide insights into designing of the new inspired coumarin derivatives as therapeutic agents against many life threatening diseases. PMID:23724004

  6. 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

  7. Determination on the binding of thiadiazole derivative to human serum albumin: a spectroscopy and computational approach.

    PubMed

    Karthikeyan, Subramani; Bharanidharan, Ganesan; Mani, Karthik Ananth; Srinivasan, Narasimhan; Kesherwani, Manish; Velmurugan, Devadasan; Aruna, Prakasarao; Ganesan, Singaravelu

    2017-03-01

    4-[3-acetyl-5-(acetylamino)-2,3-dihydro-1,3,4-thiadiazole-2-yl]phenyl benzoate from the family of thiadiazole derivative has been newly synthesized. It has good anticancer activity as well as antibacterial and less toxic in nature, its binding characteristics are therefore of huge interest for understanding pharmacokinetic mechanism of the drug. The binding of thiadiazole derivative to human serum albumin (HSA) has been investigated by studying its quenching mechanism, binding kinetics and the molecular distance, r between the donor (HSA) and acceptor (thiadiazole derivative) was estimated according to Forster's theory of non-radiative energy transfer. The Gibbs free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) changes of temperature-dependent Kb was calculated, which explains that the reaction is spontaneous and exothermic. The microenvironment of HSA have also been studied using synchronous fluorescence spectroscopy, and the feature of thiadiazole derivative-induced structural changes of HSA have been carried using Fourier transform infrared spectroscopy and the Molecular modelling simulations explore the hydrophobic and hydrogen bonding interactions.

  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. 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.

  10. Fast gradient HPLC method to determine compounds binding to human serum albumin. Relationships with octanol/water and immobilized artificial membrane lipophilicity.

    PubMed

    Valko, Klara; Nunhuck, Shenaz; Bevan, Chris; Abraham, Michael H; Reynolds, Derek P

    2003-11-01

    A fast gradient HPLC method (cycle time 15 min) has been developed to determine Human Serum Albumin (HSA) binding of discovery compounds using chemically bonded protein stationary phases. The HSA binding values were derived from the gradient retention times that were converted to the logarithm of the equilibrium constants (logK HSA) using data from a calibration set of molecules. The method has been validated using literature plasma protein binding data of 68 known drug molecules. The method is fully automated, and has been used for lead optimization in more than 20 company projects. The HSA binding data obtained for more than 4000 compounds were suitable to set up global and project specific quantitative structure binding relationships that helped compound design in early drug discovery. The obtained HSA binding of known drug molecules were compared to the Immobilized Artificial Membrane binding data (CHI IAM) obtained by our previously described HPLC-based method. The solvation equation approach has been used to characterize the normal binding ability of HSA, and this relationship shows that compound lipophilicity is a significant factor. It was found that the selectivity of the "baseline" lipophilicity governing HSA binding, membrane interaction, and octanol/water partition are very similar. However, the effect of the presence of positive or negative charges have very different effects. It was found that negatively charged compounds bind more strongly to HSA than it would be expected from the lipophilicity of the ionized species at pH 7.4. Several compounds showed stronger HSA binding than can be expected from their lipophilicity alone, and comparison between predicted and experimental binding affinity allows the identification of compounds that have good complementarities with any of the known binding sites.

  11. Binding properties of the natural red dye carthamin with human serum albumin: Surface plasmon resonance, isothermal titration microcalorimetry, and molecular docking analysis.

    PubMed

    He, Jiawei; Li, Shanshan; Xu, Kailin; Tang, Bin; Yang, Hongqin; Wang, Qing; Li, Hui

    2017-04-15

    The interaction between carthamin and human serum albumin (HSA) was investigated by multiple spectroscopic analyses, surface plasmon resonance (SPR), isothermal titration microcalorimetry (ITC), and molecular docking studies. Fluorescence lifetime measurements implied that carthamin quenched the intrinsic fluorescence of HSA with the formation of a new complex via static mode. Binding affinities regarding this interaction were obtained from SPR analysis. Results demonstrated that carthamin could form a 1:1 complex with HSA at the binding affinity of KD=8.726×10(-5)M and that a high temperature was unfavourable for the interaction. ITC analyses and molecular docking results illustrated that HSA shaped a proper cavity (site I) to embed the whole carthamin molecule and that the complex was formed depending on intermolecular forces, including hydrophobic interaction, hydrogen bonding, and electrostatic force. Moreover, circular dichroism and 3D fluorescence demonstrated that carthamin slightly disturbed the microenvironment of amino residues and affected the secondary structure of HSA.

  12. The molecular basis for the prolonged blood circulation of lipidated incretin peptides: Peptide oligomerization or binding to serum albumin?

    PubMed

    Wang, Ying; Lomakin, Aleksey; Kanai, Sonoko; Alex, Rainer; Belli, Sara; Donzelli, Massimiliano; Benedek, George B

    2016-11-10

    Hybrid incretin peptides are a new generation of drugs for the treatment of diabetes and obesity. Despite their biological potency, the effectiveness of these peptides as drugs is limited by their short circulation time in blood (typically within minutes). In this work, we show that lipid conjugated forms of a GLP-1/GIP/glucagon hybrid peptides stay in circulation for hours. We studied the oligomerization and albumin-binding of the unconjugated hybrid peptide as well as its lipidated variants. These lipidated peptides differ in the N-terminal mutation, the position of lipidation and the linkage to lipid. We found that these lipidated peptides form stable oligomers at concentrations above 1mg/mL. This concentration range is relevant to formulation and storage of the peptides. We observed no binding between the peptide oligomers and human serum albumin. However, at the expected therapeutic concentration range (~10-100ng/mL), the oligomers dissociate into monomers. The monomers of lipidated peptides bind to albumin. We have determined the dissociation constants of binding between the lipidated peptides and serum albumin. The dissociation constants of albumin-binding of our lipidated peptides are all very close and similar to that of the fatty acid binding of albumin. Our findings suggest that the monomeric lipidated peptides bind to HSA mainly by the fatty acid chain. Therefore, albumin binding is likely to be a universal mechanism of the prolonged circulating duration of lipidated pharmaceutical peptides.

  13. Effect of ageing of human serum albumin in vitro on surface hydrophobicity and binding sites of metronidazole

    NASA Astrophysics Data System (ADS)

    Równicka-Zubik, J.; Sułkowska, A.; Dubas, M.; Pożycka, J.; Maciążek-Jurczyk, M.; Bojko, B.; Sułkowski, W. W.

    2011-05-01

    The fluorescence characteristic of the "alkaline-ageing" process was performed. The quenching of the aged form of human serum albumin (AHSA) fluorescence by acrylamide (Ac) was smaller than that of native HSA, in contrast to the negatively charged anion iodide quencher. The comparison of quenching of fluorescence probes ANS and DNSA bound to aged and native forms of HSA allows for the conclusion that "alkaline-ageing process" causes an increase of hydrophobicity within the binding site located in subdomain IIA. This conclusion was confirmed by the F coefficients calculated for the emission fluorescence spectra of A- and N-forms of HSA excited at 295 nm and 275 nm which show that the increase of hydrophobicity is more significant within tyrosyl than within tryptophanyl residues. The binding constants metronidazole-HSA as well as the number of the class of binding sites were determined by the use of the Scatchard and Klotz-plot analysis. Ageing of HSA causes an increase of the quenching constant determined from the Stern-Volmer equation for λex 275 nm. However ageing does not affect the K Q value for λex 295 nm. The influence of ageing of human serum albumin on its surface hydrophobicity was also studied with the use of 8-anilino-1-naphthalenesulfonic acid (ANS) as the fluorescence probe. At the ANS fluorescence excitation wavelength λex 360 nm the change in surface hydrophobicity is not observed for both N- and A-forms of HSA. The increase of surface hydrophobicity of the A-form in comparison with that of the native form at λex 295 nm indicates that within subdomain IIA an alteration of HSA conformation takes place.

  14. Human Serum Albumin and HER2-Binding Affibody Fusion Proteins for Targeted Delivery of Fatty Acid-Modified Molecules and Therapy.

    PubMed

    Dong, Daoyuan; Xia, Guanjun; Li, Zhijun; Li, Zhiyu

    2016-10-03

    Human epidermal growth factor receptor 2 (HER2) is a well-studied therapeutic target as well as a biomarker of breast cancer. HER2-targeting affibody (ZHER2:342) is a novel small scaffold protein with an extreme high affinity against HER2 screened by phage display. However, the small molecular weight of ZHER2:342 has limited its pharmaceutical application. Human serum albumin (HSA) and ZHER2:342 fusion protein may not only extend the serum half-life of ZHER2:342 but also preserve the biological function of HSA to bind and transport fatty acids, which can be used to deliver fatty acid-modified therapeutics to HER2-positive cancer cells. Two HSA and ZHER2:342 fusion proteins, one with a single ZHER2:342 domain fused to the C terminus of HSA (rHSA-ZHER2) and another with two tandem copies of ZHER2:342 fused to the C terminus of HSA (rHSA-(ZHER2)2), have been constructed, expressed, and purified. Both fusion proteins possessed the HER2 and fatty acid (FA) binding abilities demonstrated by in vitro assays. Interestingly, rHSA-(ZHER2)2, not rHSA-ZHER2, was able to inhibit the proliferation of SK-BR-3 cells at a relatively low concentration, and the increase of HER2 and ERK1/2 phosphorylation followed by rHSA-(ZHER2)2 treatment has been observed. HSA fusion proteins are easy and economical to express, purify, and formulate. As expected, HSA fusion proteins and fusion protein-bound fatty acid-modified FITC could be efficiently taken up by cells. These results proved the feasibility of using HSA fusion proteins as therapeutic agents as well as carriers for targeted drug delivery.

  15. Binding of volatile anesthetics to serum albumin: measurements of enthalpy and solvent contributions.

    PubMed

    Sawas, Abdul H; Pentyala, Srinivas N; Rebecchi, Mario J

    2004-10-05

    This study directly examines the enthalpic contributions to binding in aqueous solution of closely related anesthetic haloethers (desflurane, isoflurane, enflurane, and sevoflurane), a haloalkane (halothane), and an intravenous anesthetic (propofol) to bovine and human serum albumin (BSA and HSA) using isothermal titration calorimetry. Binding to serum albumin is exothermic, yielding enthalpies (DeltaH(obs)) of -3 to -6 kcal/mol for BSA with a rank order of apparent equilibrium association constants (K(a) values): desflurane > isoflurane approximately enflurane > halothane >or= sevoflurane, with the differences being largely ascribed to entropic contributions. Competition experiments indicate that volatile anesthetics, at low concentrations, share the same sites in albumin previously identified in crystallographic and photo-cross-linking studies. The magnitude of the observed DeltaH increased linearly with increased reaction temperature, reflecting negative changes in heat capacities (DeltaC(p)). These -DeltaC(p) values significantly exceed those calculated for burial of each anesthetic in a hydrophobic pocket. The enhanced stabilities of the albumin/anesthetic complexes and -DeltaC(p) are consistent with favorable solvent rearrangements that promote binding. This idea is supported by substitution of D(2)O for H(2)O that significantly reduces the favorable binding enthalpy observed for desflurane and isoflurane, with an opposing increase of DeltaS(obs). From these results, we infer that solvent restructuring, resulting from release of water weakly bound to anesthetic and anesthetic-binding sites, is a dominant and favorable contributor to the enthalpy and entropy of binding to proteins.

  16. Binding of fluphenazine with human serum albumin in the presence of rutin and quercetin: An evaluation of food-drug interaction by spectroscopic techniques.

    PubMed

    Jing, Jiao-Jiao; Liu, Bin; Wang, Xin; Wang, Xin; He, Ling-Ling; Guo, Xue-Yuan; Xu, Ming-Ling; Li, Qian-Yu; Gao, Bo; Dong, Bo-Yang

    2017-04-04

    The interactions between human serum albumin (HSA) and fluphenazine (FPZ) in the presence or absence of rutin or quercetin were studied by fluorescence, absorption and circular dichroism (CD) spectroscopy and molecular modeling. The results showed that the fluorescence quenching mechanism was static quenching by the formation of an HSA-FPZ complex. Entropy change (ΔS(0) ) and enthalpy change (ΔH(0) ) values were 68.42 J/(mol⋅K) and -4.637 kJ/mol, respectively, which indicated that hydrophobic interactions and hydrogen bonds played major roles in the acting forces. The interaction process was spontaneous because the Gibbs free energy change (ΔG(0) ) values were negative. The results of competitive experiments demonstrated that FPZ was mainly located within HSA site I (sub-domain IIA). Molecular docking results were in agreement with the experimental conclusions of the thermodynamic parameters and competition experiments. Competitive binding to HSA between flavonoids and FPZ decreased the association constants and increased the binding distances of FPZ binding to HSA. The results of absorption, synchronous fluorescence, three-dimensional fluorescence, and CD spectra showed that the binding of FPZ to HSA caused conformational changes in HSA and simultaneous effects of FPZ and flavonoids induced further HSA conformational changes.

  17. 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.

  18. Binding of human serum albumin to single-walled carbon nanotubes activated neutrophils to increase production of hypochlorous acid, the oxidant capable of degrading nanotubes.

    PubMed

    Lu, Naihao; Li, Jiayu; Tian, Rong; Peng, Yi-Yuan

    2014-06-16

    Previous studies have shown that carboxylated single-walled carbon nanotubes (SWCNTs) can be catalytically biodegraded by hypochlorite (OCl-) and reactive radical intermediates of the human neutrophil enzyme myeloperoxidase (MPO). However, the importance of protein-SWCNT interactions in the biodegradation of SWCNTs was not stressed. Here, we used both experimental and theoretical approaches to investigate the interactions of SWCNTs with human serum albumin (HSA, one of the most abundant proteins in blood circulation) and found that the binding was involved in the electrostatic interactions of positively charged Arg residues of HSA with the carboxyls on the nanotubes, along with the π-π stacking interactions between SWCNTs and aromatic Tyr residues in HSA. Compared with SWCNTs, the binding of HSA could result in a reduced effect for OCl- (or the human MPO system)-induced SWCNTs degradation in vitro. However, the HSA-SWCNT interactions would enhance cellular uptake of nanotubes and stimulate MPO release and OCl- generation in neutrophils, thereby creating the conditions favorable for the degradation of the nanotubes. Upon zymosan stimulation, both SWCNTs and HSA-SWCNTs were significantly biodegraded in neutrophils, and the degree of biodegradation was more for HSA-SWCNTs under these relevant in vivo conditions. Our findings suggest that the binding of HSA may be an important determinant for MPO-mediated SWCNT biodegradation in human inflammatory cells and therefore shed light on the biomedical and biotechnological applications of safe carbon nanotubes by comprehensive preconsideration of their interactions with human serum proteins.

  19. 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.

  20. Fluorescence quenching studies on binding fluoreno-9-spiro-oxazolidinedione to human serum albumin.

    PubMed

    Gonzalez-Jimenez, J; Jacquotte, H; Cayre, I

    1992-11-16

    Human serum albumin fluorescence quenching by fluorene-9-spiro-oxazolidinedione has been analyzed as a function of temperature. Such temperature dependence suggests that the mechanism of the quenching process is static in origin. This type of quenching implies that a non-fluorescent complex between oxazolidinedione and serum albumin has been formed and following the Stern-Volmer relationship we have calculated the binding constant. Thermodynamic parameters were also determined. The positive and large values of entropy and the negative value for enthalpy suggest that both hydrophobic and electrostatic interactions may play an important role in the stabilization of the complex. Finally, the irreversible changes in the spectral properties of HSA are interpreted in binding terms.

  1. Studies of the Interaction between Isoimperatorin and Human Serum Albumin by Multispectroscopic Method: Identification of Possible Binding Site of the Compound Using Esterase Activity of the Protein

    PubMed Central

    Ranjbar, Samira; Shokoohinia, Yalda; Ghobadi, Sirous; Gholamzadeh, Saeed; Moradi, Nastaran; Ashrafi-Kooshk, Mohammad Reza; Aghaei, Abbas

    2013-01-01

    Isoimperatorin is one of the main components of Prangos ferulacea as a linear furanocoumarin and used as anti-inflammatory, analgesic, antispasmodic, and anticancer drug. Human serum albumin (HSA) is a principal extracellular protein with a high concentration in blood plasma and carrier for many drugs to different molecular targets. Since the carrying of drug by HSA may affect on its structure and action, we decided to investigate the interaction between HSA and isoimperatorin using fluorescence and UV spectroscopy. Fluorescence data indicated that isoimperatorin quenches the intrinsic fluorescence of the HSA via a static mechanism and hydrophobic interaction play the major role in the drug binding. The binding average distance between isoimperatorin and Trp 214 of HSA was estimated on the basis of the theory of Förster energy transfer. Decrease of protein surface hydrophobicity (PSH) was also documented upon isoimperatorin binding. Furthermore, the synchronous fluorescence spectra show that the microenvironment of the tryptophan residues does not have obvious changes. Site marker compettive and fluorescence experiments revealed that the binding of isoimperatorin to HSA occurred at or near site I. Finally, the binding details between isoimperatorin and HSA were further confirmed by molecular docking and esterase activity inhibition studies which revealed that drug was bound at subdomain IIA. PMID:24319355

  2. DNA binding, DNA cleavage and HSA interaction of several metal complexes containing N-(2-hydroxyethyl)-N'-benzoylthiourea and 1,10-phenanthroline ligands.

    PubMed

    Peng, Bo; Gao, Zhuantao; Li, Xibo; Li, Tingting; Chen, Guorong; Zhou, Min; Zhang, Ji

    2016-10-01

    Four novel ternary metal complexes of the type [M(Phen)(L1)2)] [phen = 1,10-phenanthroline, L1 = N-(2-hydroxyethyl)-N'-benzoylthiourea, M = Ni(II)(1), Co(II) (2), Cu(II) (3), Pd(II) (4)] were synthesized. The organic ligands and their corresponding organometallic complexes have been characterized using UV-vis absorption spectroscopy, element analysis, infrared radiation spectroscopy and fluorescence spectra. DNA binding and cleavage studies of these complexes were conducted in detail. In vitro DNA-binding properties were studied by electronic absorption spectra and fluorescence spectra methods. The results indicate that all of the ternary metal complexes can efficiently bind to DNA via intercalation mode. The DNA-binding constants for all ternary compounds are around 4 × 10(6) M(-1). The binding propensity of the complexes to human serum albumin (HSA) was also investigated. Agarose gel electrophoresis study revealed that the metal complexes could cleave super-coiled pBR322 DNA to a nicked form in the absence of external agents. In vitro anti bacterial studies show that copper complex has weak antibacterial activities. Copper complex exhibits a better biological activity among all complexes. This study provides a new perspective and evaluation on the role and importance of the effect factors on the medicinal properties of benzoylthiourea compounds. Synchronous fluorescence spectra of HSA (10 μM) as a function of concentration of the complexes 1-4.

  3. Cubilin is an albumin binding protein important for renal tubular albumin reabsorption.

    PubMed

    Birn, H; Fyfe, J C; Jacobsen, C; Mounier, F; Verroust, P J; Orskov, H; Willnow, T E; Moestrup, S K; Christensen, E I

    2000-05-01

    Using affinity chromatography and surface plasmon resonance analysis, we have identified cubilin, a 460-kDa receptor heavily expressed in kidney proximal tubule epithelial cells, as an albumin binding protein. Dogs with a functional defect in cubilin excrete large amounts of albumin in combination with virtually abolished proximal tubule reabsorption, showing the critical role for cubilin in the uptake of albumin by the proximal tubule. Also, by immunoblotting and immunocytochemistry we show that previously identified low-molecular-weight renal albumin binding proteins are fragments of cubilin. In addition, we find that mice lacking the endocytic receptor megalin show altered urinary excretion, and reduced tubular reabsorption, of albumin. Because cubilin has been shown to colocalize and interact with megalin, we propose a mechanism of albumin reabsorption mediated by both of these proteins. This process may prove important for understanding interstitial renal inflammation and fibrosis caused by proximal tubule uptake of an increased load of filtered albumin.

  4. Electrokinetic chromatographic estimation of the enantioselective binding of nomifensine to human serum albumin and total plasma proteins.

    PubMed

    Asensi-Bernardi, Lucía; Martín-Biosca, Yolanda; Sagrado, Salvador; Medina-Hernández, María J

    2012-11-01

    This report is the first evidence of enantioselective binding of nomifensine to human serum albumin (HSA) and plasma proteins. The overall process with HSA included: (i) consistent experimental design along two independent sessions; (ii) incubation of nomifensine-HSA designed mixtures; (iii) ultrafiltration for separating the unbound enantiomers fraction; (iv) electrokinetic chromatography (EKC) using heptakis-2,3,6-tri-O-methyl-β-cyclodextrin as chiral selector to provide experimental data for enantiomers (first, E1, and second, E2, eluted ones); and (v) a recent direct equation allowing univariate tests and robust statistics to provide consistent parameters and uncertainty. A significant enantioselectivity to HSA (2.7 ± 0.1) was encountered, related to a 1:1 stoichiometry and log affinity constants of 3.24 ± 0.10 and 3.67 ± 0.08 for E1 and E2, respectively. The protein binding (PB) estimated at physiological concentration levels was 40 ± 5 and 63 ± 4% for E1 and E2, respectively. The use of synthetic human sera allowed in vitro estimation of the total plasma PB for the racemate (61 ± 5%; coincident with in vivo values), and its enantiomers (58 ± 7 and 64 ± 4% for E1 and E2, respectively). Comparison allowed the relative importance of HSA respect to other plasma proteins for binding nomifensine to be established.

  5. Chromatographic analysis of the effects of fatty acids and glycation on binding by probes for Sudlow sites I and II to human serum albumin.

    PubMed

    Anguizola, Jeanethe; Debolt, Erin; Suresh, D; Hage, David S

    2016-05-15

    The primary endogenous ligands of human serum albumin (HSA) are non-esterified fatty acids, with 0.1-2mol of fatty acids normally being bound to HSA. In type II diabetes, fatty acid levels in serum are often elevated, and the presence of high glucose results in an increase in the non-enzymatic glycation of HSA. High-performance affinity chromatography (HPAC) was used to examine the combined effects of glycation and the presence of long chain fatty acids on the binding of HSA with R-warfarin and l-tryptophan (i.e., probes for Sudlow sites I and II, the major sites for drugs on this protein). Zonal elution competition studies were used to examine the interactions of myristic acid, palmitic acid and stearic acid with these probes on HSA. It was found that all these fatty acids had direct competition with R-warfarin at Sudlow site I of normal HSA and glycated HSA, with the glycated HSA typically having stronger binding for the fatty acids at this site. At Sudlow site II, direct competition was observed for all the fatty acids with l-tryptophan when using normal HSA, while glycated HSA gave no competition or positive allosteric interactions between these fatty acids and l-tryptophan. These data indicated that glycation can alter the interactions of drugs and fatty acids at specific binding sites on HSA. The results of this study should lead to a better understanding of how these interactions may change during diabetes and demonstrate how HPAC can be used to examine drug/solute-protein interactions in complex systems.

  6. A combined spectroscopic, docking and molecular dynamics simulation approach to probing binding of a Schiff base complex to human serum albumin

    NASA Astrophysics Data System (ADS)

    Fani, N.; Bordbar, A. K.; Ghayeb, Y.

    2013-02-01

    The molecular mechanism of a Schiff base complex ((E)-((E)-2-(3-((E)-((E)-3(mercapto (methylthio) methylene)cyclopentylidene) amino) propylimino) cyclopentylidene) (methylthio) methanethiol) binding to Human Serum Albumin (HSA) was investigated by fluorescence quenching, absorption spectroscopy, molecular docking and molecular dynamics (MD) simulation procedures. The fluorescence emission of HSA was quenched by this Schiff base complex that has been analyzed for estimation of binding parameters. The titration of Schiff base solution by various amount of HSA was also followed by UV-Vis absorption spectroscopy and the corresponding data were analyzed by suitable models. The results revealed that this Schiff base has an ability to bind strongly to HSA and formed 1:1 complex. Energy transfer mechanism of quenching was discussed and the value of 5.45 ± 0.06 nm was calculated as the mean distance between the bound complex and the Trp residue. This is implying the high possibility of energy transfer from HSA to this Schiff base complex. Molecular docking results indicated that the main active binding site for this Schiff base complex is site III in subdomain IB. Moreover, MD simulation results suggested that this Schiff base complex can interact with HSA, without affecting the secondary structure of HSA but probably with a slight modification of its tertiary structure. MD simulations, molecular docking and experimental data reciprocally supported each other.

  7. A combined spectroscopic, docking and molecular dynamics simulation approach to probing binding of a Schiff base complex to human serum albumin.

    PubMed

    Fani, N; Bordbar, A K; Ghayeb, Y

    2013-02-15

    The molecular mechanism of a Schiff base complex ((E)-((E)-2-(3-((E)-((E)-3(mercapto (methylthio) methylene)cyclopentylidene) amino) propylimino) cyclopentylidene) (methylthio) methanethiol) binding to Human Serum Albumin (HSA) was investigated by fluorescence quenching, absorption spectroscopy, molecular docking and molecular dynamics (MD) simulation procedures. The fluorescence emission of HSA was quenched by this Schiff base complex that has been analyzed for estimation of binding parameters. The titration of Schiff base solution by various amount of HSA was also followed by UV-Vis absorption spectroscopy and the corresponding data were analyzed by suitable models. The results revealed that this Schiff base has an ability to bind strongly to HSA and formed 1:1 complex. Energy transfer mechanism of quenching was discussed and the value of 5.45 ± 0.06 nm was calculated as the mean distance between the bound complex and the Trp residue. This is implying the high possibility of energy transfer from HSA to this Schiff base complex. Molecular docking results indicated that the main active binding site for this Schiff base complex is site III in subdomain IB. Moreover, MD simulation results suggested that this Schiff base complex can interact with HSA, without affecting the secondary structure of HSA but probably with a slight modification of its tertiary structure. MD simulations, molecular docking and experimental data reciprocally supported each other.

  8. 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.

  9. Competitive binding of (-)-epigallocatechin-3-gallate and 5-fluorouracil to human serum albumin: A fluorescence and circular dichroism study

    NASA Astrophysics Data System (ADS)

    Yuan, Lixia; Liu, Min; Liu, Guiqin; Li, Dacheng; Wang, Zhengping; Wang, Bingquan; Han, Jun; Zhang, Min

    2017-02-01

    Combination therapy with more than one therapeutic agent can improve therapeutic efficiency and decrease drug resistance. In this study, the interactions of human serum albumin (HSA) with individual or combined anticancer drugs, (-)-epigallocatechin-3-gallate (EGCG) and 5-fluorouracil (FU), were investigated by fluorescence and circular dichroism (CD) spectroscopy. The results demonstrated that the interaction of EGCG or FU with HSA is a process of static quenching and EGCG formed a more stable complex. The competitive experiments of site markers suggested that both anti-carcinogens mainly bound to site I (subdomain IIA). The interaction forces which play important roles in the binding process were discussed based on enthalpy and entropy changes. Moreover, the competition binding model for a ternary system was proposed so as to precisely calculate the binding parameters. The results demonstrated that one drug decreased the binding affinity of another drug with HSA, resulting in the increasing free drug concentration at the action sites. CD studies indicated that there was an alteration in HSA secondary structure due to the binding of EGCG and FU. It can be concluded that the combination of EGCG with FU may enhance anticancer efficacy. This finding may provide a theoretical basis for clinical treatments.

  10. 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.

  11. Molecular Analysis of AFP and HSA Interactions with PTEN Protein.

    PubMed

    Zhu, Mingyue; Lin, Bo; Zhou, Peng; Li, Mengsen

    2015-01-01

    Human cytoplasmic alpha-fetoprotein (AFP) has been classified as a member of the albuminoid gene family. The protein sequence of AFP has significant homology to that of human serum albumin (HSA), but its biological characteristics are vastly different from HSA. The AFP functions as a regulator in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway, but HSA plays a key role as a transport protein. To probe their molecular mechanisms, we have applied colocalization, coimmunoprecipitation (co-IP), and molecular docking approaches to analyze the differences between AFP and HSA. The data from colocalization and co-IP displayed a strong interaction between AFP and PTEN (phosphatase and tensin homolog), demonstrating that AFP did bind to PTEN, but HSA did not. The molecular docking study further showed that the AFP domains I and III could contact with PTEN. In silicon substitutions of AFP binding site residues at position 490M/K and 105L/R corresponding to residues K490 and R105 in HSA resulted in steric clashes with PTEN residues R150 and K46, respectively. These steric clashes may explain the reason why HSA cannot bind to PTEN. Ultimately, the experimental results and the molecular modeling data from the interactions of AFP and HSA with PTEN will help us to identify targets for designing drugs and vaccines against human hepatocellular carcinoma.

  12. Spectral characterization of the binding and conformational changes of serum albumins upon interaction with an anticancer drug, anastrozole

    NASA Astrophysics Data System (ADS)

    Punith, Reeta; Seetharamappa, J.

    2012-06-01

    The present study employed different optical spectroscopic techniques viz., fluorescence, FTIR, circular dichroism (CD) and UV-vis absorption spectroscopy to investigate the mechanism of interaction of an anticancer drug, anastrozole (AZ) with transport proteins viz., bovine serum albumin (BSA) and human serum albumin (HSA). The drug, AZ quenched the intrinsic fluorescence of protein and the analysis of results revealed the presence of dynamic quenching mechanism. The binding characteristics of drug-protein were computed. The thermodynamic parameters, enthalpy change (ΔH°) and entropy change (ΔS°) were calculated to be +92.99 kJ/mol and +159.18 J/mol/K for AZ-BSA and, +99.43 kJ/mol and +159.19 J/mol/K for AZ-HSA, respectively. These results indicated that the hydrophobic forces stabilized the interaction between the drug and protein. CD, FTIR, absorption, synchronous and 3D fluorescence results indicated that the binding of AZ to protein induced structural perturbation in both serum albumins. The distance, r between the drug and protein was calculated based on the theory of Förster's resonance energy transfer and found to be 5.9 and 6.24 nm, respectively for AZ-BSA and AZ-HSA.

  13. Drug binding to human serum albumin: abridged review of results obtained with high-performance liquid chromatography and circular dichroism.

    PubMed

    Ascoli, Giorgio A; Domenici, Enrico; Bertucci, Carlo

    2006-09-01

    The drug binding to plasma and tissue proteins are fundamental factors in determining the overall pharmacological activity of a drug. Human serum albumin (HSA), together with alpha1-acid glycoprotein (AGP), are the most important plasma proteins, which act as drug carriers, with drug pharmacokinetic implications, resulting in important clinical impacts for drugs that have a relatively narrow therapeutic index. This review focuses on the combination of biochromatography and circular dichroism as an effective approach for the characterization of albumin binding sites and their enantioselectivity. Furthermore, their applications to the study of changes in the binding properties of the protein arising by the reversible or covalent binding of drugs are discussed, and examples of physiological relevance reported. Perspectives of these studies reside in supporting the development of new drugs, which require miniaturization to facilitate the screening of classes of compounds for their binding to the target protein, and a deeper characterization of the mechanisms involved in the molecular recognition processes.

  14. 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.

  15. The automatic use of capillary isoelectric focusing with whole column imaging detection for carbamazepine binding to human serum albumin.

    PubMed

    Maciążek-Jurczyk, Małgorzata; Pawliszyn, Janusz

    2016-08-05

    The binding of the anticonvulsant drug carbamazepine (CBZ) to human serum albumin, both without (dHSA) and in the presence of fatty acids (HSA) was studied in real time by capillary isoelectric focusing with whole column imaging detection (cIEF-WCID). Reaction mixtures at different CBZ:HSA and CBZ:dHSA molar ratios (0:1/25:1) were prepared in phosphate buffer saline (PBS) solution at a physiological pH (7.4), and incubated for 0-72h at 37°C in a water bath. Application of the cIEF-WCID method allowed for observations on the impact of increasing CBZ:serum albumin molar ratios on isoelectric point (pI) shifts, as well as changes in peak area and absorbance, which serve as evidence of structural alterations occurring in the protein in the presence of CBZ. The obtained cIEF-WCID results indicated that the dynamic process of complex formation is not dependent on incubation time. The presented work allowed for recognition of different types of interactions, as well as for the calculation of association constants that demonstrate the stability of the complex. This study was also designed to examine the possible impact of fatty acids (FAs) on protein stability and drug delivery in blood.

  16. Fatty Acid Saturation of Albumin Used in Resuscitation Fluids Modulates Cell Damage in Shock: In Vitro Results Using a Novel Technique to Measure Fatty Acid Binding Capacity.

    PubMed

    Penn, Alexander H; Dubick, Michael A; Torres Filho, Ivo P

    2017-03-21

    The use of albumin for resuscitation has not proven as beneficial in human trials as expected from numerous animal studies. One explanation could be the practice of adding fatty acid (FA) during manufacture of pharmaceutical albumin. During ischemia, unbound free FAs (FFA) in the circulation could potentially induce cellular damage. We hypothesized that albumins with higher available binding capacities (ABC) for FFAs may prevent that damage. Therefore, we developed a technique to measure ABC, determined if pharmaceutical human serum albumin (HSA) has decreased ABC compared to FA-free bovine serum albumin (BSA), and if binding capacity would affect hemolysis when blood is mixed with exogenous FFA at levels similar to those observed in shock. The new assay used exogenous oleic acid (OA), glass fiber filtration, and a FFA assay kit. RBC hemolysis was determined by mixing 0-5 mM OA with PBS, HSA, FA-free BSA, or FA-saturated BSA and measuring plasma hemoglobin after incubation with human blood. 5% HSA contained 4.7±0.2 mM FFA, leaving an ABC of 5.0 ± 0.6 mM, compared to FA-free BSA's ABC of 7.0 ± 1.3 mM (P < 0.024). Hemolysis after OA was reduced with FA-free BSA but increased with FA-saturated BSA. HSA provided intermediate results. 25% solutions of FA-free BSA and HSA were more protective, while 25% FA-saturated BSA was more damaging than 5% solutions. These findings suggest that increased FA saturation may reverse albumin's potential benefit to lessen cellular damage and may explain, at least in part, its failure in human trauma studies.

  17. 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.

  18. 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

  19. Specific albumin binding to microvascular endothelium in culture

    SciTech Connect

    Schnitzer, J.E.; Carley, W.W.; Palade, G.E. )

    1988-03-01

    The specific binding of rat serum albumin (RSA) to confluent microvascular endothelial cells in culture derived from the vasculature of the rat epididymal fat pad was studied at 4{degree}C by radioassay and immunocytochemistry. Radioiodinated RSA ({sup 125}I-RSA) binding to the cells reached equilibrium at {approximately} 20 min incubation. Albumin binding was a slowly saturating function over concentrations ranging from 0.01 to 50 mg/ml. Specific RSA binding with a moderate apparent affinity constant of 1.0 mg/ml and with a maximum binding concentration of 90 ng/cm{sup 2} was immunolocalized with anti-RSA antibody to the outer (free) side of the enothelium. Scatchard analysis of the binding yielded a nonlinear binding curve with a concave-upward shape. Dissociation rate analysis supports negative cooperativity of albumin binding, but multiple binding sites may also be present. Albumin binding fulfilled many requirements for ligand specificity including saturability, reversibility, competibility, and dependence on both cell type and cell number. The results are discussed in terms of past in situ investigations on the localization of albumin binding to vascular endothelium and its effect on transendothelial molecular transport.

  20. Polypharmacotherapy in rheumatology: 1H NMR analysis of binding of phenylbutazone and methotrexate to serum albumin

    NASA Astrophysics Data System (ADS)

    Maciążek-Jurczyk, M.; Sułkowska, A.; Równicka-Zubik, J.; Bojko, B.; Szkudlarek-Haśnik, A.; Knopik, M.; Sułkowski, W. W.

    2011-05-01

    The influence of phenylbutazone (Phe) and methotrexate (MTX) on binding of MTX and Phe to human (HSA) and bovine (BSA) serum albumin in the low-affinity binding sites is investigated. The strength and kind of interactions between serum albumin (SA) and drugs used in combination therapy were found using 1H NMR spectroscopy. A stoichiometric molar ratios for Phe-SA and MTX-SA complexes are 36:1 and 31:1, respectively. It appeared these molar ratios are higher for the ternary systems than it were in the binary ones. The presence of the additional drug (MTX or Phe) causes the increase of an affinity of albumin towards Phe and MTX. It was found that the aliphatic groups of MTX are more resistant to the influence of Phe on the MTX-SA complex than the aromatic rings. The results showed the important impact of another drug (MTX or Phe) on the affinity of SA towards Phe and MTX in the low-affinity binding sites. This work is a subsequent part of the spectroscopic study on Phe-MTX-SA interactions (Maciążek-Jurczyk, 2009 [1]).

  1. Albumin binds self-assembling dyes as specific polymolecular ligands.

    PubMed

    Stopa, Barbara; Rybarska, Janina; Drozd, Anna; Konieczny, Leszek; Król, Marcin; Lisowski, Marek; Piekarska, Barbara; Roterman, Irena; Spólnik, Paweł; Zemanek, Grzegorz

    2006-12-15

    Self-assembling dyes with a structure related to Congo red (e.g. Evans blue) form polymolecular complexes with albumin. The dyes, which are lacking a self-assembling property (Trypan blue, ANS) bind as single molecules. The supramolecular character of dye ligands bound to albumin was demonstrated by indicating the complexation of dye molecules outnumbering the binding sites in albumin and by measuring the hydrodynamic radius of albumin which is growing upon complexation of self-assembling dye in contrast to dyes lacking this property. The self-assembled character of Congo red was also proved using it as a carrier introducing to albumin the intercalated nonbonding foreign compounds. Supramolecular, ordered character of the dye in the complex with albumin was also revealed by finding that self-assembling dyes become chiral upon complexation. Congo red complexation makes albumin less resistant to low pH as concluded from the facilitated N-F transition, observed in studies based on the measurement of hydrodynamic radius. This particular interference with protein stability and the specific changes in digestion resulted from binding of Congo red suggest that the self-assembled dye penetrates the central crevice of albumin.

  2. Copper(I)-Phosphine Polypyridyl Complexes: Synthesis, Characterization, DNA/HSA Binding Study, and Antiproliferative Activity.

    PubMed

    Villarreal, Wilmer; Colina-Vegas, Legna; Visbal, Gonzalo; Corona, Oscar; Corrêa, Rodrigo S; Ellena, Javier; Cominetti, Marcia Regina; Batista, Alzir Azevedo; Navarro, Maribel

    2017-04-03

    A series of copper(I)-phosphine polypyridyl complexes have been investigated as potential antitumor agents. The complexes [Cu(PPh3)2dpq]NO3 (2), [Cu(PPh3)2dppz]NO3 (3), [Cu(PPh3)2dppa]NO3 (4), and [Cu(PPh3)2dppme]NO3 (5) were synthesized by the reaction of [Cu(PPh3)2NO3] with the respective planar ligand under mild conditions. These copper complexes were fully characterized by elemental analysis, molar conductivity, FAB-MS, and NMR, UV-vis, and IR spectroscopies. Interactions between these copper(I)-phosphine polypyridyl complexes and DNA have been investigated using various spectroscopic techniques and analytical methods, such as UV-vis titrations, thermal denaturation, circular dichroism, viscosity measurements, gel electrophoresis, and competitive fluorescent intercalator displacement assays. The results of our studies suggest that these copper(I) complexes interact with DNA in an intercalative way. Furthermore, their high protein binding affinities toward human serum albumin were determined by fluorescence studies. Additionally, cytotoxicity analyses of all complexes against several tumor cell lines (human breast, MCF-7; human lung, A549; and human prostate, DU-145) and non-tumor cell lines (Chinese hamster lung, V79-4; and human lung, MRC-5) were performed. The results revealed that copper(I)-phosphine polypyridyl complexes are more cytotoxic than the corresponding planar ligand and also showed to be more active than cisplatin. A good correlation was observed between the cytostatic activity and lipophilicity of the copper(I) complexes studied here.

  3. 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.

  4. Identification of albumin-binding proteins in capillary endothelial cells

    PubMed Central

    1988-01-01

    Isolated fat tissue microvessels and lung, whose capillary endothelia express in situ specific binding sites for albumin, were homogenized and subjected to SDS-gel electrophoresis and electroblotting. The nitrocellulose strips were incubated with either albumin-gold (Alb-Au) and directly visualized, or with [125I]albumin (monomeric or polymeric) and autoradiographed. The extracts of both microvascular endothelium and the lung express albumin-binding proteins (ABPs) represented by two pairs of polypeptides with major components of molecular mass 31 and 18 kD. The ABP peptides have pIs 8.05 to 8.75. Rabbit aortic endothelium, used as control, does not express detectable amounts of ABPs. The ABPs subjected to electrophoresis bind specifically and with high affinity (Kd = approximately 60 X 10(-9)M) both monomeric and polymeric albumin: the binding is saturable at approximately 80 nM concentration and 50% inhibition is reached at 5.5 micrograms/ml albumin concentration. Sulfhydryl-reducing agents beta-mercaptoethanol and dithiothreitol do not markedly affect the ABPs electrophoretic mobility and binding properties. As indicated by cell surface iodination of isolated capillary endothelium followed by electroblotting, autoradiography, and incubation with Alb-Au, the bands specifically stained by this ligand are also labeled with radioiodine. PMID:2839518

  5. Application of phenol red as a marker ligand for bilirubin binding site at subdomain IIA on human serum albumin.

    PubMed

    Sochacka, Jolanta

    2015-10-01

    The drug-bilirubin interaction for all drugs administered especially to infants with hyperbilirubinemia should be evaluated for their ability to displace bilirubin and vice versa. In order to examine whether phenol red (PhRed) can be used as a marker for bilirubin binding site located in subdomain IIA the interaction between PhRed and human serum albumin (HSA) in buffer solution or in normal and pathological sera solutions with different HSA:bilirubin molar ratio was investigated using absorption/absorption difference spectroscopy and molecular docking method. Six sulfonamides representing the binding site in the subdomain IIA and known to influence the binding of bilirubin were used for the PhRed displacement studies. The absorption spectra for PhRed completely bound to HSA showed significant differences in the spectral characteristic relative to the spectral profile of free PhRed. The intensity of the peak originating from the bivalent anionic form of dye was strongly reduced and the maximum peak position was red-shifted by 12 nm. The binding constant (K) of the bivalent anionic form of PhRed, calculated from absorbance data, was 1.61 · 10(4) L mol(-1). The variations of the absorption and absorption difference spectra of PhRed in the presence of HSA-bilirubin complex were indicative of the inhibition of PhRed binding process by bilirubin. Binding of PhRed carried out in the presence of sulfonamides showed that drugs and PhRed have a common site which also involves bilirubin. In agreement with the results of the spectroscopic analysis and molecular docking it was concluded that PhRed may be applied as a marker in the study of the binding of drugs to high-affinity bilirubin binding site.

  6. 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

  7. Location and binding mechanism of an ESIPT probe 3-hydroxy-2-naphthoic acid in unsaturated fatty acid bound serum albumins.

    PubMed

    Ghorai, Shyamal Kr; Tripathy, Debi Ranjan; Dasgupta, Swagata; Ghosh, Sanjib

    2014-02-05

    The binding site and the binding mechanism of 3-hydroxy-2-naphthoic acid (3HNA) in oleic acid (OA) bound serum albumins (bovine serum albumin (BSA) and human serum albumin (HSA)) have been determined using steady state and time resolved emission of tryptophan residues (Trp) in proteins and the ESIPT emission of 3HNA. Time resolved anisotropy of the probe 3HNA and low temperature phosphorescence of Trp residues of BSA in OA bound BSA at 77K reveals a drastic change of the binding site of 3HNA in the ternary system compared to that in the free protein. 3HNA binds near Trp213 in the ternary system whereas 3HNA binds near Trp134 in the free protein. The structure of OA bound BSA generated using docking methodology exhibits U-bend configuration of all bound OA. The docked pose of 3HNA in the free protein and in OA bound albumins (ternary systems) and the concomitant perturbation of the structure of proteins around the binding region of 3HNA corroborate the enhanced ESIPT emission of 3HNA and the energy transfer efficiency from the donor Trp213 of BSA to 3HNA acceptor in 3HNA-OA-BSA system.

  8. Initial Study of Radiological and Clinical Efficacy Radioembolization Using 188Re-Human Serum Albumin (HSA) Microspheres in Patients with Progressive, Unresectable Primary or Secondary Liver Cancers

    PubMed Central

    Nowicki, Mirosław L.; Ćwikła, Jarosław B.; Sankowski, Artur J.; Shcherbinin, Sergey; Grimes, Josh; Celler, Anna; Buscombe, John R.; Bator, Andrzej; Pech, Maciej; Mikołajczak, Renata; Pawlak, Dariusz

    2014-01-01

    Background The aim of this initial study was to evaluate the clinical and radiological effectiveness of radioembolization (RE) using 188Re-Human Serum Albumin (HSA) microspheres in patients with advanced, progressive, unresectable primary or secondary liver cancers, not suitable to any other form of therapy. Material/Methods Overall, we included 13 patients with 20 therapy sessions. Clinical and radiological responses were assessed at 6 weeks after therapy, and then every 3 months. The objective radiological response was classified according to Response Evaluation Criteria in Solid Tumors (RECIST) v.1.0 by sequential MRI. Adverse events were evaluated using NCI CTCAE v.4.03. Results There were 4 patients with hepatocellular carcinoma (HCC), 6 with metastatic colorectal cancer (mCRC), 2 with neuroendocrine carcinoma (NEC), and 1 patient with ovarian carcinoma. Mean administered activity of 188Re HSA was 7.24 GBq (range 3.8–12.4) A high microspheres labeling efficacy of over 97±2.1% and low urinary excretion of 188Re (6.5±2.3%) during first 48-h follow-up. Median overall survival (OS) for all patients was 7.1 months (CI 6.2–13.3) and progression-free survival (PFS) was 5.1 months (CI 2.4–9.9). In those patients who had a clinical partial response (PR), stable disease (SD), and disease progression (DP) as assessed 6 weeks after therapy, the median OS was 9/5/4 months, respectively, and PFS was 5/2/0 months, respectively. The treatment adverse events (toxicity) were at an acceptable level. Initially and after 6 weeks, the CTC AE was grade 2, while after 3 months it increased to grade 3 in 4 subjects. This effect was mostly related to rapid cancer progression in this patient subgroup. Conclusions The results of this preliminary study indicate that RE using 188Re HSA is feasible and a viable option for palliative therapy in patients with extensive progressive liver cancer. It was well tolerated by most patients, with a low level of toxicity during the 3 months of

  9. Fluorescence analysis of competition of phenylbutazone and methotrexate in binding to serum albumin in combination treatment in rheumatology

    NASA Astrophysics Data System (ADS)

    Maciążek-Jurczyk, M.; Sułkowska, A.; Bojko, B.; Równicka, J.; Sułkowski, W. W.

    2009-04-01

    Combination of several drugs is often necessary especially during long-them therapy. The competition between drugs can cause a decrease of the amount of a drug bound to albumin. This results in an increase of the free, biological active fraction of the drug. The aim of the presented study was to describe the competition between phenylbutazone (Phe) and methotrexate (MTX), two drugs recommended for the treatment of rheumatology in binding to bovine (BSA) and human (HSA) serum albumin in the high affinity binding site. Fluorescence analysis was used to estimate the effect of drugs on the protein fluorescence and to define the binding and quenching properties of drugs-serum albumin complexes. The effect of the displacement of one drug from the complex of the other with serum albumin has been described on the basis of the comparison of the quenching curves and binding constants for the binary and ternary systems. The conclusion that both Phe and MTX form a binding site in the same subdomain (IIA) points to the necessity of using a monitoring therapy owning to the possible increase of the uncontrolled toxic effects.

  10. 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.

  11. Evaluation of enantioselective binding of antihistamines to human serum albumin by ACE.

    PubMed

    Martínez-Gómez, María Amparo; Villanueva-Camañas, Rosa M; Sagrado, Salvador; Medina-Hernández, María José

    2007-08-01

    The drug binding to plasma and tissue proteins is a fundamental factor in determining the overall pharmacological activity of a drug. HSA, together with alpha(1)-acid glycoprotein, are the most important plasma proteins, which act as drug carriers, with implications on the pharmacokinetic of drugs. Among plasma proteins, HSA possesses the highest enantioselectivity. In this paper, a new methodology for the study of enantiodifferentiation of chiral drugs with HSA is developed and applied to evaluate the possible enantioselective binding of four antihistamines: brompheniramine, chlorpheniramine, hydroxyzine and orphenadrine to HSA. This study includes the determination of affinity constants of drug enantiomers to HSA and the evaluation of the binding sites of antihistamines on the HSA molecule. The developed methodology includes the ultrafiltration of samples containing HSA and racemic antihistaminic drugs and the analysis of the free or bound drug fraction using the affinity EKC-partial filling technique and HSA as chiral selector. The results shown in this paper represent the first evidence of the enantioselective binding of antihistamines to HSA, the major plasmatic protein.

  12. Identification of drug-binding sites on human serum albumin using affinity capillary electrophoresis and chemically modified proteins as buffer additives.

    PubMed

    Kim, Hee Seung; Austin, John; Hage, David S

    2002-03-01

    A technique based on affinity capillary electrophoresis (ACE) and chemically modified proteins was used to screen the binding sites of various drugs on human serum albumin (HSA). This involved using HSA as a buffer additive, following the site-selective modification of this protein at two residues (tryptophan 214 or tyrosine 411) located in its major binding regions. The migration times of four compounds (warfarin, ibuprofen, suprofen and flurbiprofen) were measured in the presence of normal or modified HSA. These times were then compared and the mobility shifts observed with the modified proteins were used to identify the binding regions of each injected solute on HSA. Items considered in optimizing this assay included the concentration of protein placed into the running buffer, the reagents used to modify HSA, and the use of dextran as a secondary additive to adjust protein mobility. The results of this method showed good agreement with those of previous reports. The advantages and disadvantages of this approach are examined, as well as its possible extension to other solutes.

  13. Biothermodynamic characterization of monocarboxylic and dicarboxylic aliphatic acids binding to human serum albumin: a flow microcalorimetric study.

    PubMed

    Aki, H; Yamamoto, M

    1993-02-01

    Thermodynamic parameters have been evaluated for the binding of unbranched monocarboyxlic aliphatic acids (MCAs) of 4 to 16 carbons (MC4 to MC16) and dicarboxylic aliphatic acids (DCAs) of 4 to 16 carbons (DC4 to DC16) to human serum albumin (HSA) on the basis of microcalorimetric measurement at pH 7.4 and 37 degrees C by computer-fitting to single- and two-class binding models. Long-chain MCAs (MC10 to MC16) and DCAs (DC14 and DC16) had the first class of binding sites with high affinity (large binding constant) of 10(5) to 10(6) M-1 and the second class with lower affinity and high capacity (large numbers of binding sites). Short- or medium-chain MCAs and DCAs bound to HSA at some low affinity binding sites. The binding constants of MCAs were ten times larger than those of DCAs. All the relationships between the thermodynamic parameters and alkyl-chain length of the acids showed clear-cut inflections in their plots around eight or nine methylene units. The free energy change of the first class of binding sites (- delta G1) became more negative with an increment of -1.0 kJ mol-1 CH2(-1) as the alkyl-chain length increased, but there were steep rises between MC9 and MC11 with -2.90 kJ mol-1 CH2(-1) and between DC9 and DC12 with -2.02 kJ mol-1 CH2(-1). The enthalpy change (- delta H) increased at the rate of -7.4 kJ mol-1 CH2(-1) to the maximum at MC9 and DC10, then decreased due to hydrophobicity of the alkyl-chains. From compensation analyses (delta H vs. delta S and delta G), HSA binding sites were characterized into three groups.

  14. 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.

  15. Hyphenated affinity capillary electrophoresis with a high-sensitivity cell for the simultaneous binding study of retinol and retinoic acid in nanomolars with serum albumins.

    PubMed

    El-Hady, D Abd; Albishri, H M

    2012-12-12

    Retinol and retinoic acid are Vitamin A components that are critical for many biological processes. Both of them are strongly complexing with serum albumins giving constants of the order of 10(5)Lmol(-1) or higher. With respect to this fact, affinity capillary electrophoresis (ACE) is not applicable in its commonly used form. Therefore, for the first time, the hyphenated ACE with a high-sensitivity cell was developed and employed to investigate the binding of retinol and retinoic acid in nanomolars with human serum albumin (HSA) and bovine serum albumin (BSA) under physiological conditions. ACE/high-sensitivity coupled cell had contributed to fast the association and dissociation rates of the complexes in nanomolar scale of analytes ensuring the establishment of a dynamic equilibrium within a short electrophoresis time. In addition, this hyphenation led to reduce the concentrations of serum albumins as additives in background electrolyte making a sense beside the proper rinsing protocol for the negligible possibility of their adsorption. The mobility ratio based on nonlinear regression analysis was used to deduce precise binding constants of analytes with serum albumins. The binding constants (K, Lmol(-1)) of retinol were 1.28×10(5) and 5.25×10(6) and retinoic acid were 3.29×10(5) and 2.27×10(6) with HSA and BSA, respectively. The displacement and reciprocal competitive binding of analytes were investigated and indicated that retinoic acid was able to replace retinol from HSA and vice versa in the case of BSA.

  16. Separate and simultaneous binding effects through a non-cooperative behavior between cyclophosphamide hydrochloride and fluoxymesterone upon interaction with human serum albumin: Multi-spectroscopic and molecular modeling approaches

    NASA Astrophysics Data System (ADS)

    Zohoorian-Abootorabi, Toktam; Sanee, Hamideh; Iranfar, Hediyeh; Saberi, Mohammad Reza; Chamani, Jamshidkhan

    2012-03-01

    This study was designed to examine the interaction of two anti-breast cancer drugs, i.e., fluoxymesterone (FLU) and cyclophosphamide (CYC), with human serum albumin (HSA) using different kinds of spectroscopic, zeta potential and molecular modeling techniques under imitated physiological conditions. The RLS technique was utilized to investigate the effect of the two anticancer drugs on changes of the protein conformation, both separately and simultaneously. Our study suggested that the enhancement in RLS intensity was attributed to the formation of a new complex between the two drugs and the protein. Both drugs demonstrated a powerful ability to quench the fluorescence of HSA, and the fluorescence quenching action was much stronger when the two drugs coexisted. The quenching mechanism was suggested to be static as confirmed by time-resolved fluorescence spectroscopy results. The effect of both drugs on the conformation of HSA was analyzed using synchronous fluorescence spectroscopy. Our results revealed that the fluorescence quenching of HSA originated from the Trp and Tyr residues, and demonstrated a conformational change of HSA with the addition of both drugs. The binding distances between HSA and the drugs were estimated by the Förster theory, and it was revealed that nonradiative energy transfer from HSA to both drugs occurred with a high probability. According to CD measurements, the influence of both drugs on the secondary structure of HSA in aqueous solutions was also investigated and illustrated that the α-helix content of HSA decreased with increasing drug concentration in both systems. Moreover, the zeta-potential experiments revealed that both drugs induced conformational changes on HSA. Docking studies were also performed and demonstrated that a reduction of the binding affinity between the drugs and HSA occurred in the presence of both drugs.

  17. The investigation of the binding behavior between ethyl maltol and human serum albumin by multi-spectroscopic methods and molecular docking

    NASA Astrophysics Data System (ADS)

    Yue, Yuanyuan; Liu, Jianming; Yao, Meihuan; Yao, Xiaojun; Fan, Jing; Ji, Hanxuan

    2012-10-01

    This paper was designed to investigate the interaction of ethyl maltol with human serum albumin (HSA) under physiological condition by fluorescence, synchronous fluorescence, three-dimensional fluorescence, Fourier transformation infrared spectra, and molecular docking method. Spectroscopic analysis of the emission quenching at different temperatures revealed that the quenching mechanism of HSA by ethyl maltol was static quenching mechanism. The binding constants of ethyl maltol-HSA complexes were observed to be 2.59, 1.88, 1.54, 1.13 × 104 M-1 at 289, 296, 303 and 310 K, respectively. The thermodynamic parameters, ΔH0 and ΔS0 were calculated to be -28.61 kJ mol-1 and -14.59 J mol-1 K-1. Energy transfer from tryptophan to ethyl maltol occurred by a FRET mechanism, and the donor-acceptor distance (3.04 nm) had been determined according to Förster's theory. Molecular docking studies revealed that ethyl maltol situated within subdomain IIA (site I) of HSA. Fluorescence displacement experiments also proved the binding sites between ethyl maltol and HSA.

  18. The investigation of the binding behavior between ethyl maltol and human serum albumin by multi-spectroscopic methods and molecular docking.

    PubMed

    Yue, Yuanyuan; Liu, Jianming; Yao, Meihuan; Yao, Xiaojun; Fan, Jing; Ji, Hanxuan

    2012-10-01

    This paper was designed to investigate the interaction of ethyl maltol with human serum albumin (HSA) under physiological condition by fluorescence, synchronous fluorescence, three-dimensional fluorescence, Fourier transformation infrared spectra, and molecular docking method. Spectroscopic analysis of the emission quenching at different temperatures revealed that the quenching mechanism of HSA by ethyl maltol was static quenching mechanism. The binding constants of ethyl maltol-HSA complexes were observed to be 2.59, 1.88, 1.54, 1.13×10(4) M(-1) at 289, 296, 303 and 310 K, respectively. The thermodynamic parameters, ΔH(0) and ΔS(0) were calculated to be -28.61 kJ mol(-1) and -14.59 J mol(-1) K(-1). Energy transfer from tryptophan to ethyl maltol occurred by a FRET mechanism, and the donor-acceptor distance (3.04 nm) had been determined according to Förster's theory. Molecular docking studies revealed that ethyl maltol situated within subdomain IIA (site I) of HSA. Fluorescence displacement experiments also proved the binding sites between ethyl maltol and HSA.

  19. Reply to commentary on "Spectral characterization of the binding and conformational changes of serum albumins upon interaction with an anticancer drug, anastrozole"

    NASA Astrophysics Data System (ADS)

    Punith, Reeta; Seetharamappa, J.

    2015-03-01

    The study by Punith and Seetharamappa [1] presents the effect of an anticancer drug, anastrozole (AZ), on the secondary structure of human serum albumin (HSA) and bovine serum albumin (BSA). Spectroscopic methods used in the study are absorption, CD, FTIR, synchronous and 3D fluorescence. The study reports that one molecule of AZ spontaneously bind to the protein on one site with hydrophobic interactions. As a result, almost 30% of the secondary structure of HSA is altered. This last conclusion is based on the results from CD and FTIR spectroscopy. Although this commentary is mainly intended to point out the misuse of FTIR data, there are also questions raised regarding other sections of the study.

  20. Binding sites of retinol and retinoic acid with serum albumins.

    PubMed

    Belatik, A; Hotchandani, S; Bariyanga, J; Tajmir-Riahi, H A

    2012-02-01

    Retinoids are effectively transported in the bloodstream via serum albumins. We report the complexation of bovine serum albumin (BSA) with retinol and retinoic acid at physiological conditions, using constant protein concentration and various retinoid contents. FTIR, CD and fluorescence spectroscopic methods and molecular modeling were used to analyze retinoid binding site, the binding constant and the effects of complexation on BSA stability and secondary structure. Structural analysis showed that retinoids bind BSA via hydrophilic and hydrophobic interactions with overall binding constants of K(Ret)(-BSA) = 5.3 (±0.8) × 10(6) M(-1) and K(Retac-BSA) = 2.3 (±0.4) × 10(6) M(-1). The number of bound retinoid molecules (n) was 1.20 (±0.2) for retinol and 1.8 (±0.3) for retinoic acid. Molecular modeling showed the participation of several amino acids in retinoid-BSA complexes stabilized by H-bonding network. The retinoid binding altered BSA conformation with a major reduction of α-helix from 61% (free BSA) to 36% (retinol-BSA) and 26% (retinoic acid-BSA) with an increase in turn and random coil structures indicating a partial protein unfolding. The results indicate that serum albumins are capable of transporting retinoids in vitro and in vivo.

  1. Human serum albumin binding to silica nanoparticles--effect of protein fatty acid ligand.

    PubMed

    Ang, Joo Chuan; Henderson, Mark J; Campbell, Richard A; Lin, Jhih-Min; Yaron, Peter N; Nelson, Andrew; Faunce, Thomas; White, John W

    2014-06-07

    Neutron reflectivity shows that fatted (F-HSA) and defatted (DF-HSA) versions of human serum albumin behave differently in their interaction with silica nanoparticles premixed in buffer solutions although these proteins have close to the same surface excess when the silica is absent. In both cases a silica containing film is quickly established at the air-water interface. This film is stable for F-HSA at all relative protein-silica concentrations measured. This behaviour has been verified for two small silica nanoparticle radii (42 Å and 48 Å). Contrast variation and co-refinement have been used to find the film composition for the F-HSA-silica system. The film structure changes with protein concentration only for the DF-HSA-silica system. The different behaviour of the two proteins is interpreted as a combination of three factors: increased structural stability of F-HSA induced by the fatty acid ligand, differences in the electrostatic interactions, and the higher propensity of defatted albumin to self-aggregate. The interfacial structures of the proteins alone in buffer are also reported and discussed.

  2. 1HNMR study of methotrexate serum albumin (MTX SA) binding in rheumatoid arthritis

    NASA Astrophysics Data System (ADS)

    Sułkowska, A.; Maciążek-Jurczyk, M.; Bojko, B.; Równicka, J.; Sułkowski, W. W.

    2008-11-01

    Rheumatoid arthritis (RA) is an immunologically depended disease. It is characterized by a chronic, progressive inflammatory process. Methotrexate (4-amino-10-methylfolic acid, MTX) is the modifying drug used to treat RA. The aim of the presented studies is to determine the low affinity binding site of MTX in bovine (BSA) and human (HSA) serum albumin with the use of proton nuclear magnetic resonance ( 1HNMR) spectroscopy. The analysis of 1HNMR spectra of MTX in the presence of serum albumin (SA) allows us to observe the interactions between aromatic rings of the drug and the rings of amino acids located in the hydrophobic subdomains of the protein. On the basis of the chemical shifts σ [ppm] and the relaxation times T1 [s] of drug protons the hydrophobic interaction between MTX-SA and the stoichiometric molar ratio of the complex was evaluated. This work is a part of a spectroscopic study on MTX-SA interactions [A. Sułkowska, M. Maciążek, J. Równicka, B. Bojko, D. Pentak, W.W. Sułkowski, J. Mol. Struct. 834-836 (2007) 162-169].

  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. The metallomics approach: use of Fe(II) and Cu(II) footprinting to examine metal binding sites on serum albumins.

    PubMed

    Duff, Michael R; Kumar, Challa V

    2009-11-01

    Metal binding to serum albumins is examined by oxidative protein-cleavage chemistry, and relative affinities of multiple metal ions to particular sites on these proteins were identified using a fast and reliable chemical footprinting approach. Fe(ii) and Cu(ii), for example, mediate protein cleavage at their respective binding sites on serum albumins, in the presence of hydrogen peroxide and ascorbate. This metal-mediated protein-cleavge reaction is used to evaluate the binding of metal ions, Na(+), Mg(2+), Ca(2+), Al(3+), Cr(3+), Mn(2+), Co(2+), Ni(2+), Zn(2+), Cd(2+), Hg(2+), Pb(2+), and Ce(3+) to albumins, and the relative affinities (selectivities) of the metal ions are rapidly evaluated by examining the extent of inhibition of protein cleavage. Four distinct systems Fe(II)/BSA, Cu(II)/BSA, Fe(II)/HSA and Cu(II)/HSA are examined using the above strategy. This metallomics approach is novel, even though the cleavage of serum albumins by Fe(II)/Cu(II) has been reported previously by this laboratory and many others. The protein cleavage products were analyzed by SDS PAGE, and the intensities of the product bands quantified to evaluate the extent of inhibition of the cleavage and thereby evaluate the relative binding affinities of specific metal ions to particular sites on albumins. The data show that Co(II) and Cr(III) showed the highest degree of inhibition, across the table, followed by Mn(II) and Ce(III). Alakali metal ions and alkaline earth metal ions showed very poor affinity for these metal sites on albumins. Thus, metal binding profiles for particular sites on proteins can be obtained quickly and accurately, using the metallomics approach.

  5. Simple bioconjugate chemistry serves great clinical advances: albumin as a versatile platform for diagnosis and precision therapy

    PubMed Central

    2017-01-01

    Albumin is the most abundant circulating protein in plasma and has recently emerged as a versatile protein carrier for drug targeting and for improving the pharmacokinetic profile of peptide or protein based drugs. Three drug delivery technologies related to albumin have been developed, which include the coupling of low-molecular weight drugs to exogenous or endogenous albumin, conjugating bioactive proteins by albumin fusion technology (AFT), and encapsulation of drugs into albumin nanoparticles. This review article starts with a brief introduction of human serum albumin (HSA), and then summarizes the mainstream chemical strategies of developing HSA binding molecules for coupling with drug molecules. Moreover, we also concisely condense the recent progress of the most important clinical applications of HSA-binding platforms, and specify the current challenges that need to be met for a bright future of HSA-binding. PMID:26771036

  6. 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.

  7. Application of capillary electrophoresis-frontal analysis for comparative evaluation of the binding interaction of captopril with human serum albumin in the absence and presence of hydrochlorothiazide.

    PubMed

    Liu, Ting-Ting; Xiang, Li-Li; Wang, Jian-Ling; Chen, Dong-Ying

    2015-11-10

    The application of capillary electrophoresis-frontal analysis for comparative evaluation of the binding interaction between antihypertensive drug captopril and human serum albumin in the absence and presence of diuretic drug hydrochlorothiazide was presented in this work. At near-physiological conditions (67mM phosphate buffer, pH 7.4, I=0.17, 37°C), the individual solution of 100μM captopril and the co-binding solution with 60μM hydrochlorothiazide added were pre-equilibrated with series concentrations of HSA (10-475μM) respectively, introducing hydrodynamically into an uncoated fused silica capillary (35cm×50μm I.D. with 26.5cm effective length). The values of number of binding sites, the binding constant for captopril and hydrochlorothiazide binding to HSA were obtained, respectively. It can be found that both drugs exhibit moderate binding properties towards HSA and there does not exist significant competitive binding effects between them.

  8. Interaction of amphiphilic drugs with human and bovine serum albumins

    NASA Astrophysics Data System (ADS)

    Khan, Abbul Bashar; Khan, Javed Masood; Ali, Mohd. Sajid; Khan, Rizwan Hasan; Kabir-ud-Din

    2012-11-01

    To know the interaction of amphiphilic drugs nortriptyline hydrochloride (NOT) and promazine hydrochloride (PMZ) with serum albumins (i.e., human serum albumin (HSA) and bovine serum albumin (BSA)), techniques of UV-visible, fluorescence, and circular dichroism (CD) spectroscopies are used. The binding affinity is more in case of PMZ with both the serum albumins. The quenching rate constant (kq) values suggest a static quenching process for all the drug-serum albumin interactions. The UV-visible results show that the change in protein conformation of PMZ-serum albumin interactions are more prominent as compared to NOT-serum albumin interactions. The CD results also explain the conformational changes in the serum albumins on binding with the drugs. The increment in %α-helical structure is slightly more for drug-BSA complexes as compared to drug-HSA complexes.

  9. Interaction of amphiphilic drugs with human and bovine serum albumins.

    PubMed

    Khan, Abbul Bashar; Khan, Javed Masood; Ali, Mohd Sajid; Khan, Rizwan Hasan; Kabir-Ud-Din

    2012-11-01

    To know the interaction of amphiphilic drugs nortriptyline hydrochloride (NOT) and promazine hydrochloride (PMZ) with serum albumins (i.e., human serum albumin (HSA) and bovine serum albumin (BSA)), techniques of UV-visible, fluorescence, and circular dichroism (CD) spectroscopies are used. The binding affinity is more in case of PMZ with both the serum albumins. The quenching rate constant (k(q)) values suggest a static quenching process for all the drug-serum albumin interactions. The UV-visible results show that the change in protein conformation of PMZ-serum albumin interactions are more prominent as compared to NOT-serum albumin interactions. The CD results also explain the conformational changes in the serum albumins on binding with the drugs. The increment in %α-helical structure is slightly more for drug-BSA complexes as compared to drug-HSA complexes.

  10. Structures of bovine, equine and leporine serum albumin.

    PubMed

    Bujacz, Anna

    2012-10-01

    Serum albumin first appeared in early vertebrates and is present in the plasma of all mammals. Its canonical structure supported by a conserved set of disulfide bridges is maintained in all mammalian serum albumins and any changes in sequence are highly correlated with evolution of the species. Previous structural investigations of mammalian serum albumins have only concentrated on human serum albumin (HSA), most likely as a consequence of crystallization and diffraction difficulties. Here, the crystal structures of serum albumins isolated from bovine, equine and leporine blood plasma are reported. The structure of bovine serum albumin (BSA) was determined at 2.47 Å resolution, two crystal structures of equine serum albumin (ESA) were determined at resolutions of 2.32 and 2.04 Å, and that of leporine serum albumin (LSA) was determined at 2.27 Å resolution. These structures were compared in detail with the structure of HSA. The ligand-binding pockets in BSA, ESA and LSA revealed different amino-acid compositions and conformations in comparison to HSA in some cases; however, much more significant differences were observed on the surface of the molecules. BSA, which is one of the most extensively utilized proteins in laboratory practice and is used as an HSA substitute in many experiments, exhibits only 75.8% identity compared with HSA. The higher resolution crystal structure of ESA highlights the binding properties of this protein because it includes several bound compounds from the crystallization solution that provide additional structural information about potential ligand-binding pockets.

  11. Bilirubin-albumin binding, bilirubin/albumin ratios, and free bilirubin levels: where do we stand?

    PubMed

    Hulzebos, Christian V; Dijk, Peter H

    2014-11-01

    Treatment for unconjugated hyperbilirubinemia is predominantly based on one parameter, i.e., total serum bilirubin (TSB) levels. Yet, overt kernicterus has been reported in preterm infants at relatively low TSB levels, and it has been repeatedly shown that free unconjugated bilirubin (freeUCB) levels, or bilirubin/albumin (B/A) ratios for that matter, are more closely associated with bilirubin neurotoxicity. In this article, we review bilirubin-albumin binding, UCBfree levels, and B/A ratios in addition to TSB levels to individualize and optimize treatment especially in preterm infants. Methods to measure bilirubin-albumin binding or UCBfree are neither routinely performed in Western clinical laboratories nor incorporated in current management guidelines on unconjugated hyperbilirubinemia. For bilirubin-albumin binding, this seems justified because several of these methods have been challenged, and sufficiently powered prospective trials on the clinical benefits are lacking. Technological advances in the measurement of UCBfree may provide a convenient means for integrating UCBfree measurements into routine clinical management of jaundiced infants. A point-of-care method, as well as determination of UCBfree levels in various newborn populations, is desirable to learn more about variations in time and how various clinical pathophysiological conditions affect UCBfree levels. This will improve the estimation of approximate UCBfree levels associated with neurotoxicity. To delineate the role of UCBfree in the management of jaundiced (preterm) infants, trials are needed using UCBfree as treatment parameter. The additional use of the B/A ratio in jaundiced preterms has been evaluated in the Bilirubin Albumin Ratio Trial (BARTrial; Clinical Trials: ISRCTN74465643) but failed to demonstrate better neurodevelopmental outcome in preterm infants <32 weeks assigned to the study group. Awaiting a study in which infants are assigned to be managed solely on the basis of their B

  12. Comparison of Posttranslational Modification and the Functional Impairment of Human Serum Albumin in Commercial Preparations.

    PubMed

    Miyamura, Shigeyuki; Imafuku, Tadashi; Anraku, Makoto; Taguchi, Kazuaki; Yamasaki, Keishi; Tominaga, Yuna; Maeda, Hitoshi; Ishima, Yu; Watanabe, Hiroshi; Otagiri, Masaki; Maruyama, Toru

    2016-03-01

    On account of its long circulating half-life, human serum albumin (HSA) is susceptible to posttranslational modifications that can alter its functions. Here, we comprehensively compared the degree of posttranslational modifications with the functional impairment of HSA derived from 5 different commercially available albumin preparations and clarified their relationships. We used electrospray ionization-time of flight mass spectrometry to evaluate the degree of posttranslational modification of the entire HSA molecule that was associated with disease development and found that the fraction of Cys34-cysteinylated HSA (Cys-Cys34-HSA), a major form of oxidative modification, varied substantially among the albumin preparations. Meanwhile, no remarkable difference was found in the degree of glycated or N-terminal truncated HSA among the preparations tested. The nonosmotic pressure maintenance functions of HSA, such as its antioxidative and ligand-binding activities significantly differed among the preparations. Interestingly, the alternations of these functions showed a significantly negative correlation only with the Cys-Cys34-HSA fraction. These findings suggest that the Cys-Cys34-HSA fraction, as estimated by electrospray ionization-time of flight mass spectrometry can be used as a predictive marker for the functional impairment of albumin preparations and that it would be preferable to use albumin preparations with higher contents of functionally effective albumin that correspond to a lower degree of cysteinylation of Cys34 in clinical practice.

  13. Fluorescence spectroscopy and molecular simulation on the interaction of caffeic acid with human serum albumin.

    PubMed

    Xiang, Yuhong; Duan, Lili; Ma, Qiang; Lv, Zizheng; Ruohua, Zhu; Zhang, Zhuoyong

    2016-12-01

    Fluorescence spectroscopy and molecular simulation were explored to study the interaction between caffeic acid and human serum albumin (HSA). The experimental results indicated that the fluorescence quenching mechanism between caffeic acid and HSA is a static quenching, which was proved again by the analysis of fluorescence lifetime by time-correlated single photon counting. The binding process is spontaneous and the hydrophobic force is the main force between caffeic acid and HSA. In addition, the binding of caffeic acid to HSA was modeled by molecular dynamics simulations. The root mean square deviations, root mean square fluctuations, radius of gyration and the number of hydrogen bonds of the molecular dynamic (MD) simulation process were analyzed. Both experimental and modeling results demonstrated strong binding between HSA and caffeic acid. HSA had a slight conformational change when it binds with caffeic acid. The obtained information is useful for HSA drug design. Copyright © 2016 John Wiley & Sons, Ltd.

  14. 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.

  15. 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.

  16. 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.

  17. Investigations on the interactions of diclofenac sodium with HSA and ctDNA using molecular modeling and multispectroscopic methods

    NASA Astrophysics Data System (ADS)

    Cui, Yanrui; Hao, Erjun; Hui, Guangquan; Guo, Wei; Cui, Fengling

    2013-06-01

    A tentative study on interaction of diclofenac sodium (DF-Na) with human serum albumin (HSA) and calf thymus DNA (ctDNA) was conducted by using multi-spectroscopic and molecular modeling techniques under simulative physiological conditions. The results of spectroscopic measurements suggested that the quenching mechanisms were static quenching. Three-dimensional fluorescence spectroscopy clearly demonstrated the occurrence of conformational changes of HSA with addition of DF-Na. In addition, competitive studies with ethidium bromide (EB) have shown that DF-Na can bind to ctDNA relatively strong via groove binding. Based on the values of thermodynamic parameters and the results of molecular modeling, it was confirmed that hydrophobic forces and hydrogen bond were the mainly binding forces in DF-Na-HSA and DF-Na-DNA systems. The binding distance between DF-Na and HSA was also determined using the theory of the Förster energy transference.

  18. Binding of furosemide to albumin isolated from human fetal and adult serum.

    PubMed

    Viani, A; Cappiello, M; Silvestri, D; Pacifici, G M

    1991-01-01

    Albumin was isolated from pooled fetal serum from 58 placentas obtained at normal delivery at term and from pooled adult plasma from 8 individuals. Albumin isolation was carried out by means of PEG precipitation followed by ion-exchange chromatography on DEAE-Sephadex A 50 and then on SP-Sephadex C 50. The electrophoresis on SDS-polyacrylamide gels showed only one spot that comigrated with commercial human albumin. Binding to albumin was measured by equilibrium dialysis of an aliquot of albumin solution (0.7 ml) against the same volume of 0.13 M sodium orthophosphate buffer (pH 7.4). At a total concentration of 2 micrograms/ml (therapeutic range), the unbound fraction of furosemide was 2.71% (fetal albumin) and 2.51% (adult albumin). Two classes of binding sites for furosemide were observed in fetal and adult albumin. The number of binding sites (moles of furosemide per mole of albumin) was 1.22 (fetal albumin) and 1.58 (adult albumin) for the high-affinity site and 2.97 (fetal albumin) and 3.25 (adult albumin) for the low-affinity site. The association constants (M-1) were 3.1 X 10(4) (fetal albumin) and 2.6 X 10(4) (adult albumin) for the high-affinity set of sites and 0.83 X 10(4) (fetal albumin) and 1.0 X 10(4) (adult albumin) low-affinity site. The displacement of furosemide from albumin was studied with therapeutic concentrations of several drugs. Valproic acid, salicylic acid, azapropazone and tolbutamide had the highest displacing effects which were significantly higher with fetal than with adult albumin.

  19. A comparative study of capillary electrophoresis and isothermal titration calorimetry for the determination of binding constant of human serum albumin to monoclonal antibody.

    PubMed

    Andrási, Melinda; Lehoczki, Gábor; Nagy, Zoltán; Gyémánt, Gyöngyi; Pungor, András; Gáspár, Attila

    2015-06-01

    This paper focuses on the investigation of the interactions between the anti-HSA-mAb and its protein antigen using CZE, ACE, and isothermal titration calorimetry. The CZE revealed the formation of the anti-HSA-mAb·HSA and anti-HSA-mAb·(HSA)2 complexes and the binding constants determined by plotting the amount of the bound anti-HSA-mAb as a function of the concentration of HSA. The ACE provided information on the binding strength from the change in effective electrophoretic mobility of the anti-HSA-mAb. These two separation techniques estimated the presence of two binding sites. The equilibrium dissociation constant values obtained by CZE and ACE were found to be 2.26 × 10(-6) M for anti-HSA-mAb·HSA, 1.22 × 10(-6) M for anti-HSA-mAb·(HSA)2 and 4.45 × 10(-8) M for anti-HSA-mAb·HSA, 1.08 × 10(-7) M for anti-HSA-mAb·(HSA)2 , respectively. The dissociation constant data obtained by ACE were in congruence with the values obtained by isothermal titration calorimetry (2.74 × 10(-8) M, 1.04 × 10(-7) M).

  20. 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.

  1. Potentiating the cellular targeting and anti-tumor activity of Dp44mT via binding to human serum albumin: two saturable mechanisms of Dp44mT uptake by cells.

    PubMed

    Merlot, Angelica M; Sahni, Sumit; Lane, Darius J R; Fordham, Ashleigh M; Pantarat, Namfon; Hibbs, David E; Richardson, Vera; Doddareddy, Munikumar R; Ong, Jennifer A; Huang, Michael L H; Richardson, Des R; Kalinowski, Danuta S

    2015-04-30

    Di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) demonstrates potent anti-cancer activity. We previously demonstrated that 14C-Dp44mT enters and targets cells through a carrier/receptor-mediated uptake process. Despite structural similarity, 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT) and pyridoxal isonicotinoyl hydrazone (PIH) enter cells via passive diffusion. Considering albumin alters the uptake of many drugs, we examined the effect of human serum albumin (HSA) on the cellular uptake of Dp44mT, Bp4eT and PIH. Chelator-HSA binding studies demonstrated the following order of relative affinity: Bp4eT≈PIH>Dp44mT. Interestingly, HSA decreased Bp4eT and PIH uptake, potentially due to its high affinity for the ligands. In contrast, HSA markedly stimulated Dp44mT uptake by cells, with two saturable uptake mechanisms identified. The first mechanism saturated at 5-10 µM (B(max):1.20±0.04 × 10⁷ molecules/cell; K(d):33±3 µM) and was consistent with a previously identified Dp44mT receptor/carrier. The second mechanism was of lower affinity, but higher capacity (B(max):2.90±0.12 × 10⁷ molecules/cell; K(d):65±6 µM), becoming saturated at 100 µM and was only evident in the presence of HSA. This second saturable Dp44mT uptake process was inhibited by excess HSA and had characteristics suggesting it was mediated by a specific binding site. Significantly, the HSA-mediated increase in the targeting of Dp44mT to cancer cells potentiated apoptosis and could be important for enhancing efficacy.

  2. Chemically synthesized glycosides of hydroxylated flavylium ions as suitable models of anthocyanins: binding to iron ions and human serum albumin, antioxidant activity in model gastric conditions.

    PubMed

    Al Bittar, Sheiraz; Mora, Nathalie; Loonis, Michèle; Dangles, Olivier

    2014-12-11

    Polyhydroxylated flavylium ions, such as 3',4',7-trihydroxyflavylium chloride (P1) and its more water-soluble 7-O-β-d-glucopyranoside (P2), are readily accessible by chemical synthesis and suitable models of natural anthocyanins in terms of color and species distribution in aqueous solution. Owing to their catechol B-ring, they rapidly bind FeIII, weakly interact with FeII and promote its autoxidation to FeIII. Both pigments inhibit heme-induced lipid peroxidation in mildly acidic conditions (a model of postprandial oxidative stress in the stomach), the colorless (chalcone) forms being more potent than the colored forms. Finally, P1 and P2 are moderate ligands of human serum albumin (HSA), their likely carrier in the blood circulation, with chalcones having a higher affinity for HSA than the corresponding colored forms.

  3. Albumin binding as a potential biomarker of exposure to moderately low levels of organophosphorus pesticides.

    PubMed

    Tarhoni, Mabruka H; Lister, Timothy; Ray, David E; Carter, Wayne G

    2008-06-01

    We have evaluated the potential of plasma albumin to provide a sensitive biomarker of exposure to commonly used organophosphorus pesticides in order to complement the widely used measure of acetylcholinesterase (AChE) inhibition. Rat or human plasma albumin binding by tritiated-diisopropylfluorophosphate ((3)H-DFP) was quantified by retention of albumin on glass microfibre filters. Preincubation with unlabelled pesticide in vitro or dosing of F344 rats with pesticide in vivo resulted in a reduction in subsequent albumin radiolabelling with (3)H-DFP, the decrease in which was used to quantify pesticide binding. At pesticide exposures producing approximately 30% inhibition of AChE, rat plasma albumin binding in vitro by azamethiphos (oxon), chlorfenvinphos (oxon), chlorpyrifos-oxon, diazinon-oxon and malaoxon was reduced from controls by 9+/-1%, 67+/-2%, 56+/-2%, 54+/-2% and 8+/-1%, respectively. After 1 h of incubation with 19 microM (3)H-DFP alone, the level of binding to rat or human plasma albumins reached 0.011 or 0.039 moles of DFP per mole of albumin, respectively. This level of binding could be further increased by raising the concentration of (3)H-DFP, increasing the (3)H-DFP incubation time, or by substitution of commercial albumins for native albumin. Pesticide binding to albumin was presumed covalent since it survived 24 h dialysis. After dosing rats with pirimiphos-methyl (dimethoxy) or chlorfenvinphos (oxon) (diethoxy) pesticides, the resultant albumin binding were still significant 7 days after dosing. As in vitro, dosing of rats with malathion did not result in significant albumin binding in vivo. Our results suggest albumin may be a useful additional biomonitor for moderately low-level exposures to several widely used pesticides, and that this binding differs markedly between pesticides.

  4. Water participation in molecular recognition and protein-ligand association: Probing the drug binding site "Sudlow I" in human serum albumin

    NASA Astrophysics Data System (ADS)

    Al-Lawatia, Najla; Steinbrecher, Thomas; Abou-Zied, Osama K.

    2012-03-01

    Human serum albumin (HSA) plays an important role in the transport and disposition of endogenous and exogenous ligands present in blood. Its capacity to reversibly bind a large variety of drugs results in its prevailing role in drug pharmacokinetics and pharmacodynamics. In this work, we used 7-hydroxyquinoline (7HQ) as a probe to study the binding nature of one of the major drug binding sites of HSA (Sudlow I) and to reveal the local environment around the probe in the binding site. The interaction between 7HQ and HSA at a physiological pH of 7.2 was investigated using steady-state and lifetime spectroscopic measurements, molecular docking and molecular dynamics (MD) simulations methods. The fluorescence results indicate a selective interaction between 7HQ and the Trp214 residue. The reduction in both the intensity and lifetime of the Trp214 fluorescence upon probe binding indicates the dominant role of static quenching. Molecular docking and MD simulations show that 7HQ binds in Sudlow site I close to Trp214, confirming the experimental results, and pinpoint the dominant role of hydrophobic interaction in the binding site. Electrostatic interactions were also found to be important in which two water molecules form strong hydrogen bonds with the polar groups of 7HQ. Detection of water in the binding site agrees with the absorption and fluorescence results that show the formation of a zwitterion tautomer of 7HQ. The unique spectral signatures of 7HQ in water make this molecule a potential probe for detecting the presence of water in nanocavities of proteins. Interaction of 7HQ with water in the binding site shows that water molecules can be crucial for molecular recognition and association in protein binding sites.

  5. 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

  6. Determination of free bilirubin and its binding capacity by HSA using a microfluidic chip-capillary electrophoresis device with a multi-segment circular-ferrofluid-driven micromixing injection.

    PubMed

    Sun, Hui; Nie, Zhou; Fung, Ying Sing

    2010-09-01

    A PMMA microfluidic chip-CE device with a multi-segment circular-ferrofluid-driven micromixing injector has been developed for the determination of free bilirubin and its binding capacity by HSA at equilibrium. The design of the device and its fabrication by a low cost CO(2) laser are discussed for intended applications. Under optimized conditions, the total binding capacity of HSA for bilirubin was determined as 16.3±1.4 mg/l00 mL human serum (n=3) and residual binding capacity for bilirubin 9.8 mg/100 mL (n=3) in normal infants. To assess risk of hyperbilirubinemia, free bilirubin and residual binding capacity by HSA provide a better indicator than total bilirubin, as neonates with impaired bilirubin binding capacity could be detected. In addition, residual binding capacity provides an advanced indicator to predict the onset of hyperbilirubinemia before the appearance of free bilirubin. HSA down to 94 nL is used in each titration and a full assay of four titrations takes up 376 nL HSA, sufficient for newborns with HSA in microliter range. The device has shown capable to provide adequate margin of protection to detect an early rising level of bilirubin and impaired binding capacity prior to the onset of jaundice condition.

  7. Biogenic and synthetic polyamines bind bovine serum albumin.

    PubMed

    Dubeau, S; Bourassa, P; Thomas, T J; Tajmir-Riahi, H A

    2010-06-14

    Biogenic polyamines are found to modulate protein synthesis at different levels, while polyamine analogues have shown major antitumor activity in multiple experimental models, including breast cancer. The aim of this study was to examine the interaction of bovine serum albumin (BSA) with biogenic polyamines, spermine and spermidine, and polyamine analogues 3,7,11,15-tetrazaheptadecane x 4 HCl (BE-333) and 3,7,11,15,19-pentazahenicosane x 5 HCl (BE-3333) in aqueous solution at physiological conditions. FTIR, UV-visible, CD, and fluorescence spectroscopic methods were used to determine the polyamine binding mode and the effects of polyamine complexation on protein stability and secondary structure. Structural analysis showed that polyamines bind BSA via both hydrophilic and hydrophobic interactions. Stronger polyamine-protein complexes formed with biogenic than synthetic polyamines with overall binding constants of K(spm) = 3.56 (+/-0.5) x 10(5) M(-1), K(spmd) = 1.77 (+/-0.4) x 10(5) M(-1), K(BE-333) = 1.11 (+/-0.3) x 10(4) M(-1) and K(BE-3333) = 3.90 (+/-0.7) x 10(4) M(-1) that correlate with their positively charged amino group contents. Major alterations of protein conformation were observed with reduction of alpha-helix from 63% (free protein) to 55-33% and increase of turn 12% (free protein) to 28-16% and random coil from 6% (free protein) to 24-17% in the polyamine-BSA complexes, indicating a partial protein unfolding. These data suggest that serum albumins might act as polyamine carrier proteins in delivering polyamine analogues to target tissues.

  8. M-M-R(®)II manufactured using recombinant human albumin (rHA) and M-M-R(®)II manufactured using human serum albumin (HSA) exhibit similar safety and immunogenicity profiles when administered as a 2-dose regimen to healthy children.

    PubMed

    Wiedmann, Richard T; Reisinger, Keith S; Hartzel, Jonathan; Malacaman, Edgardo; Senders, Shelly D; Giacoletti, Katherine E D; Shaw, Eric; Kuter, Barbara J; Schödel, Florian; Musey, Luwy K

    2015-04-27

    Prior to 2006, M-M-R(®)II (measles, mumps, and rubella virus vaccine live) was manufactured using human serum albumin (HSA) and each dose of the vaccine contained a relatively small amount (≤0.3mg) of HSA. Because of specific regulatory requirements and limited suppliers of HSA acceptable for human use, there was a need to replace HSA with recombinant human albumin (rHA) to mitigate any potential risk to the availability of M-M-R(®)II. Two different formulations of M-M-R(®)II manufactured using either rHA or HSA were clinically evaluated for safety and immunogenicity when administered as a 2-dose regimen to healthy children 12-18 months and 3-4 years of age. Adverse events, including those indicative of a possible hypersensitivity reaction, were collected for 42 days after each dose. Antibodies to measles, mumps, and rubella were measured before and approximately 6 weeks after dose 1. Antibodies to rHA were measured before and approximately 6 weeks after dose 1 and dose 2. Antibody seroconversion rates to measles, mumps, and rubella were 97.0%, 99.5%, and 99.7%, respectively, for recipients of M-M-R(®)II with rHA and 97.2%, 97.9%, and 99.6%, respectively, for recipients of M-M-R(®)II with HSA, and geometric mean titers to all 3 vaccine viral antigens were comparable between the 2 vaccination groups. The proportions of subjects who reported adverse events, including those suggestive of hypersensitivity reactions, after each dose of study vaccine were comparable between the 2 vaccination groups. No subject had detectable antibodies to rHA immediately prior to or following receipt of either the first or second dose of study vaccine. Given the comparable immunogenicity and safety profiles of both formulations, rHA is an acceptable replacement for HSA in the manufacture of M-M-R(®)II.

  9. 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.

  10. 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.

  11. A quantitative binding study of fibrinogen and human serum albumin to metal oxide nanoparticles by surface plasmon resonance.

    PubMed

    Canoa, Pilar; Simón-Vázquez, Rosana; Popplewell, Jonathan; González-Fernández, África

    2015-12-15

    The interaction of plasma proteins with metal oxide nanoparticles (NPs) is important due to the potential biomedical application of these NPs. In this study, new approaches were applied to measure quantitatively the kinetics and affinities of fibrinogen and human serum albumin (HSA) for TiO2, CeO2, Al2O3 and ZnO NPs immobilized on a sensor chip. Real-time surface plasmon resonance (SPR) measurements showed that fibrinogen interacted with TiO2 and CeO2 NPs with high affinity (135 and 40 pM, respectively) and to Al2O3 NPs with moderate affinity (15 nM). The data fitted well to the Langmuir model describing a 1:1 interaction. In contrast, HSA interacted with TiO2, CeO2 and Al2O3 NPs with lower affinity (80 nM, 37 nM and 2 µM, respectively) with the data fitting better to the conformational change model. TiO2 and CeO2 NPs had fast association rate constants with fibrinogen (1×10(6) M(-1) s(-1)) and Al2O3 NPs had a slower association rate constant (1×10(4) M(-1) s(-1)). By contrast, HSA had markedly slower association rate constants (1×10(3)-1×10(4) M(-1) s(-1)). The binding of the proteins was reversible, thus allowing the rapid capture of data for replicates. The occurrence of matrix effects was evaluated by using surfaces with different chemistries to capture the NPs, namely alginate, NeutrAvidin and bare gold. The affinity values determined for the NP-protein interactions were largely independent of the underlying surface used to capture the NPs.

  12. Binding of diazepam, salicylic acid and digitoxin to albumin isolated from fetal and adult serum.

    PubMed

    Viani, A; Cappiello, M; Pacifici, G M

    1991-01-01

    Albumin was isolated from pooled fetal serum obtained at normal delivery at term and from pooled adult plasma. Albumin isolation was carried out by means of PEG precipitation followed by ion exchange chromatography on DEAE-Sephadex A 50 and then on SP-Sephadex C 50. The binding of diazepam (1 microM), salicylic acid (2 mM) and digitoxin (6 nM) to albumin (40 g/l) was measured by equilibrium dialysis at 37 degrees C. The unbound fraction (mean +/- SD) for fetal and adult albumin of diazepam was 1.86 +/- 0.24 and 1.82 +/- 0.15% (NS), that of digitoxin was 3.18 +/- 0.27 and 3.36 +/- 0.04% (NS) and that of salicylic acid was 11.65 +/- 0.99 and 9.47 +/- 0.75% (p less than 0.05), respectively. With both fetal and adult albumin, a single class of binding sites was observed for diazepam and digitoxin, whereas two classes of binding sites were observed for salicylic acid. The number of binding sites (n, moles of drug per mole of albumin) for fetal and adult albumin was 0.83 and 1.02 for diazepam and 0.014 and 0.018 for digitoxin, respectively. For salicylic acid, n was 1.45 (fetal albumin) and 1.55 (adult albumin) for the higher affinity site, and 3.06 (fetal albumin) and 3.27 (adult albumin) for the lower affinity site. The association constant (Ka, M-1) for diazepam was 1.36 x 10(5) (fetal albumin) and 1.00 x 10(5) (adult albumin) and that for digitoxin was 4.12 x 10(6) (fetal albumin) and 2.7 x 10(6) (adult albumin). For salicylic acid, Ka was 38.4 x 10(3) (fetal albumin) and 35.8 x 10(3) (adult albumin) for the higher affinity site, and 2.7 x 10(3) (fetal albumin) and 4.3 x 10(3) (adult albumin) for the lower affinity site. This work shows that fetal and adult albumin have similar binding properties and corroborates our previous findings with furosemide.

  13. Peculiar reactivity of a di-imine copper(II) complex regarding its binding to albumin protein.

    PubMed

    Silveira, Vivian C; Abbott, Mariana P; Cavicchioli, Maurício; Gonçalves, Marcos B; Petrilli, Helena M; de Rezende, Leandro; Amaral, Antonia T; Fonseca, David E P; Caramori, Giovanni F; Ferreira, Ana M da Costa

    2013-05-14

    A set of four di-imine copper(II) complexes containing pyridine, pyrazine and/or imidazole moieties, [Cu(apyhist)H2O](2+) 1 (apyhist = 2-(1H-imidazol-4-yl)-N-(1-(pyridin-2-yl)ethylidene)ethanamine), [Cu(apzhist)OH](+) 2 (apzhist = 2-(1H-imidazol-4-yl)-N-(1-(pyrazin-2-yl)ethylidene)ethanamine), [Cu(apyepy)OH](+) 3 (apyepy = 2-(pyridin-2-yl)-N-(1-(pyridin-2-yl)ethylidene)ethanamine), and [Cu(apzepy)H2O](2+) 4 (apzepy = N-(1-(pyrazin-2-yl)ethylidene)-2-(pyridin-2-yl)ethanamine), were investigated regarding their capability of interacting with serum albumin (human, HSA and bovine, BSA), by using spectroscopic techniques, CD, UV/Vis and EPR. Like other similar di-imine copper(II) complexes, most of them showed an expected preferential insertion of the metal ion at the primary N-terminal site of the protein, very selective for copper and characterized by a CD band at 560 nm. Further insertion of the copper ion at a secondary site is expected when using an excess of the metal. However, one of these studied complexes, [Cu(apyhist)H2O](2+) 1, exhibited anomalous behaviour interacting only at this secondary metal binding site of albumin, characterized by a CD band at 370 nm, and attributed to the coordination of copper at the Cys34 pocket. Analogous experiments with HSA previously treated with N-ethyl-maleimide (NEM), that oxidizes the protein Cys34 residue and obstructs the metal coordination, verified these results. Additional data obtained by EPR spectroscopy complemented those results. DFT calculations, considering some structural and electronic characteristics of such series of di-imine ligands and of the corresponding copper complexes, suggested molecular recognition of the apyhist ligand at the protein cavity as a feasible explanation for this unexpected and peculiar behaviour of complex 1.

  14. Review: Glycation of human serum albumin

    PubMed Central

    Anguizola, Jeanethe; Matsuda, Ryan; Barnaby, Omar S.; Joseph, K.S.; Wa, Chunling; DeBolt, Erin; Koke, Michelle; Hage, David S.

    2013-01-01

    Glycation involves the non-enzymatic addition of reducing sugars and/or their reactive degradation products to amine groups on proteins. This process is promoted by the presence of elevated blood glucose concentrations in diabetes and occurs with various proteins that include human serum albumin (HSA). This review examines work that has been conducted in the study and analysis of glycated HSA. The general structure and properties of HSA are discussed, along with the reactions that can lead to modification of this protein during glycation. The use of glycated HSA as a short-to-intermediate term marker for glycemic control in diabetes is examined, and approaches that have been utilized for measuring glycated HSA are summarized. Structural studies of glycated HSA are reviewed, as acquired for both in vivo and in vitro glycated HSA, along with data that have been obtained on the rate and thermodynamics of HSA glycation. In addition, this review considers various studies that have investigated the effects of glycation on the binding of HSA with drugs, fatty acids and other solutes and the potential clinical significance of these effects. PMID:23891854

  15. [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

  16. New tungstenocenes containing 3-hydroxy-4-pyrone ligands: antiproliferative activity on HT-29 and MCF-7 cell lines and binding to human serum albumin studied by fluorescence spectroscopy and molecular modeling methods

    PubMed Central

    Domínguez-García, Moralba; Ortega-Zúñiga, Carlos; Meléndez, Enrique

    2012-01-01

    Three new water-soluble tungstenocene derivatives were synthesized and characterized using 3-hydroxy-4-pyrone ligands, which provide aqueous stability to the complexes. The antiproliferative activities of the complexes on HT-29 colon cancer and MCF-7 breast cancer cell lines were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and showed the new tungstenocene derivatives have higher antiproliferative action than tungstenocene dichloride (Cp2WCl2, where Cp is cyclopentadienyl). The binding interactions of the tungstenocenes with human serum albumin (HSA) were investigated using fluorescence spectroscopy and molecular modeling methods. Analysis of the fluorescence quenching spectra indicates that the tungstenocene complexes bind HSA by hydrophobic interactions and hydrogen bonding at fatty acid binding site 6 and drug binding site II. Docking studies provided a description of the hydrophobic interactions and hydrogen bonding by which the tungstenocenes become engaged with HSA. It was determined that the binding affinity of the tungstenoecenes for HSA is in the order Cp2WCl2 < [Cp2W(ethyl maltolato)]Cl < [Cp2W (maltolato)]Cl < [Cp2W(kojato)]Cl, consistent with the hydrophobic interactions and the number of hydrogen bonds involved. PMID:23212785

  17. Synthesis, characterization, crystal structure, DNA- and HSA-binding studies of a dinuclear Schiff base Zn(II) complex derived from 2-hydroxynaphtaldehyde and 2-picolylamine

    NASA Astrophysics Data System (ADS)

    Kazemi, Zahra; Rudbari, Hadi Amiri; Mirkhani, Valiollah; Sahihi, Mehdi; Moghadam, Majid; Tangestaninejad, Sharam; Mohammadpoor-Baltork, Iraj

    2015-09-01

    A tridentate Schiff base ligand NNO donor (HL: 1-((E)-((pyridin-2-yl)methylimino)methyl)naphthalen-2-ol was synthesized from condensation of 2-hydroxynaphtaldehyde and 2-picolylamine. Zinc complex, Zn2L2(NO3)2, was prepared from reaction of Zn(NO3)2 and HL at ambient temperature. The ligand and complex were characterized by FT-IR, 1H NMR, 13C NMR and elemental analysis (CHN). Furthermore, the structure of dinuclear Zn(II) complex was determined by single crystal X-ray analysis. The complex, Zn2L2(NO3)2, is centrosymmetric dimer in which deprotonated phenolates bridge the two Zn(II) atoms and link the two halves of the dimer. In the structure, Zinc(II) ions have a highly distorted six-coordinate structure bonded to two oxygen atoms from a bidentate nitrate group, the pyridine nitrogen, an amine nitrogen and phenolate oxygens. The interaction of dinuclear Zn(II) complex with fish sperm DNA (FS-DNA) and HSA was investigated under physiological conditions using fluorescence quenching, UV-Vis spectroscopy, molecular dynamics simulation and molecular docking methods. The estimated binding constants for the DNA-complex and HSA-complex were (3.60 ± 0.18) × 104 M-1 and (1.35 ± 0.24) × 104 M-1, respectively. The distance between dinuclear Zn(II) complex and HSA was obtained based on the Förster's theory of non-radiative energy transfer. Molecular docking studies revealed the binding of dinuclear Zn(II) complex to the major groove of FS-DNA and IIA site of protein by formation of hydrogen bond, π-cation and hydrophobic interactions.

  18. Relations between high-affinity binding sites of markers for binding regions on human serum albumin.

    PubMed Central

    Kragh-Hansen, U

    1985-01-01

    Binding of warfarin, digitoxin, diazepam, salicylate and Phenol Red, individually or in different pair combinations, to defatted human serum albumin at ligand/protein molar ratios less than 1:1 was studied at pH 7.0. The binding was determined by ultrafiltration. Some of the experiments were repeated with the use of equilibrium dialysis in order to strengthen the results. Irrespective of the method used, all ligands bind to one high-affinity binding site with an association constant in the range 10(4)-10(6) M-1. High-affinity binding of the following pair of ligands took place independently: warfarin-Phenol Red, warfarin-diazepam, warfarin-digitoxin and digitoxin-diazepam. Simultaneous binding of warfarin and salicylate led to a mutual decrease in binding of one another, as did simultaneous binding of digitoxin and Phenol Red. Both effects could be accounted for by a coupling constant. The coupling constant is the factor by which the primary association constants are affected; in these examples of anti-co-operativity the factor has a value between 0 and 1. In the first example it was calculated to be 0.8 and in the latter 0.5. Finally, digitoxin and salicylate were found to compete for a common high-affinity binding site. The present findings support the proposal of four separate primary binding sites for warfarin, digitoxin (and salicylate), diazepam and Phenol Red. An attempt to correlate this partial binding model for serum albumin with other models in the literature is made. PMID:3977850

  19. A comparative analysis on the binding characteristics of various mammalian albumins towards a multitherapeutic agent, pinostrobin.

    PubMed

    Feroz, Shevin R; Sumi, Rumana A; Malek, Sri N A; Tayyab, Saad

    2015-01-01

    The interaction of pinostrobin (PS), a multitherapeutic agent with serum albumins of various mammalian species namely, goat, bovine, human, porcine, rabbit, sheep and dog was investigated using fluorescence quench titration and competitive drug displacement experiments. Analysis of the intrinsic fluorescence quenching data revealed values of the association constant, K(a) in the range of 1.49 - 6.12 × 10(4) M(-1), with 1:1 binding stoichiometry. Based on the PS-albumin binding characteristics, these albumins were grouped into two classes. Ligand displacement studies using warfarin as the site I marker ligand correlated well with the binding data. Albumins from goat and bovine were found to be closely similar to human albumin on the basis of PS binding characteristics.

  20. Interaction of mycotoxin zearalenone with human serum albumin.

    PubMed

    Poór, Miklós; Kunsági-Máté, Sándor; Bálint, Mónika; Hetényi, Csaba; Gerner, Zsófia; Lemli, Beáta

    2017-03-27

    Zearalenone (ZEN) is a mycotoxin produced mainly by Fusarium species. Fungal contamination of cereals and plants can result in the formation of ZEN, leading to its presence in different foods, animal feeds, and drinks. Because ZEN is an endocrine disruptor, it causes reproductive disorders in farm animals and hyperoestrogenic syndromes in humans. Despite toxicokinetic properties of ZEN were studied in more species, we have no information regarding the interaction of ZEN with serum albumin. Since albumin commonly plays an important role in the toxicokinetics of different toxins, interaction of ZEN with albumin has of high biological importance. Therefore the interaction of ZEN with human serum albumin (HSA) was investigated using spectroscopic methods, ultrafiltration, and molecular modeling studies. Fluorescence spectroscopic studies demonstrate that ZEN forms complex with HSA. Binding constant (K) of ZEN-HSA complex was quantified with fluorescence quenching technique. The determined binding constant (logK=5.1) reflects the strong interaction of ZEN with albumin suggesting the potential biological importance of ZEN-HSA complex formation. Based on the results of the investigations with site markers as well as docking studies, ZEN occupies a non-conventional binding site on HSA. Considering the above listed observations, we should keep in mind this interaction if we would like to precisely understand the toxicokinetic behavior of ZEN.

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

    PubMed

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

    2016-01-15

    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 (1)H 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.

  2. 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.

  3. 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

  4. 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

  5. Fluorescence Spectroscopic Studies on the Complexation of Antidiabetic Drugs with Glycosylated Serum Albumin

    NASA Astrophysics Data System (ADS)

    Seedher, N.; Kanojia, M.

    2013-11-01

    Glycosylation decreases the association constant values and hence the binding affinity of human serum albumin (HSA) for the antidiabetic drugs under study. The percentage of HAS-bound drug at physiological temperature was only about 21-38 % as compared to 46-74 % for non-glycosylated HSA. Thus the percentage of free drug available for an antihyperglycemic effect was about double (62-79 %) compared to the values for non-glycosylated HSA. Much higher free drug concentrations available for pharmacological effect can lead to the risk of hypoglycemia. Hydrophobic interactions were predominantly involved in the binding. In the binding of gliclazide, hydrogen bonding and electrostatic interactions were involved. Site specificity for glycosylated HSA was the same as that for non-glycosylated HSA; gliclazide and repaglinide bind only at site II whereas glimepiride and glipizide bind at both sites I and II. Glycosylation, however, caused conformational changes in albumin, and the binding region within site II was different for glycosylated and non-glycosylated albumin. Stern-Volmer analysis also indicated the conformational changes in albumin as a result of glycosylation and showed that the dynamic quenching mechanism was valid for fluorescence of both glycosylated and non-glycosylated HSA.

  6. Interaction of cromolyn sodium with human serum albumin: a fluorescence quenching study.

    PubMed

    Hu, Yan-Jun; Liu, Yi; Pi, Zhen-Bang; Qu, Song-Sheng

    2005-12-15

    The interaction between cromolyn sodium (CS) and human serum albumin (HSA) was investigated using tryptophan fluorescence quenching. In the discussion of the mechanism, it was proved that the fluorescence quenching of HSA by CS is a result of the formation of a CS-HSA complex. Quenching constants were determined using the Sterns-Volmer equation to provide a measure of the binding affinity between CS and HSA. The thermodynamic parameters DeltaG, DeltaH, and DeltaS at different temperatures were calculated. The distance r between donor (Trp214) and acceptor (CS) was obtained according to fluorescence resonance energy transfer (FRET). Furthermore, synchronous fluorescence spectroscopy data and UV-vis absorbance spectra have suggested that the association between CS and HSA changed the molecular conformation of HSA and the electrostatic interactions play a major role in CS-HSA association.

  7. 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.

  8. The location of the high- and low-affinity bilirubin-binding sites on serum albumin: ligand-competition analysis investigated by circular dichroism.

    PubMed

    Goncharova, Iryna; Orlov, Sergey; Urbanová, Marie

    2013-01-01

    The locations of three bilirubin (BR)-binding sites with different affinities were identified as subdomains IB, IIA and IIIA for five mammalian serum albumins (SAs): human (HSA), bovine (BSA), rat, (RSA), rabbit (RbSA) and sheep (SSA). The stereoselectivity of a high-affinity BR-binding site was identified in the BR/SA=1/1 system by circular dichroism (CD) spectroscopy, the sites with low affinity to BR were analyzed using difference CD. Site-specific ligand-competition experiments with ibuprofen (marker for subdomain IIIA) and hemin (marker for subdomain IB) did not reveal any changes for the BR/SA=1/1 system and showed a decrease of the bound BR at BR/SA=3/1. Both sites were identified as sites with low affinity to BR. The correlation between stereoselectivity and the arrangement of Arg-Lys residues indicated similarity between the BR-binding sites in subdomain IIIA for all of the SAs studied. Subdomain IB in HSA, BSA, SSA and RbSA has P-stereoselectivity while in RSA it has M-selectivity toward BR. A ligand-competition experiment with gossypol shows a decrease of the CD signal of bound BR for the BR/SA=1/1 system as well as for BR/SA=3/1. Subdomain IIA was assigned as a high-affinity BR-binding site. The P-stereoselectivity of this site in HSA (and RSA, RbSA) was caused by the right-hand localization of charged residues R257/R218-R222, whereas the left-hand orientation of R257/R218-R199 led to the M-stereoselectivity of the primary binding site in BSA (and SSA).

  9. Conjugation of a dipicolyl chelate to polypeptide conjugates increases binding affinities for human serum albumin and survival times in human serum.

    PubMed

    Balliu, Aleksandra; Baltzer, Lars

    2017-03-16

    The affinity for human serum albumin (HSA) of a series of 2-5 kDa peptides covalently linked to 3,5-bis[[bis(2-pyridylmethyl)amino]methyl]benzoic acid, a dipicolyl chelator with μM affinity for Zn2+, was found by surface plasmon resonance to increase in the presence of 1μM ZnCl2 at physiological pH. The dependence on polypeptide hydrophobicity was found to be minor, suggesting that the conjugates bound to the metal binding site and not to the fatty acid binding site. The affinity of the conjugates increased strongly with the positive charge of the polypeptides suggesting proximity to the negatively charged protein surface surrounding the metal binding site. The survival times of the peptides in human serum were extended as a consequence of stronger binding to HSA suggesting that Zn2+ ion chelating agents may provide a general route to increased survival times of peptides in serum in therapeutic and diagnostic applications without significantly increasing their molecular weights.

  10. Fatty acid binding sites of human and bovine albumins: Differences observed by spin probe ESR

    NASA Astrophysics Data System (ADS)

    Muravsky, Vladimir; Gurachevskaya, Tatjana; Berezenko, Stephen; Schnurr, Kerstin; Gurachevsky, Andrey

    2009-09-01

    Bovine and human serum albumins and recombinant human albumin, all non-covalently complexed with 5- and 16-doxyl stearic acids, were investigated by ESR spectroscopy in solution over a range of pH values (5.5-8.0) and temperatures (25-50 °C), with respect to the allocation and mobility of fatty acid (FA) molecules bound to the proteins and conformation of the binding sites. In all proteins bound FA undergo a permanent intra-albumin migration between the binding sites and inter-domain residence. Nature identity of the recombinant human albumin to its serum-derived analog was observed. However, the binding sites of bovine albumin appeared shorter in length and wider in diameter than those of human albumin. Presumably, less tightly folded domains in bovine albumin allow better penetration of water molecules in the interior of the globule that resulted in higher activation energy of FA dissociation from the binding site. Thus, the sensitive technique based on ESR non-covalent spin labeling allowed quantitative analysis and reliable comparison of the fine features of binding proteins.

  11. Study on the interaction of sulforaphane with human and bovine serum albumins.

    PubMed

    Abassi, Parvane; Abassi, Farzane; Yari, Faramarz; Hashemi, Mehrdad; Nafisi, Shohreh

    2013-05-05

    Sulforaphane; [1-isothiocyanato-4-(methylsulfinyl) butane], (SFN) is an isothiocyanate derived from glucoraphanin present in cruciferous vegetables and has a variety of potential chemopreventive actions. This study was designed to examine the interaction of sulforaphane with HSA and BSA. FTIR, UV-Vis spectroscopic methods as well as molecular modeling were used to determine the drug binding mode, binding constant and the effect of drug complexation on serum albumins stability and conformation. Structural analysis showed that SFN bind HSA and BSA via polypeptide polar groups with overall binding constants of KSFN-HSA=6.54×10(4) and KSFN-BSA=8.55×10(4) M(-1). HSA and BSA conformations were altered by a major reduction of α-helix upon SFN interaction. These results suggest that serum albumins might act as carrier proteins for SFN in delivering them to target tissues.

  12. β-Carotene and astaxanthin with human and bovine serum albumins.

    PubMed

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

    2015-07-15

    β-Carotene and astaxanthin are two carotenoids with powerful antioxidant properties. In this study, the interaction of these two carotenoids with human serum albumin (HSA) and bovine serum albumin (BSA) under physiological conditions was investigated using several spectroscopic techniques. The experimental results indicate the quenching mechanism of HSA/BSA, by the two carotenoids, is a static process. The binding constants and number of binding sites were evaluated at different temperatures. Thermodynamic investigations revealed the interaction between the two carotenoids and HSA/BSA is synergistically driven by enthalpy and entropy, and hydrophobic forces and electrostatic attraction have a significant role in the reactions. Binding site I was found to be the primary binding site for β-carotene and astaxanthin. In addition, as shown by synchronous fluorescence spectroscopy and FT-IR, the two carotenoids may induce conformational and micro-environmental changes in HSA/BSA.

  13. Spectroscopic investigations of the interaction of the anti‑hypertension drug valsartan with human serum albumin.

    PubMed

    Jing, Jian; Qu, Xin; Tu, Zhi; Zheng, Chenhoo; Zheng, Zhicheng

    2014-06-01

    The aim of the present study was to investigate the interaction between valsartan, an anti-hypertension drug, and human serum albumin (HSA) using spectroscopic techniques, including fluorescence, ultraviolet-visible absorption, synchronous fluorescence and circular dichroism (CD). The results demonstrated that valsartan and HSA form a complex and that a static quenching mechanism occurs. In addition, the binding constant and the number of binding sites for valsartan on HSA were analyzed. Hydrophobic interactions and hydrogen bonds were the predominant forces in the association reaction based on thermodynamic parameters. The distance between the donor (HSA) and the acceptor (valsartan) was 1.994 nm as derived from Forster's theory. Alterations in the secondary structure of HSA in the presence of valsartan were assessed using synchronous fluorescence and CD. This study provides an enhanced understanding of the pharmacodynamic effects of valsartan on the physiologically important protein HSA.

  14. 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.

  15. Elucidation of structural and functional properties of albumin bound to gold nanoparticles.

    PubMed

    Mariam, Jessy; Sivakami, S; Dongre, P M

    2017-02-01

    Nanoparticle-albumin complexes are being designed for targeted drug delivery and imaging. However, the changes in the functional properties of albumin due to adsorption on nanoparticles remain elusive. Thus, the objective of this work was to elucidate the structural and functional properties of human and bovine serum albumin bound to negatively charged gold nanoparticles (GNPs). Fluorescence data demonstrated static quenching of albumin by GNP with the quenching of buried as well as surface tryptophan in BSA. The binding process was enthalpy and entropy-driven in HSA and BSA, respectively. At lower concentrations of GNP there was a higher affinity for tryptophan, whereas at higher concentrations both tryptophan and tyrosine participated in the interaction. Synchronous fluorescence spectra revealed that the microenvironment of tryptophan in HSA turned more hydrophilic upon exposure to GNP. The α-helical content of albumin was unaltered by GNP. Approximately 37 and 23% reduction in specific activity of HSA and BSA was observed due to GNP binding. In presence of warfarin and ibuprofen the binding constants of albumin-GNP complexes were altered. A very interesting observation not reported so far is the retained antioxidant activity of albumin in presence of GNP i.e. we believe that GNPs did not bind to the free sulfhydryl groups of albumin. However enhanced levels of copper binding were observed. We have also highlighted the differential response in albumin due to gold and silver nanoparticles which could be attributed to differences in the charge of the nanoparticle.

  16. Improving the pharmacokinetic properties of biologics by fusion to an anti-HSA shark VNAR domain.

    PubMed

    Müller, Mischa R; Saunders, Kenneth; Grace, Christopher; Jin, Macy; Piche-Nicholas, Nicole; Steven, John; O'Dwyer, Ronan; Wu, Leeying; Khetemenee, Lam; Vugmeyster, Yulia; Hickling, Timothy P; Tchistiakova, Lioudmila; Olland, Stephane; Gill, Davinder; Jensen, Allan; Barelle, Caroline J

    2012-01-01

    Advances in recombinant antibody technology and protein engineering have provided the opportunity to reduce antibodies to their smallest binding domain components and have concomitantly driven the requirement for devising strategies to increase serum half-life to optimise drug exposure, thereby increasing therapeutic efficacy. In this study, we adopted an immunization route to raise picomolar affinity shark immunoglobulin new antigen receptors (IgNARs) to target human serum albumin (HSA). From our model shark species, Squalus acanthias, a phage display library encompassing the variable binding domain of IgNAR (VNAR) was constructed, screened against target, and positive clones were characterized for affinity and specificity. N-terminal and C-terminal molecular fusions of our lead hit in complex with a naïve VNAR domain were expressed, purified and exhibited the retention of high affinity binding to HSA, but also cross-selectivity to mouse, rat and monkey serum albumin both in vitro and in vivo. Furthermore, the naïve VNAR had enhanced pharmacokinetic (PK) characteristics in both N- and C-terminal orientations and when tested as a three domain construct with naïve VNAR flanking the HSA binding domain at both the N and C termini. Molecules derived from this platform technology also demonstrated the potential for clinical utility by being available via the subcutaneous route of delivery. This study thus demonstrates the first in vivo functional efficacy of a VNAR binding domain with the ability to enhance PK properties and support delivery of multifunctional therapies.

  17. Biophysical study on the interaction between eperisone hydrochloride and human serum albumin using spectroscopic, calorimetric, and molecular docking analysis.

    PubMed

    Rabbani, Gulam; Baig, Mohammad Hassan; Lee, Eun Ju; Cho, Won Kyung; Ma, Jin Yeul; Choi, Inho

    2017-04-05

    Eperisone hydrochloride (EH) is a widely used as a muscle relaxant for patients with muscular contracture, low back pain, or spasticity. Human serum albumin (HSA), a highly soluble negatively charged, endogenous and abundant plasma protein ascribed with the ligand binding and transport properties. The current study was undertaken to explore the interaction between EH and the serum transport protein, HSA. Study of the interaction between HSA and EH was carried by UV-vis, fluorescence quenching, circular dichroism (CD) spectroscopy, FRET, and ITC. Tryptophan fluorescence intensity of HSA was strongly quenched by EH. The binding constants (Kb) were obtained by fluorescence quenching and results shows that the EH-HSA interaction revealed a static mode of quenching, with binding constant Kb ~104 reflecting high affinity of EH for HSA. The negative ΔGº value for binding indicated that HSA-EH interaction is a spontaneous process. Thermodynamic analysis shows HSA-EH complex formation occurs primarily due to hydrophobic interactions and hydrogen bonds were facilitate the binding of EH. EH binding induces α-helix of HSA as obtained by far-UV CD and FTIR spectroscopy. In addition, the distance between EH (acceptor) and Trp residue of HSA (donor) was calculated 2.18 nm using Förster's resonance energy transfer theory. Furthermore, molecular docking results revealed EH binds with HSA, and binding site is positioned in Sudlow Site I of HSA (subdomain IIA). This work provides a useful experimental strategy for studying the interaction of myorelaxant with HSA, helping to understand the activity and mechanism of drug binding.

  18. Fusion to a highly stable consensus albumin binding domain allows for tunable pharmacokinetics.

    PubMed

    Jacobs, Steven A; Gibbs, Alan C; Conk, Michelle; Yi, Fang; Maguire, Diane; Kane, Colleen; O'Neil, Karyn T

    2015-10-01

    A number of classes of proteins have been engineered for high stability using consensus sequence design methods. Here we describe the engineering of a novel albumin binding domain (ABD) three-helix bundle protein. The resulting engineered ABD molecule, called ABDCon, is expressed at high levels in the soluble fraction of Escherichia coli and is highly stable, with a melting temperature of 81.5°C. ABDCon binds human, monkey and mouse serum albumins with affinity as high as 61 pM. The solution structure of ABDCon is consistent with the three-helix bundle design and epitope mapping studies enabled a precise definition of the albumin binding interface. Fusion of a 10 kDa scaffold protein to ABDCon results in a long terminal half-life of 60 h in mice and 182 h in cynomolgus monkeys. To explore the link between albumin affinity and in vivo exposure, mutations were designed at the albumin binding interface of ABDCon yielding variants that span an 11 000-fold range in affinity. The PK properties of five such variants were determined in mice in order to demonstrate the tunable nature of serum half-life, exposure and clearance with variations in albumin binding affinity.

  19. Albumin Binding Function: The Potential Earliest Indicator for Liver Function Damage

    PubMed Central

    Ge, Penglei; Yang, Huayu; Lu, Jingfen; Liao, Wenjun; Du, Shunda; Xu, Yingli; Xu, Haifeng; Lu, Xin; Sang, Xinting; Zhong, Shouxian; Huang, Jiefu

    2016-01-01

    Background. Currently there is no indicator that can evaluate actual liver lesion for early stages of viral hepatitis, nonalcoholic fatty liver disease (NAFLD), and cirrhosis. Aim of this study was to investigate if albumin binding function could better reflect liver function in these liver diseases. Methods. An observational study was performed on 193 patients with early NAFLD, viral hepatitis, and cirrhosis. Cirrhosis patients were separated according to Child-Pugh score into A, B, and C subgroup. Albumin metal ion binding capacity (Ischemia-modified albumin transformed, IMAT) and fatty acid binding capacity (total binding sites, TBS) were detected. Results. Both IMAT and TBS were significantly decreased in patients with NAFLD and early hepatitis. In hepatitis group, they declined prior to changes of liver enzymes. IMAT was significantly higher in cirrhosis Child-Pugh class A group than hepatitis patients and decreased in Child-Pugh class B and class C patients. Both IMAT/albumin and TBS/albumin decreased significantly in hepatitis and NAFLD group patients. Conclusions. This is the first study to discover changes of albumin metal ion and fatty acid binding capacities prior to conventional biomarkers for liver damage in early stage of liver diseases. They may become potential earliest sensitive indicators for liver function evaluation. PMID:28101103

  20. On the zopiclone enantioselective binding to human albumin and plasma proteins. An electrokinetic chromatography approach.

    PubMed

    Asensi-Bernardi, L; Martín-Biosca, Y; Medina-Hernández, M J; Sagrado, S

    2011-05-20

    In this work, a methodology for the chiral separation of zopiclone (ZPC) by electrokinetic chromatography (EKC) using carboxymethylated-β-cyclodextrin as chiral selector has been developed and applied to the evaluation of the enantioselective binding of ZPC enantiomers to HSA and total plasma proteins. Two mathematical approaches were used to estimate protein binding (PB), affinity constants (K(1)) and enantioselectivity (ES) for both enantiomers of ZPC. Contradictory results in the literature, mainly related to plasma protein binding reported data, suggest that this is an unresolved matter and that more information is needed. Discrepancies and coincidences with previous data are highlighted.

  1. Glycation alters ligand binding, enzymatic, and pharmacological properties of human albumin.

    PubMed

    Baraka-Vidot, Jennifer; Planesse, Cynthia; Meilhac, Olivier; Militello, Valeria; van den Elsen, Jean; Bourdon, Emmanuel; Rondeau, Philippe

    2015-05-19

    Albumin, the major circulating protein in blood plasma, can be subjected to an increased level of glycation in a diabetic context. Albumin exerts crucial pharmacological activities through its drug binding capacity, i.e., ketoprofen, and via its esterase-like activity, allowing the conversion of prodrugs into active drugs. In this study, the impact of the glucose-mediated glycation on the pharmacological and biochemical properties of human albumin was investigated. Aggregation product levels and the redox state were quantified to assess the impact of glycation-mediated changes on the structural properties of albumin. Glucose-mediated changes in ketoprofen binding properties and esterase-like activity were evaluated using fluorescence spectroscopy and p-nitrophenyl acetate hydrolysis assays, respectively. With the exception of oxidative parameters, significant dose-dependent alterations in biochemical and functional properties of in vitro glycated albumin were observed. We also found that the dose-dependent increase in levels of glycation and protein aggregation and average molecular mass changes correlated with a gradual decrease in the affinity of albumin for ketoprofen and its esterase-like property. In parallel, significant alterations in both pharmacological properties were also evidenced in albumin purified from diabetic patients. Partial least-squares regression analyses established a significant correlation between glycation-mediated changes in biochemical and pharmacological properties of albumin, highlighting the important role for glycation in the variability of the drug response in a diabetic situation.

  2. Albumin binding, relaxivity, and water exchange kinetics of the diastereoisomers of MS-325, a gadolinium(III)-based magnetic resonance angiography contrast agent.

    PubMed

    Caravan, Peter; Parigi, Giacomo; Chasse, Jaclyn M; Cloutier, Normand J; Ellison, Jeffrey J; Lauffer, Randall B; Luchinat, Claudio; McDermid, Sarah A; Spiller, Marga; McMurry, Thomas J

    2007-08-06

    The amphiphilic gadolinium complex MS-325 ((trisodium-{(2-(R)-[(4,4-diphenylcyclohexyl) phosphonooxymethyl] diethylenetriaminepentaacetato) (aquo)gadolinium(III)}) is a contrast agent for magnetic resonance angiography (MRA). MS-325 consists of two slowly interconverting diastereoisomers, A and B (65:35 ratio), which can be isolated at pH > 8.5 (TyeklAr, Z.; Dunham, S. U.; Midelfort, K.; Scott, D. M.; Sajiki, H.; Ong, K.; Lauffer, R. B.; Caravan, P.; McMurry, T. J. Inorg. Chem. 2007, 46, 6621-6631). MS-325 binds to human serum albumin (HSA) in plasma resulting in an extended plasma half-life, retention of the agent within the blood compartment, and an increased relaxation rate of water protons in plasma. Under physiological conditions (37 degrees C, pH 7.4, phosphate buffered saline (PBS), 4.5% HSA, 0.05 mM complex), there is no statistical difference in HSA affinity or relaxivity between the two isomers (A 88.6 +/- 0.6% bound, r1 = 42.0 +/- 1.0 mM(-1) s(-1) at 20 MHz; B 90.2 +/- 0.6% bound, r1 = 38.3 +/- 1.0 mM(-1) s(-1) at 20 MHz; errors represent 1 standard deviation). At lower temperatures, isomer A has a higher relaxivity than isomer B. The water exchange rates in the absence of HSA at 298 K, kA298 = 5.9 +/- 2.8 x 10(6) s(-1), kB298 = 3.2 +/- 1.8 x 10(6) s(-1), and heats of activation, DeltaHA = 56 +/- 8 kJ/mol, DeltaHB = 59 +/- 11 kJ/mol, were determined by variable-temperature 17O NMR at 7.05 T. Proton nuclear magnetic relaxation dispersion (NMRD) profiles were recorded over the frequency range of 0.01-50 MHz at 5, 15, 25, and 35 degrees C in a 4.5% HSA in PBS solution for each isomer (0.1 mM). Differences in the relaxivity in HSA between the two isomers could be attributed to the differing water exchange rates.

  3. Recombinant HSA-CMG2 Is a Promising Anthrax Toxin Inhibitor

    PubMed Central

    Li, Liangliang; Guo, Qiang; Liu, Ju; Zhang, Jun; Yin, Ying; Dong, Dayong; Fu, Ling; Xu, Junjie; Chen, Wei

    2016-01-01

    Anthrax toxin is the major virulence factor produced by Bacillus anthracis. Protective antigen (PA) is the key component of the toxin and has been confirmed as the main target for the development of toxin inhibitors. The inhibition of the binding of PA to its receptor, capillary morphogenesis protein-2 (CMG2), can effectively block anthrax intoxication. The recombinant, soluble von Willebrand factor type A (vWA) domain of CMG2 (sCMG2) has demonstrated potency against anthrax toxin. However, the short half-life of sCMG2 in vivo is a disadvantage for its development as a new anthrax drug. In the present study, we report that HSA-CMG2, a protein combining human serum albumin (HSA) and sCMG2, produced in the Pichia pastoris expression system prolonged the half-life of sCMG2 while maintaining PA binding ability. The IC50 of HSA-CMG2 is similar to those of sCMG2 and CMG2-Fc in in vitro toxin neutralization assays, and HSA-CMG2 completely protects rats from lethal doses of anthrax toxin challenge; these same challenge doses exceed sCMG2 at a sub-equivalent dose ratio and overwhelm CMG2-Fc. Our results suggest that HSA-CMG2 is a promising inhibitor of anthrax toxin and may contribute to the development of novel anthrax drugs. PMID:26805881

  4. Synthesis and crystal structure elucidation of new copper(II)-based chemotherapeutic agent coupled with 1,2-DACH and orthovanillin: Validated by in vitro DNA/HSA binding profile and pBR322 cleavage pathway.

    PubMed

    Zaki, Mehvash; Afzal, Mohd; Ahmad, Musheer; Tabassum, Sartaj

    2016-08-01

    New copper(II)-based complex (1) was synthesized and characterized by analytical, spectroscopic and single crystal X-ray diffraction. The in vitro binding studies of complex 1 with CT DNA and HSA have been investigated by employing biophysical techniques to examine the binding propensity of 1 towards DNA and HSA. The results showed that 1 avidly binds to CT DNA via electrostatic mode along with the hydrogen bonding interaction of NH2 and CN groups of Schiff base ligand with the base pairs of DNA helix, leads to partial unwinding and destabilization of the DNA double helix. Moreover, the CD spectral studies revealed that complex 1 binds through groove binding interaction that stabilizes the right-handed B-form of DNA. Complex 1 showed an impressive photoinduced nuclease activity generating single-strand breaks in comparison with the DNA cleavage activity in presence of visible light. The mechanistic investigation revealed the efficiency of 1 to cleave DNA strands by involving the generation of reactive oxygen species. Furthermore, the time dependent DNA cleavage activity showed that there was gradual increase in the amount of NC DNA on increasing the photoexposure time. However, the interaction of 1 and HSA showed that the change of intrinsic fluorescence intensity of HSA was induced by the microenvironment of Trp residue.

  5. 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.

  6. Interaction of sulpiride and serum albumin: Modeling from spectrofluorimetric data

    NASA Astrophysics Data System (ADS)

    Fragoso, Viviane Muniz da Silva; Silva, Dilson

    2015-12-01

    We have applied the fluorescence quenching modeling to study the process of interaction of sulpiride with human serum albumin (HSA) and bovine (BSA). Albumin is more abundant protein in blood and it emits fluorescence when excited by 260-295 nm. Sulpiride is an atypical antipsychotic used in the treatment of many psychiatric disorders. As sulpiride is fluorescent, we developed a mathematical model to analyzing the interaction of two fluorescent substances. This model was able to separate the albumin fluorescence from the quencher fluorescence. Results have shown that sulpiride quenches the fluorescence of both albumins by a static process, due to the complex formation drugalbumin. The association constants calculated for sulpiride-HSA was 2.20 (± 0.08) × 104 M-1 at 37° C, and 5.46 (± 0.20) × 104 M-1, 25 ° C, and the primary binding site to sulpiride in the albumin is located closer to the subdomain IB.

  7. Estimation of postmortem interval through albumin in CSF by simple dye binding method.

    PubMed

    Parmar, Ankita K; Menon, Shobhana K

    2015-12-01

    Estimation of postmortem interval is a very important question in some medicolegal investigations. For the precise estimation of postmortem interval, there is a need of a method which can give accurate estimation. Bromocresol green (BCG) is a simple dye binding method and widely used in routine practice. Application of this method in forensic practice may bring revolutionary changes. In this study, cerebrospinal fluid was aspirated from cisternal puncture from 100 autopsies. A study was carried out on concentration of albumin with respect to postmortem interval. After death, albumin present in CSF undergoes changes, after 72 h of death, concentration of albumin has become 0.012 mM, and this decrease was linear from 2 h to 72 h. An important relationship was found between albumin concentration and postmortem interval with an error of ± 1-4h. The study concludes that CSF albumin can be a useful and significant parameter in estimation of postmortem interval.

  8. Interaction of anticancer drug clofarabine with human serum albumin and human α-1 acid glycoprotein. Spectroscopic and molecular docking approach.

    PubMed

    Ajmal, Mohammad Rehan; Nusrat, Saima; Alam, Parvez; Zaidi, Nida; Khan, Mohsin Vahid; Zaman, Masihuz; Shahein, Yasser E; Mahmoud, Mohamed H; Badr, Gamal; Khan, Rizwan Hasan

    2017-02-20

    The binding interaction between clofarabine, an important anticancer drug and two important carrier proteins found abundantly in human plasma, Human Serum Albumin (HSA) and α-1 acid glycoprotein (AAG) was investigated by spectroscopic and molecular modeling methods. The results obtained from fluorescence quenching experiments demonstrated that the fluorescence intensity of HSA and AAG is quenched by clofarabine and the static mode of fluorescence quenching is operative. UV-vis spectroscopy deciphered the formation of ground state complex between anticancer drug and the two studied proteins. Clofarabine was found to bind at 298K with both AAG and HSA with the binding constant of 8.128×10(3) and 4.120×10(3) for AAG and HSA, respectively. There is stronger interaction of clofarabine with AAG as compared to HSA. The Gibbs free energy change was found to be negative for the interaction of clofarabine with AAG and HSA indicating that the binding process is spontaneous. Binding of clofarabine with HSA and AAG induced ordered structures in both proteins and lead to molecular compaction. Clofarabine binds to HSA near to drug site II. Hydrogen bonding and hydrophobic interactions were the main bonding forces between HSA-clofarabine and AAG-clofarabine as revealed by docking results. This study suggests the importance of binding of anticancer drug to AAG spatially in the diseases like cancers where the plasma concentration of AAG increases many folds. Design of drug dosage can be adjusted accordingly to achieve optimal treatment outcome.

  9. 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.

  10. Characterization of minor site probes for human serum albumin by high-performance affinity chromatography.

    PubMed

    Sengupta, A; Hage, D S

    1999-09-01

    This study used high-performance affinity chromatography (HPAC) and immobilized human serum albumin (HSA) columns to examine the specificity and cross-reactivity of various compounds that have been proposed as markers for the minor binding sites of HSA. These agents included acetyldigitoxin and digitoxin as probes for the digitoxin site, phenol red as a probe for the bilirubin site, and cisor trans-clomiphene as markers for the tamoxifen site. None of these probes showed any significant binding at HSA's indole-benzodiazepine site. However, phenol red did bind at the warfarin-azapropazone site of HSA, and cis/trans-clomiphene gave positive allosteric effects caused by the binding of warfarin to HSA. Digitoxin and acetyldigitoxin were found to bind to a common, unique region on HSA; cis- and trans-clomiphene also appeared to interact at a unique site, although trans-clomiphene displayed additional direct competition with phenol red. From these results it was possible to develop a model that described the general relationship between these binding regions on HSA. This information should be useful in future studies that employ HPAC for characterizing the binding of HSA to other drugs or clinical agents.

  11. 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.

  12. Drugs modulate allosterically heme-Fe-recognition by human serum albumin and heme-fe-mediated reactivity.

    PubMed

    di Masi, Alessandra; Leboffe, Loris; Trezza, Viviana; Fanali, Gabriella; Coletta, Massimo; Fasano, Mauro; Ascenzi, Paolo

    2015-01-01

    Human serum albumin (HSA) represents an important determinant of plasma oncotic pressure and a relevant factor that modulates fluid distribution between the body compartments. Moreover, HSA (i) represents the depot and transporter of several compounds, both endogenous and exogenous, (ii) affects the pharmacokinetics of many drugs, (iii) regulates chemical modifications of some ligands, (iv) shows (pseudo-)enzymatic properties, (v) inactivates some toxic compounds, and (vi) displays anti-oxidant properties. HSA binding and (pseudo-)enzymatic properties are regulated competitively, allosterically, and by covalent modifications. While competitive inhibition of HSA binding properties is evident, allosteric mechanisms and covalent modifications affecting HSA reactivity are less clear. In several pathological conditions in which free heme-Fe levels increase, the buffering capacity of plasma hemopexin is overwhelmed and most of heme-Fe binds to the fatty acid site 1 of HSA. HSA-heme-Fe displays globin-like properties; in turn, heme-Fe modulates competitively and allosterically HSA binding and reactivity properties. Remarkably, heme-Fe-mediated HSA properties are time-dependent, representing a case for "chronosteric effects". Here, we review the drug-based modulation of (i) heme-Fe-recognition by HSA and (ii) heme-Fe-mediated reactivity.

  13. Binding of benzodiazepine drugs to bovine serum albumin: A second derivative spectrophotometric study

    NASA Astrophysics Data System (ADS)

    Omran, Ahmed A.; El-Sayed, Abdel-Aziz; Shehata, Ahmed

    2011-12-01

    The binding constants ( K values) of three benzodiazepine drugs to bovine serum albumin were determined by a second derivative spectrophotometric method. Despite the sample and reference samples were prepared in the same way to maintain the same albumin content in each sample and reference pair, the absorption spectra show that the baseline compensation was incomplete because of the strong background signals caused by bovine serum albumin. Accordingly, further quantitative spectral information could not be obtained from these absorption spectra. On the other hand, the calculated second derivative spectra clearly show isosbestic points indicating the complete removal of the residual background signal effects. Using the derivative intensity differences (Δ D values) of the studied benzodiazepine drugs before and after the addition of albumin, the binding constants were calculated and obtained with R.S.D. of less than 8%. The interactions of drugs with bovine serum albumin were investigated using Scatchard's plot. In addition, the consistency between the fractions of bound benzodiazepine calculated from the obtained K values and the experimental values were established. The results indicate that the second derivative method can be advantageously applicable to the determination of binding constants of drugs to serum albumin without prior separation. Moreover, the validity of the proposed method was confirmed.

  14. Interaction of tetramethylpyrazine with two serum albumins by a hybrid spectroscopic method

    NASA Astrophysics Data System (ADS)

    Cheng, Zhengjun

    The interactions of tetramethylpyrazine (TMPZ) with bovine serum albumin (BSA) and human serum albumin (HSA) have been investigated by various spectroscopic techniques. Fluorescence tests showed that TMPZ could bind to BSA/HSA to form complexes. The binding constants of TMPZ-BSA and TMPZ-HSA complexes were observed to be 1.442 × 104 and 3.302 × 104 M-1 at 298 K, respectively. The thermodynamic parameters (ΔG, ΔH and ΔS) calculated on the basis of different temperatures revealed that the binding of TMPZ-HSA was mainly depended on hydrophobic interaction, and yet the binding of TMPZ-BSA might involve hydrophobic interaction strongly and electrostatic interaction. The results of synchronous fluorescence, three-dimensional fluorescence, UV-vis absorption, FT-IR and CD spectra showed that the conformations of both BSA and HSA altered with the addition of TMPZ. The binding average distance between TMPZ and BSA/HSA was evaluated according to Föster non-radioactive energy transfer theory. In addition, with the aid of site markers (such as, phenylbutazone, ibuprofen and digitoxin), TMPZ primarily bound to tryptophan residues of BSA/HSA within site I (sub-domain II A).

  15. Study on the interactions of mapenterol with serum albumins using multi-spectroscopy and molecular docking.

    PubMed

    Bi, Shuyun; Zhao, Tingting; Wang, Yu; Zhou, Huifeng

    2016-03-01

    The interactions of mapenterol with bovine serum albumin (BSA) and human serum albumin (HSA) have been investigated systematically using fluorescence spectroscopy, absorption spectroscopy, circular dichroism (CD) and molecular docking techniques. Mapenterol has a strong ability to quench the intrinsic fluorescence of BSA and HSA through static quenching procedures. At 291 K, the binding constants, Ka, were 1.93 × 10(3) and 2.73 × 10(3) L/mol for mapenterol-BSA and mapenterol-HAS, respectively. Electrostatic forces and hydrophobic interactions played important roles in stabilizing the mapenterol-BSA/has complex. Using site marker competitive studies, mapenterol was found to bind at Sudlow site I on BSA/HSA. There was little effect of K(+), Ca(2+), Cu(2+), Zn(2+) and Fe(3+) on the binding. The conformation of BSA/HSA was changed by mapenterol, as seen from the synchronous fluorescence spectra. The CD spectra showed that the binding of mapenterol to BSA/HSA changed the secondary structure of BSA/HSA. Molecular docking further confirmed that mapenterol could bind to Sudlow site I of BSA/HSA. According to Förster non-radiative energy transfer theory (FRET), the distances r0 between the donor and acceptor were calculated as 3.18 and 2.75 nm for mapenterol-BSA and mapenterol-HAS, respectively.

  16. Physicochemical aspects of the energetics of binding of sulphanilic acid with bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Banipal, Tarlok S.; Kaur, Amandeep; Banipal, Parampaul K.

    2017-01-01

    The thermodynamic study of the binding of sulphanilic acid with model transport protein bovine serum albumin is a promising approach in the area of synthesizing new sulfa drugs with improved therapeutic effect. Thus, such binding studies play an important role in the rational drug design process. The binding between sulphanilic acid and bovine serum albumin has been studied using calorimetry, light scattering in combination with spectroscopic and microscopic techniques. The calorimetric data reveals the presence of two sequential nature of binding sites where the first binding site has stronger affinity ( 104 M- 1) and second binding site has weaker affinity ( 103 M- 1). However, the spectroscopic (absorption and fluorescence) results suggest the presence of single low affinity binding site ( 103 M- 1) on protein. The contribution of polar and non-polar interactions to the binding process has been explored in the presence of various additives. It is found that sulphanilic acid binds with high affinity at Sudlow site II and with low affinity at Sudlow site I of protein. Light scattering and circular dichroism measurements have been used to study the effect on the molecular topology and conformation of protein, respectively. Thus these studies provide important insights into the binding of sulphanilic acid with bovine serum albumin both quantitatively and qualitatively.

  17. Bioactivity of albumins bound to silver nanoparticles.

    PubMed

    Mariam, Jessy; Sivakami, S; Kothari, D C; Dongre, P M

    2014-06-01

    The last decade has witnessed a tremendous rise in the proposed applications of nanomaterials in the field of medicine due to their very attractive physiochemical properties and novel actions such as the ability to reach previously inaccessible targets such as brain. However biological activity of functional molecules bound to nanoparticles and its physiological consequences is still unclear and hence this area requires immediate attention. The functional properties of Human Serum Albumin (HSA) and Bovine Serum Albumin (BSA) bound to silver nanoparticles (~60 nm) have been studied under physiological environment. Esterase activity, binding of drugs (warfarin and ibuprofen), antioxidant activity and copper binding by albumins was evaluated. The catalytic efficiencies of HSA and BSA diminished upon binding to silver nanoparticles. Perturbation in binding of warfarin and ibuprofen, loss of free sulphydryls, antioxidant activity and enhancement of copper binding were observed in albumins bound to nanoparticles. These alterations in functional activity of nanoparticle bound albumins which will have important consequences should be taken into consideration while using nanoparticles for diagnostic and therapeutic purposes.

  18. Probing the mechanism of interaction of metoprolol succinate with human serum albumin by spectroscopic and molecular docking analysis.

    PubMed

    Pawar, Suma K; Jaldappagari, Seetharamappa

    2017-02-24

    In the present work, the mechanism of the interaction between a β1 receptor blocker, metoprolol succinate (MS) and human serum albumin (HSA) under physiological conditions was investigated by spectroscopic techniques, namely fluorescence, Fourier transform infra-red spectroscopy (FT-IR), fluorescence lifetime decay and circular dichroism (CD) as well as molecular docking and cyclic voltammetric methods. The fluorescence and lifetime decay results indicated that MS quenched the intrinsic intensity of HSA through a static quenching mechanism. The Stern-Volmer quenching constants and binding constants for the MS-HSA system at 293, 298 and 303 K were obtained from the Stern-Volmer plot. Thermodynamic parameters for the interaction of MS with HSA were evaluated; negative values of entropy change (ΔG°) indicated the spontaneity of the MS and HSA interaction. Thermodynamic parameters such as negative ΔH° and positive ΔS° values revealed that hydrogen bonding and hydrophobic forces played a major role in MS-HSA interaction and stabilized the complex. The binding site for MS in HSA was identified by competitive site probe experiments and molecular docking studies. These results indicated that MS was bound to HSA at Sudlow's site I. The efficiency of energy transfer and the distance between the donor (HSA) and acceptor (MS) was calculated based on the theory of Fosters' resonance energy transfer (FRET). Three-dimensional fluorescence spectra and CD results revealed that the binding of MS to HSA resulted in an obvious change in the conformation of HSA. Cyclic voltammograms of the MS-HSA system also confirmed the interaction between MS and HSA. Furthermore, the effects of metal ions on the binding of MS to HSA were also studied.

  19. Spectroscopic studies on the interaction between tetrandrine and two serum albumins by chemometrics methods

    NASA Astrophysics Data System (ADS)

    Cheng, Zhengjun; Liu, Rong; jiang, Xiaohui

    2013-11-01

    The binding interactions of tetrandrine (TETD) with bovine serum albumin (BSA) and human serum albumin (HSA) have been investigated by spectroscopic methods. These experimental data were further analyzed using multivariate curve resolution-alternating least squares (MCR-ALS) method, and the concentration profiles and pure spectra for three species (BSA/HSA, TETD and TETD-BSA/HSA) existed in the interaction procedure, as well as, the apparent equilibrium constants Kapp were evaluated. The binding sites number n and the binding constants K were obtained at various temperatures. The binding distance between TETD and BSA/HSA was 1.455/1.451 nm. The site markers competitive experiments indicated that TETD primarily bound to the tryptophan residue of BSA/HSA within site I. The thermodynamic parameters (ΔG, ΔH and ΔS) calculated on the basis of different temperatures revealed that the binding of TETD-BSA was mainly depended on the hydrophobic interaction strongly and electrostatic interaction, and yet the binding of TETD-HSA was strongly relied on the hydrophobic interaction. The results of synchronous fluorescence, 3D fluorescence and FT-IR spectra show that the conformation of proteins has altered in the presence of TETD. In addition, the effect of some common ions on the binding constants between TETD and proteins were also discussed.

  20. Spectroscopic studies on the interaction between tetrandrine and two serum albumins by chemometrics methods.

    PubMed

    Cheng, Zhengjun; Liu, Rong; Jiang, Xiaohui

    2013-11-01

    The binding interactions of tetrandrine (TETD) with bovine serum albumin (BSA) and human serum albumin (HSA) have been investigated by spectroscopic methods. These experimental data were further analyzed using multivariate curve resolution-alternating least squares (MCR-ALS) method, and the concentration profiles and pure spectra for three species (BSA/HSA, TETD and TETD-BSA/HSA) existed in the interaction procedure, as well as, the apparent equilibrium constants Kapp were evaluated. The binding sites number n and the binding constants K were obtained at various temperatures. The binding distance between TETD and BSA/HSA was 1.455/1.451nm. The site markers competitive experiments indicated that TETD primarily bound to the tryptophan residue of BSA/HSA within site I. The thermodynamic parameters (ΔG, ΔH and ΔS) calculated on the basis of different temperatures revealed that the binding of TETD-BSA was mainly depended on the hydrophobic interaction strongly and electrostatic interaction, and yet the binding of TETD-HSA was strongly relied on the hydrophobic interaction. The results of synchronous fluorescence, 3D fluorescence and FT-IR spectra show that the conformation of proteins has altered in the presence of TETD. In addition, the effect of some common ions on the binding constants between TETD and proteins were also discussed.

  1. 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

    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

    2016-10-01

    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.

  2. HDM-PAMPA to predict gastrointestinal absorption, binding percentage, equilibrium and kinetics constants with human serum albumin and using 2 end-point measurements.

    PubMed

    Bujard, Alban; Petit, Charlotte; Carrupt, Pierre-Alain; Rudaz, Serge; Schappler, Julie

    2017-01-15

    The parallel artificial membrane permeability assay (PAMPA) is a high-throughput screening (HTS) technique developed to predict passive permeability through numerous different biological membranes, such as the gastrointestinal tract (GIT), the blood brain barrier (BBB), and the dermal layer. PAMPA is based on an artificial membrane, such as hexadecane (HDM), which separates two compartments (i.e., a donor and an acceptor compartment). In the present study, an HDM-PAMPA method was developed with human serum albumin (HSA) under iso-pH and gradient-pH conditions to predict the percentage of binding, dissociation/association constants (Kd and Ka, respectively) and dissociation/association kinetic rates (koff and kon, respectively) between a given drug and HSA. Thanks to the kinetic properties of PAMPA, a two end-point assay was implemented to obtain all three properties. The assay was used to measure basic, acidic, and amphoteric compounds. The protein was free in solution, allowing a direct comparison between this assay and equilibrium dialysis (ED). The developed PAMPA enabled screening of up to 96 compounds in a single run, generating valuable information on absorption and distribution in a high-throughput and high-repeatable manner.

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

    PubMed

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

    2014-03-04

    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.

  4. Fatty acid-binding site environments of serum vitamin D-binding protein and albumin are different

    PubMed Central

    Swamy, Narasimha; Ray, Rahul

    2008-01-01

    Vitamin D-binding protein (DBP) and albumin (ALB) are abundant serum proteins and both possess high-affinity binding for saturated and unsaturated fatty acids. However, certain differences exist. We surmised that in cases where serum albumin level is low, DBP presumably can act as a transporter of fatty acids. To explore this possibility we synthesized several alkylating derivatives of 14C-palmitic acid to probe the fatty acid binding pockets of DBP and ALB. We observed that N-ethyl-5-phenylisooxazolium-3′-sulfonate-ester (WRK ester) of 14C-palmitic acid specifically labeled DBP; but p-nitrophenyl- and N-hydroxysuccinimidyl-esters failed to do so. However, p-nitrophenyl ester of 14C-palmitic acid specifically labeled bovine ALB, indicating that the micro-environment of the fatty acid-binding domains of DBP and ALB may be different; and DBP may not replace ALB as a transporter of fatty acids. PMID:18374965

  5. Structural studies of bovine, equine, and leporine serum albumin complexes with naproxen.

    PubMed

    Bujacz, Anna; Zielinski, Kamil; Sekula, Bartosz

    2014-09-01

    Serum albumin, a protein naturally abundant in blood plasma, shows remarkable ligand binding properties of numerous endogenous and exogenous compounds. Most of serum albumin binding sites are able to interact with more than one class of ligands. Determining the protein-ligand interactions among mammalian serum albumins is essential for understanding the complexity of this transporter. We present three crystal structures of serum albumins in complexes with naproxen (NPS): bovine (BSA-NPS), equine (ESA-NPS), and leporine (LSA-NPS) determined to 2.58 Å (C2), 2.42 Å (P61), and 2.73 Å (P2₁2₁2₁) resolutions, respectively. A comparison of the structurally investigated complexes with the analogous complex of human serum albumin (HSA-NPS) revealed surprising differences in the number and distribution of naproxen binding sites. Bovine and leporine serum albumins possess three NPS binding sites, but ESA has only two. All three complexes of albumins studied here have two common naproxen locations, but BSA and LSA differ in the third NPS binding site. None of these binding sites coincides with the naproxen location in the HSA-NPS complex, which was obtained in the presence of other ligands besides naproxen. Even small differences in sequences of serum albumins from various species, especially in the area of the binding pockets, influence the affinity and the binding mode of naproxen to this transport protein.

  6. Detergents as probes of hydrophobic binding cavities in serum albumin and other water-soluble proteins.

    PubMed Central

    Kragh-Hansen, U; Hellec, F; de Foresta, B; le Maire, M; Møller, J V

    2001-01-01

    As an extension of our studies on the interaction of detergents with membranes and membrane proteins, we have investigated their binding to water-soluble proteins. Anionic aliphatic compounds (dodecanoate and dodecylsulfate) were bound to serum albumin with high affinity at nine sites; related nonionic detergents (C12E8 and dodecylmaltoside) were bound at seven to eight sites, many in common with those of dodecanoate. The compounds were also bound in the hydrophobic cavity of beta-lactoglobulin, but not to ovalbumin. In addition to the generally recognized role of the Sudlow binding region II of serum albumin (localized at the IIIA subdomain) in fatty acid binding, quenching of the fluorescence intensity of tryptophan-214 by 7,8-dibromododecylmaltoside and 12-bromododecanoate also implicate the Sudlow binding region I (subdomain IIA) as a locus for binding of aliphatic compounds. Our data document the usefulness of dodecyl amphipathic compounds as probes of hydrophobic cavities in water-soluble proteins. In conjunction with recent x-ray diffraction analyses of fatty acid binding as the starting point we propose a new symmetrical binding model for the location of nine high-affinity sites on serum albumin for aliphatic compounds. PMID:11371462

  7. Calorimetric investigation of diclofenac drug binding to a panel of moderately glycated serum albumins.

    PubMed

    Indurthi, Venkata S K; Leclerc, Estelle; Vetter, Stefan W

    2014-08-01

    Glycation alters the drug binding properties of serum proteins and could affect free drug concentrations in diabetic patients with elevated glycation levels. We investigated the effect of bovine serum albumin glycation by eight physiologically relevant glycation reagents (glucose, ribose, carboxymethyllysine, acetoin, methylglyoxal, glyceraldehyde, diacetyl and glycolaldehyde) on diclofenac drug binding. We used this non-steroidal anti-inflammatory drug diclofenac as a paradigm for acidic drugs with high serum binding and because of its potential cardiovascular risks in diabetic patients. Isothermal titration calorimetry showed that glycation reduced the binding affinity Ka of serum albumin and diclofenac 2 to 6-fold by reducing structural rigidity of albumin. Glycation affected the number of drug binding sites in a glycation reagent dependent manner and lead to a 25% decrease for most reagent, expect for ribose, with decreased by 60% and for the CML-modification, increased the number of binding sites by 60%. Using isothermal titration calorimetry and differential scanning calorimetry we derived the complete thermodynamic characterization of diclofenac binding to all glycated BSA samples. Our results suggest that glycation in diabetic patients could significantly alter the pharmacokinetics of the widely used over-the-counter NSDAI drug diclofenac and with possibly negative implications for patients.

  8. Chemical modification of human albumin at cys34 by ethacrynic acid: structural characterisation and binding properties.

    PubMed

    Bertucci, C; Nanni, B; Raffaelli, A; Salvadori, P

    1998-10-01

    Derivatization of the free cys3,4 in human albumin, which is reported to occur under physiological conditions, has been performed in vitro by reaction of the protein with ethacrynic acid. This modification has been investigated by mass spectrometry and circular dichroism. Ethacrynic acid has been proven to bind human albumin either covalently and non-covalently. This post-translational modification does not determine significant changes in the secondary structure of the protein, as shown by the comparable circular dichroism spectra of the native and the modified proteins. Furthermore, the binding properties of the human albumin samples have been investigated by circular dichroism and equilibrium dialysis. The affinity to the higher affinity binding sites does not change either for drugs binding to site I, like phenylbutazone, or to site II, like diazepam, while a small but significant increase has been observed for bilirubin, known to bind to site III. Nevertheless significant decreases of the affinity at the lower affinity binding sites of the modified protein were observed for both drugs binding to site I or to site II.

  9. Spectroscopic approach of the interaction study of amphiphilic drugs with the serum albumins.

    PubMed

    Khan, Abbul Bashar; Khan, Javed Masood; Ali, Mohd Sajid; Khan, Rizwan Hasan; Kabir-ud Din

    2011-10-15

    The interaction of the amphiphilic drugs, i.e., amitriptyline hydrochloride (AMT) and promethazine hydrochloride (PMT), with serum albumins (i.e., human serum albumin (HSA) and bovine serum albumin (BSA)), has been examined by the various spectroscopic techniques, like fluorescence, UV-vis, and circular dichroism (CD). Fluorescence results indicate that in case of HSA-drug complexes the quenching of fluorescence intensity at 280 nm is less effective as compared to at 295 nm while in case of BSA-drug complexes both have almost same effect and for most of drug-serum albumin complexes there is only one independent class of binding. For all drug-serum albumin complexes the quenching rate constant (K(q)) values suggest the static quenching procedure. The UV-vis results show that the change in protein conformation of PMT-serum albumin complexes was more prominent as compared to AMT-serum albumin complexes. The CD results also explain the conformational changes in the serum albumins on binding with drugs. The increase in α-helical structure for AMT-serum albumin complexes is found to be more as compared to PMT-serum albumin complexes. Hence, the various spectroscopic techniques provide a quantitative understanding of the binding of amphiphilic drugs with serum albumins.

  10. Denaturation of Human Serum Albumin by Cerium (iii) Chloride

    NASA Astrophysics Data System (ADS)

    Behbahani, G. Rezaei; Shalbafan, M.; Gheibi, N.; Barzegar, L.; Behbahani, H. Rezaei; Yaghdavaei, N.; Behbahani, Z. Rezaei

    2013-08-01

    Cerium (III) Chloride-induced conformational changes of human serum albumin, HSA, in phosphate buffer, 10 mM at pH 7.4 was investigated, using isothermal titration calorimetry (ITC), UV and fluorescence emission spectroscopic methods. The results indicate that CeCl3, Ce3+, induces irreversible denaturation of the HSA structure. The UV absorption intensity of HSA + Ce3+ shows a slight blueshift in the absorbance wavelength with increasing Ce3+ concentration. The fluorescence intensity was increased regularly and a slight redshift was observed in the emission wavelength. The HSA + Ce3+ complex quenches the fluorescence of HSA and changes the microenvironment of tryptophan residue. The emission intensity increases suggesting the loss of the tertiary structure of HSA. The results obtained from the ITC data are in agreement with the spectroscopic methods. The strong negative cooperativity of Ce3+ binding with HSA (Table 1) recovered from the extended solvation model, indicates that HSA has been denatured as a result of its interaction with Ce3+ ions.

  11. Interfacial structure of immobilized antibodies and perdeuterated HSA in model pregnancy tests measured with neutron reflectivity.

    PubMed

    Cowsill, Benjamin J; Zhao, Xiubo; Waigh, Thomas A; Eapen, Saji; Davies, Robert; Laux, Valerie; Haertlein, Michael; Forsyth, V Trevor; Lu, Jian R

    2014-05-27

    Experimental studies of antibody adsorption and antigen binding that mimicked pregnancy test immunoassays have been performed using neutron reflectivity studies of a model antibody/antigen system immobilized on the silica/water interface. The study revealed the nature of the antibody/antigen interaction and also the importance of a blocking protein, in this case human serum albumin (HSA), that enhances the immunoassay's specificity and efficiency. Of central importance to this study has been the use of a perdeuterated human serum albumin (d-HSA), providing contrast that highlights the orientation and position of the blocking agent within the adsorbed layer. It was found that the adsorbed HSA filled the gaps between the preadsorbed antibodies on the substrate, with decreased adsorption occurring as a function of increased antibody surface coverage. In addition, the antigen binding capacity of the adsorbed antibodies was investigated as a function of antibody surface coverage. The amount of specifically bound antigen was found to saturate at approximately 0.17 mg/m(2) and became independent of the antibody surface coverage. The ratio of bound antigen to immobilized antibody decreased with increased antibody surface coverage. These results are of importance for a full understanding of immunoassay systems that are widely used in clinical tests and in the detection of environmental contaminants.

  12. 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

  13. 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).

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

    PubMed

    Li, Yunqi; An, Lijia; Huang, Qingrong

    2014-09-04

    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.

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

    PubMed

    Li, Xiangrong; Yang, Zhenhua

    2015-05-05

    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.

  16. Fusion protein of retinol-binding protein and albumin domain III reduces liver fibrosis.

    PubMed

    Lee, Hongsik; Jeong, Hyeyeun; Park, Sangeun; Yoo, Wonbaek; Choi, Soyoung; Choi, Kyungmin; Lee, Min-Goo; Lee, Mihwa; Cha, DaeRyong; Kim, Young-Sik; Han, Jeeyoung; Kim, Wonkon; Park, Sun-Hwa; Oh, Junseo

    2015-06-01

    Activated hepatic stellate cells (HSCs) play a key role in liver fibrosis, and inactivating HSCs has been considered a promising therapeutic approach. We previously showed that albumin and its derivative designed for stellate cell-targeting, retinol-binding protein-albumin domain III fusion protein (referred to as R-III), inactivate cultured HSCs. Here, we investigated the mechanism of action of albumin/R-III in HSCs and examined the anti-fibrotic potential of R-III in vivo. R-III treatment and albumin expression downregulated retinoic acid (RA) signaling which was involved in HSC activation. RA receptor agonist and retinaldehyde dehydrogenase overexpression abolished the anti-fibrotic effect of R-III and albumin, respectively. R-III uptake into cultured HSCs was significantly decreased by siRNA-STRA6, and injected R-III was localized predominantly in HSCs in liver. Importantly, R-III administration reduced CCl4- and bile duct ligation-induced liver fibrosis. R-III also exhibited a preventive effect against CCl4-inducd liver fibrosis. These findings suggest that the anti-fibrotic effect of albumin/R-III is, at least in part, mediated by downregulation of RA signaling and that R-III is a good candidate as a novel anti-fibrotic drug.

  17. In vivo binding of 1-nitropyrene to albumin in the rat.

    PubMed

    el-Bayoumy, K; Johnson, B; Partian, S; Upadhyaya, P; Hecht, S S

    1994-01-01

    Human risk assessment from exposure to nitropolynuclear aromatic hydrocarbons (NO2-PAH) has not been clearly defined, despite the widespread occurrence of such agents in the environment and their possible involvement in the etiology of some human cancers. This study was conducted since methods to determine exposure to and uptake of metabolically activated NO2-PAH are lacking. 1-Nitropyrene (1-NP), the most abundant and most extensively studied NO2-PAH, was found to bind to rat albumin at a level of 0.04 +/- 0.01% (mean +/- SD, n = 3) of the dose administered by gavage; the binding was linear over five orders of magnitude (P < 0.01). The adducts cleared at a rate (half-life = 60 h) similar to that of the unmodified rat albumin. Chromatographic analysis revealed that albumin adducts could be resolved further into a major and a minor component. Mild acid hydrolysis of the major 1-NP-albumin adduct yielded phenolic derivatives that, when subjected to acetylation, produced a material with a mass spectrum similar to that of a synthetically prepared mixture, consisting of more than one isomer, 1-acetylamino-X,Y-diacetoxypyrenes (chromatographic separation of the individual isomers was not achieved). Thus, this phenolic material that is released upon the acid treatment of albumin adducts may be a suitable indicator(s) for monitoring exposure to and metabolic activation of 1-NP.

  18. Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

    PubMed Central

    Nilvebrant, Johan; Åstrand, Mikael; Georgieva-Kotseva, Maria; Björnmalm, Mattias; Löfblom, John; Hober, Sophia

    2014-01-01

    The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein. PMID:25089830

  19. Modeling techniques and fluorescence imaging investigation of the interactions of an anthraquinone derivative with HSA and ctDNA.

    PubMed

    Fu, Zheng; Cui, Yanrui; Cui, Fengling; Zhang, Guisheng

    2016-01-15

    A new anthraquinone derivative (AORha) was synthesized. Its interactions with human serum albumin (HSA) and calf thymus DNA (ctDNA) were investigated by fluorescence spectroscopy, UV-visible absorption spectroscopy and molecular modeling. Cell viability assay and cell imaging experiment were performed using cervical cancer cells (HepG2 cells). The fluorescence results revealed that the quenching mechanism was static quenching. At different temperatures (290, 300, 310 K), the binding constants (K) and the number of binding sites (n) were determined, respectively. The positive ΔH and ΔS values showed that the binding of AORha with HSA was hydrophobic force, which was identical with the molecular docking result. Studying the fluorescence spectra, UV spectra and molecular modeling also verified that the binding mode of AORha and ctDNA might be intercalative. When HepG2 cells were treated with AORha, the fluorescence became brighter and turned green, which could be used for bioimaging.

  20. Modeling techniques and fluorescence imaging investigation of the interactions of an anthraquinone derivative with HSA and ctDNA

    NASA Astrophysics Data System (ADS)

    Fu, Zheng; Cui, Yanrui; Cui, Fengling; Zhang, Guisheng

    2016-01-01

    A new anthraquinone derivative (AORha) was synthesized. Its interactions with human serum albumin (HSA) and calf thymus DNA (ctDNA) were investigated by fluorescence spectroscopy, UV-visible absorption spectroscopy and molecular modeling. Cell viability assay and cell imaging experiment were performed using cervical cancer cells (HepG2 cells). The fluorescence results revealed that the quenching mechanism was static quenching. At different temperatures (290, 300, 310 K), the binding constants (K) and the number of binding sites (n) were determined, respectively. The positive ΔH and ΔS values showed that the binding of AORha with HSA was hydrophobic force, which was identical with the molecular docking result. Studying the fluorescence spectra, UV spectra and molecular modeling also verified that the binding mode of AORha and ctDNA might be intercalative. When HepG2 cells were treated with AORha, the fluorescence became brighter and turned green, which could be used for bioimaging.

  1. Molecular interaction of ctDNA and HSA with sulfadiazine sodium by multispectroscopic methods and molecular modeling.

    PubMed

    Geng, Shaoguang; Liu, Guosheng; Li, Wei; Cui, Fengling

    2013-01-01

    Interactions of sulfadiazine sodium (SD-Na) with calf thymus DNA (ctDNA) and human serum albumin (HSA) were studied using fluorescence spectroscopy, UV absorption spectroscopy and molecular modeling. The fluorescence experiments showed that the processes were static quenching. The results of UV spectra and molecular modeling of the interaction between SD-Na and ctDNA indicated that the binding mode might be groove binding. In addition, the interaction of SD-Na with HSA under simulative physiological conditions was also investigated. The binding constants (K) and the number of binding sites (n) at different temperatures (292, 302, 312 K) were 5.23 × 10(3) L/mol, 2.18; 4.50 × 10(3) L/mol, 2.35; and 4.08 × 10(3) L/mol, 2.47, respectively. Thermodynamic parameters including enthalpy change (ΔH) and entropy change (ΔS) were calculated, the results suggesting that hydrophobic force played a very important role in SD-Na binding to HSA, which was in good agreement with the molecular modeling study. Moreover, the effect of SD-Na on the conformation of HSA was analyzed using three-dimensional fluorescence spectra.

  2. Advanced Glycation End Products Modulate Structure and Drug Binding Properties of Albumin.

    PubMed

    Awasthi, Saurabh; Murugan, N Arul; Saraswathi, N T

    2015-09-08

    The extraordinary ligand binding properties of albumin makes it a key player in the pharmacokinetics and pharmacodynamics of many vital drugs. Albumin is highly susceptible for nonenzymatic glycation mediated structural modifications, and there is a need to determine structural and functional impact of specific AGEs modifications. The present study was aimed toward determining the AGE mediated structure and function changes, primarily looking into the effect on binding affinity of drugs in the two major drug binding sites of albumin. The impact of the two most predominant AGEs modifications, i.e., carboxyethyllysine (CEL) and argpyrimidine (Arg-P), was studied on the basis of the combination of in vitro and in silico experiments. In vitro studies were carried out by AGEs modification of bovine serum albumin (BSA) for the formation of Arg-P and CEL followed by drug interaction studies. In silico studies involved molecular dynamics (MD) simulations and docking studies for native and AGEs modified BSAs. In particular the side chain modification was specifically carried out for the residues in the drug binding sites, i.e., Arg-194, Arg-196, Arg-198, and Arg-217, and Lys-204 (site I) and Arg-409 and Lys-413 (site II). The equilibrated structures of native BSA (n-BSA) and glycated BSA (G-BSA) as obtained from MD were used for drug binding studies using molecular docking approach. It was evident from the results of both in vitro and in silico drug interaction studies that AGEs modification results in the reduced drug binding affinity for tolbutamide (TLB) and ibuprofen (IBP) in sites I and II. Moreover, the AGEs modification mediated conformational changes resulted in the shallow binding pockets with reduced accessibility for drugs.

  3. Nickel(II)-Schiff base complex recognizing domain II of bovine and human serum albumin: Spectroscopic and docking studies

    NASA Astrophysics Data System (ADS)

    Ray, Aurkie; Koley Seth, Banabithi; Pal, Uttam; Basu, Samita

    It has been spectroscopically monitored that a mononuclear nickel(II)-Schiff base complex {[NiL]·CH3OH = NSC} exhibits greater binding affinity for bovine serum albumin (BSA) than that of its human counterpart (HSA). Moreover the modes of binding of NSC with the two serum albumins also differ significantly. Docking studies predict a relatively rare type of 'superficial binding' of NSC at domain IIB of HSA with certain mobility whereas for BSA such phenomena has not been detected. The mobile nature of NSC at domain IIB of HSA has been well correlated with the spectroscopic results. It is to be noted that thermodynamic parameters for the NSC interaction also differ for the two serum albumins. Occurrence of energy transfer between the donor (Trp of BSA and HSA) and acceptor (NSC) has been obtained by means of Förster resonance energy transfer (FRET). The protein stability on NSC binding has also been experimented by the GuHCl-induced protein unfolding studies. Interestingly it has been found that NSC-HSA interaction enhances the protein stability whereas NSC-BSA binding has no such impact. Such observations are indicative of the fact that the conformation of NSC is responsible in recognizing the two serum albumins and selectively enhancing protein stability.

  4. Nickel(II)-Schiff base complex recognizing domain II of bovine and human serum albumin: spectroscopic and docking studies.

    PubMed

    Ray, Aurkie; Seth, Banabithi Koley; Pal, Uttam; Basu, Samita

    2012-06-15

    It has been spectroscopically monitored that a mononuclear nickel(II)-Schiff base complex {[NiL]·CH(3)OH=NSC} exhibits greater binding affinity for bovine serum albumin (BSA) than that of its human counterpart (HSA). Moreover the modes of binding of NSC with the two serum albumins also differ significantly. Docking studies predict a relatively rare type of 'superficial binding' of NSC at domain IIB of HSA with certain mobility whereas for BSA such phenomena has not been detected. The mobile nature of NSC at domain IIB of HSA has been well correlated with the spectroscopic results. It is to be noted that thermodynamic parameters for the NSC interaction also differ for the two serum albumins. Occurrence of energy transfer between the donor (Trp of BSA and HSA) and acceptor (NSC) has been obtained by means of Förster resonance energy transfer (FRET). The protein stability on NSC binding has also been experimented by the GuHCl-induced protein unfolding studies. Interestingly it has been found that NSC-HSA interaction enhances the protein stability whereas NSC-BSA binding has no such impact. Such observations are indicative of the fact that the conformation of NSC is responsible in recognizing the two serum albumins and selectively enhancing protein stability.

  5. 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.

  6. Virus-Enabled Biosensor for Human Serum Albumin.

    PubMed

    Ogata, Alana F; Edgar, Joshua M; Majumdar, Sudipta; Briggs, Jeffrey S; Patterson, Shae V; Tan, Ming X; Kudlacek, Stephan T; Schneider, Christine A; Weiss, Gregory A; Penner, Reginald M

    2017-01-17

    The label-free detection of human serum albumin (HSA) in aqueous buffer is demonstrated using a simple, monolithic, two-electrode electrochemical biosensor. In this device, both millimeter-scale electrodes are coated with a thin layer of a composite containing M13 virus particles and the electronically conductive polymer poly(3,4-ethylenedioxy thiophene) or PEDOT. These virus particles, engineered to selectively bind HSA, serve as receptors in this biosensor. The resistance component of the electrical impedance, Zre, measured between these two electrodes provides electrical transduction of HSA binding to the virus-PEDOT film. The analysis of sample volumes as small as 50 μL is made possible using a microfluidic cell. Upon exposure to HSA, virus-PEDOT films show a prompt increase in Zre within 5 s and a stable Zre signal within 15 min. HSA concentrations in the range from 100 nM to 5 μM are detectable. Sensor-to-sensor reproducibility of the HSA measurement is characterized by a coefficient-of-variance (COV) ranging from 2% to 8% across this entire concentration range. In addition, virus-PEDOT sensors successfully detected HSA in synthetic urine solutions.

  7. The interaction between cepharanthine and two serum albumins: multiple spectroscopic and chemometric investigations.

    PubMed

    Cheng, Zhengjun; Liu, Rong; Jiang, Xiaohui; Xu, Qianyong

    2014-08-01

    The binding modes of cepharanthine (CEPT) with bovine serum albumin (BSA) and human serum albumin (HSA) have been established by reproducing physiological conditions, which is very important to understand the pharmacokinetics and toxicity of CEPT. These spectral data were further analyzed by the multivariate curve resolution-alternating least squares method. Moreover, the concentration profiles and pure spectra of three species (BSA/HSA, CEPT and CEPT-BSA/HSA) and the apparent equilibrium constants K(app) were evaluated. The experimental results showed that CEPT could quench the fluorescence intensity of BSA/HSA by a combined quenching (static and dynamic) procedure. The binding constant (K), the thermodynamic parameters (ΔG, ΔH and ΔS) and binding subdomain were measured, and indicated that CEPT could spontaneously bind to BSA/HSA on subdomain IIA through the hydrophobic interactions. The effect of CEPT on the secondary structure of proteins has been analyzed by circular dichroism, 3D fluorescence and Fourier transform infrared spectra. The binding distance between CEPT and tryptophan of BSA/HSA was 2.305/1.749 nm, which is based on the Förster resonance energy transfer theory.

  8. Binding of a chromen-4-one Schiff's base with bovine serum albumin: capping with β-cyclodextrin influences the binding.

    PubMed

    Chandrasekaran, Sowrirajan; Sudha, Natesan; Premnath, D; Enoch, Israel V M V

    2015-09-01

    This work deals with the synthesis of 6-methyl-3-[(4'-methylphenyl)imino]methyl-4H-chromen-4-one (MMPIMC), its binding to β-cyclodextrin, and the influence of the cyclodextrin complexation on the compound's binding to bovine serum albumin (BSA). The 1:2 stoichiometry for the complexation of MMPIMC with β-cyclodextrin is determined with the binding constant of 1.90 × 10(4) M(-2). The structure of host-guest complex plays a role in protein binding of MMPIMC. One- and two-dimensional NMR spectra are used to determine the mode of binding of the guest to β-cyclodextrin cavity and the structure of the inclusion complex is proposed. The binding of MMPIMC with BSA in the absence and the presence of β-cyclodextrin is studied. The binding strengths of MMPIMC-BSA (1.73 × 10(5) M(-1)) and β-cyclodextrin-complexed MMPIMC-BSA (9.0 × 10(4) M(-1)) show difference in magnitude. The Förster Resonance Energy Transfer efficiency and the proximity of the donor and acceptor molecules, are modulated by β-cyclodextrin. Molecular modeling is used to optimize the sites and mode of binding of MMPIMC with bovine serum albumin.

  9. Fluorescence study on the interaction of human serum albumin with bromsulphalein

    NASA Astrophysics Data System (ADS)

    Cheng, Fang-Qin; Wang, Ya-Ping; Li, Zhong-Ping; Dong, Chuan

    2006-12-01

    The binding of bromsulphalein (BSP) with human serum albumin was investigated at different temperatures, 298 and 308 K, by the fluorescence spectroscopy at pH 7.24. The binding constant was determined by Stern-Volmer equation based on the quenching of the fluorescence HSA in the presence of bromsulphalein. The effect of various metal ions on the binding constants of BSP with HSA was investigated. The thermodynamic parameters were calculated according to the dependence of enthalpy change on the temperature as follows: Δ H and Δ S possess small negative (9.3 kJ mol -1) and positive values (22.3 J K -l mol -l), respectively. The experimental results revealed that BSP has a strong ability to quench the intrinsic fluorescence of HSA through a static quenching procedure. The binding constants between BSP to HSA were remarkable and independent on temperature. The binding constants between HSA and BSP decreased in the presence of various ions, commonly decreased by 30-55%. The hydrophobic force played a major role in the interaction of BSP with HSA. All these experimental results and theoretical data clarified that BSP could bind to HSA and be effectively transported and eliminated in body, which could be a useful guideline for further drug design.

  10. Effect of direct albumin binding to sphingosylphosphorylcholine in Jurkat T cells.

    PubMed

    Han, Mijin; Kim, Yu-Lee; Sacket, Santosh J; Kim, Kyeok; Kim, Hyo-Lim; Jo, Ji-Yeong; Ha, Nam-Chul; Im, Dong-Soon

    2007-11-01

    We investigated the effects of serum on lysophospholipid-induced cytotoxicity in Jurkat T cells. We found that sphingosylphosphorylcholine (SPC, also known as lysosphingomyelin) induced cytotoxicity and that albumin in serum could protect cells by binding directly to SPC. Furthermore, we also found that SPC induced ROS generation, increased [Ca(2+)](i), and decreased MMP. However, those effects were only observed at concentrations higher than 10 microM and were only induced in albumin-free media. Therefore, SPC may be trapped by albumin in plasma and unable to exert its effects under normal conditions, although at high concentrations, SPC could induce several responses such as ROS generation, increased [Ca(2+)](i), and decreased MMP in Jurkat T cells.

  11. Bovine Serum Albumin binding to CoCrMo nanoparticles and the influence on dissolution

    NASA Astrophysics Data System (ADS)

    Simoes, T. A.; Brown, A. P.; Milne, S. J.; Brydson, R. M. D.

    2015-10-01

    CoCrMo alloys exhibit good mechanical properties, excellent biocompatibility and are widely utilised in orthopaedic joint replacements. Metal-on-metal hip implant degradation leads to the release of metal ions and nanoparticles, which persist through the implant's life and could be a possible cause of health complications. This study correlates preferential binding between proteins and metal alloy nanoparticles to the alloy's corrosion behaviour and the release of metal ions. TEM images show the formation of a protein corona in all particles immersed in albumin containing solutions. Only molybdenum release was significant in these tests, suggesting high dissolution of this element when CoCrMo alloy nanoparticles are produced as wear debris in the presence of serum albumin. The same trend was observed during extended exposure of molybdenum reference nanoparticles to albumin.

  12. Recombinant fusion protein of albumin-retinol binding protein inactivates stellate cells

    SciTech Connect

    Choi, Soyoung; Park, Sangeun; Kim, Suhyun; Lim, Chaeseung; Kim, Jungho; Cha, Dae Ryong; Oh, Junseo

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer We designed novel recombinant albumin-RBP fusion proteins. Black-Right-Pointing-Pointer Expression of fusion proteins inactivates pancreatic stellate cells (PSCs). Black-Right-Pointing-Pointer Fusion proteins are successfully internalized into and inactivate PSCs. Black-Right-Pointing-Pointer RBP moiety mediates cell specific uptake of fusion protein. -- Abstract: Quiescent pancreatic- (PSCs) and hepatic- (HSCs) stellate cells store vitamin A (retinol) in lipid droplets via retinol binding protein (RBP) receptor and, when activated by profibrogenic stimuli, they transform into myofibroblast-like cells which play a key role in the fibrogenesis. Despite extensive investigations, there is, however, currently no appropriate therapy available for tissue fibrosis. We previously showed that the expression of albumin, composed of three homologous domains (I-III), inhibits stellate cell activation, which requires its high-affinity fatty acid-binding sites asymmetrically distributed in domain I and III. To attain stellate cell-specific uptake, albumin (domain I/III) was coupled to RBP; RBP-albumin{sup domain} {sup III} (R-III) and albumin{sup domain} {sup I}-RBP-albumin{sup III} (I-R-III). To assess the biological activity of fusion proteins, cultured PSCs were used. Like wild type albumin, expression of R-III or I-R-III in PSCs after passage 2 (activated PSCs) induced phenotypic reversal from activated to fat-storing cells. On the other hand, R-III and I-R-III, but not albumin, secreted from transfected 293 cells were successfully internalized into and inactivated PSCs. FPLC-purified R-III was found to be internalized into PSCs via caveolae-mediated endocytosis, and its efficient cellular uptake was also observed in HSCs and podocytes among several cell lines tested. Moreover, tissue distribution of intravenously injected R-III was closely similar to that of RBP. Therefore, our data suggest that albumin-RBP fusion protein comprises

  13. Albumin-based microbubbles bind up-regulated scavenger receptors following vascular injury.

    PubMed

    Anderson, Daniel R; Duryee, Michael J; Anchan, Rajeev K; Garvin, Robert P; Johnston, Michael D; Porter, Thomas R; Thiele, Geoffrey M; Klassen, Lynell W

    2010-12-24

    We have shown previously that perfluorocarbon-exposed sonicated dextrose albumin (PESDA) microbubbles bind to injured vascular tissue and can be detected with ultrasound imaging techniques. Prior studies have shown that scavenger receptors (SRs) are regulators of innate and adaptive immune responses and are involved in the progression of vascular disease such as atherosclerosis. In this study, we sought to determine the molecular mechanism of PESDA binding to balloon-injured vasculature. RT-PCR analysis of angioplastied aortas demonstrated a significantly (p ≤ 0.01) increased expression of SRs. Binding to SRs was confirmed using SR-expressing CHO cells, and this binding was blocked by competitive inhibition with the SR-binding ligands oxidized LDL and malondialdehyde-acetaldehyde-modified LDL. Confocal imaging confirmed the co-localization of PESDA microbubbles to CD36, SRB-1, and Toll-like receptor 4, but not to monocytes/macrophages. This study demonstrates that PESDA binds to SRs and that this binding is in major part dependent upon the oxidized nature of PESDA microbubble shell proteins. The extent of SR mRNA expression was increased with injury and associated with microbubble retention as defined by scanning electron microscopy and immunohistochemistry. These findings clarify the mechanisms of how albumin-based microbubbles bind to injured and inflamed vasculature and further support the potential of this imaging technique to detect early vascular innate inflammatory pathophysiologic processes.

  14. Spectroscopic and molecular modelling studies of binding mechanism of metformin with bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Sharma, Deepti; Ojha, Himanshu; Pathak, Mallika; Singh, Bhawna; Sharma, Navneet; Singh, Anju; Kakkar, Rita; Sharma, Rakesh K.

    2016-08-01

    Metformin is a biguanide class of drug used for the treatment of diabetes mellitus. It is well known that serum protein-ligand binding interaction significantly influence the biodistribution of a drug. Current study was performed to characterize the binding mechanism of metformin with serum albumin. The binding interaction of the metformin with bovine serum albumin (BSA) was examined using UV-Vis absorption spectroscopy, fluorescence, circular dichroism, density functional theory and molecular docking studies. Absorption spectra and fluorescence emission spectra pointed out the weak binding of metformin with BSA as was apparent from the slight change in absorbance and fluorescence intensity of BSA in presence of metformin. Circular dichroism study implied the significant change in the conformation of BSA upon binding with metformin. Density functional theory calculations showed that metformin has non-planar geometry and has two energy states. The docking studies evidently signified that metformin could bind significantly to the three binding sites in BSA via hydrophobic, hydrogen bonding and electrostatic interactions. The data suggested the existence of non-covalent specific binding interaction in the complexation of metformin with BSA. The present study will certainly contribute to the development of metformin as a therapeutic molecule.

  15. Characterization of the binding sites for dicarboxylic acids on bovine serum albumin.

    PubMed Central

    Tonsgard, J H; Meredith, S C

    1991-01-01

    Dicarboxylic acids are prominent features of several diseases, including Reye's syndrome and inborn errors of mitochondrial and peroxisomal fatty acid oxidation. Moreover, dicarboxylic acids are potentially toxic to cellular processes. Previous studies [Tonsgard, Mendelson & Meredith (1988) J. Clin. Invest. 82, 1567-1573] demonstrated that long-chain dicarboxylic acids have a single high-affinity binding site and between one and three lower-affinity sites on albumin. Medium-chain-length dicarboxylic acids have a single low-affinity site. We further characterized dicarboxylic acid binding to albumin in order to understand the potential effects of drugs and other ligands on dicarboxylic acid binding and toxicity. Progesterone and oleate competitively inhibit octadecanedioic acid binding to the single high-affinity site. Octanoate inhibits binding to the low-affinity sites. Dansylated probes for subdomain 2AB inhibit dodecanedioic acid binding whereas probes for subdomain 3AB do not. In contrast, low concentrations of octadecanedioic acid inhibit the binding of dansylated probes to subdomain 3AB and 2AB. L-Tryptophan, which binds in subdomain 3AB, inhibits hexadecanedioic acid binding but has no effect on dodecanedioic acid. Bilirubin and acetylsalicylic acid, which bind in subdomain 2AB, inhibit the binding of medium-chain and long-chain dicarboxylic acids. Our results suggest that long-chain dicarboxylic acids bind in subdomains 2C, 3AB and 2AB. The single low-affinity binding site for medium-chain dicarboxylic acids is in subdomain 2AB. These studies suggest that dicarboxylic acids are likely to be unbound in disease states and may be potentially toxic. PMID:2064600

  16. Comparison of human serum and bovine serum albumins on oxidation dynamics induced by talaporfin sodium photosensitization reaction with albumin rich conditions: solution experiments

    NASA Astrophysics Data System (ADS)

    Kurotsu, Mariko; Nakamura, Tetsuya; Takahashi, Mei; Ogawa, Emiyu; Arai, Tsunenori

    2014-02-01

    In order to understand extracellular-photosensitization reaction (PR) using talaporfin sodium, we studied comparison of oxidation dynamics of albumin and talaporfin sodium in solution system by visible and ultraviolet absorption spectrum measurements. Almost all talaporfin sodium particles may be bound to albumin in interstitial fluid, and this binding would affect the oxidation dynamics during this PR. Bovine serum albumin (BSA) is commonly used in vitro study but its binding characteristics with talaporfin sodium are different from human serum albumin (HSA). PR was operated in a solution composed of 20 μg/ml talaporfin sodium and 1.3 mg/ml HSA or BSA to simulate myocardial extracellular PR condition. Laser radiation of 662 nm was irradiated to this solution with irradiance of 0.29 W/cm2. Absorption spectra of these solutions were measured during the PR. We estimated oxidized ratio by absorption difference around 240 nm before and after the PR. Talaporfin sodium was oxidized 100% with HSA and BSA by the PR of 100 J/cm2 in radiant exposure. On the other hand, HSA and BSA were oxidized 60% and 94%, respectively in this radiant exposure. Q-band absorption peak of talaporfin sodium with HSA was shifted to 1 nm longer wavelength increasing radiant exposure up to 100 J/cm2. This longer wavelength shift would mean binding ratio of non-oxidized talaporfin sodium to non-oxidized HSA was increased with increasing radiant exposure. Therefore it would be possible that PR with talaporfin sodium bound to HSA might present efficient PDT than PR bound to BSA.

  17. Probing the binding of (+)-catechin to bovine serum albumin by isothermal titration calorimetry and spectroscopic techniques

    NASA Astrophysics Data System (ADS)

    Li, Xiangrong; Hao, Yongbing

    2015-07-01

    In this study, the interaction between (+)-catechin and bovine serum albumin (BSA) was investigated using isothermal titration calorimetry (ITC), in combination with fluorescence spectroscopy, UV-vis absorption spectroscopy, and Fourier transform infrared (FT-IR) spectroscopy. Thermodynamic investigations reveal that the electrostatic interaction and hydrophobic interaction are the major binding forces in the binding of (+)-catechin to BSA. The binding of (+)-catechin to BSA is synergistically driven by enthalpy and entropy. Fluorescence experiments suggest that (+)-catechin can quench the fluorescence of BSA through a static quenching mechanism. The obtained binding constants and the equilibrium fraction of unbound (+)-catechin show that (+)-catechin can be stored and transported from the circulatory system to reach its target organ. Binding site I is found to be the primary binding site for (+)-catechin. Additionally, as shown by the UV-vis absorption, synchronous fluorescence spectroscopy and FT-IR, (+)-catechin may induce conformational and microenvironmental changes of BSA.

  18. Insights into the molecular interaction between two polyoxygenated cinnamoylcoumarin derivatives and human serum albumin.

    PubMed

    Khammari, Anahita; Saboury, Ali Akbar; Karimi-Jafari, Mohammad Hossein; Khoobi, Mehdi; Ghasemi, Atiyeh; Yousefinejad, Saeed; Abou-Zied, Osama K

    2017-04-03

    Ligand binding studies on human serum albumin (HSA) are crucial in determining the pharmacological properties of drug candidates. Here, two representatives of coumarin-chalcone hybrids were selected and their binding mechanism was identified via thermodynamics techniques, curve resolution analysis and computational methods at molecular levels. The binding parameters were derived using spectroscopic approaches and the results point to only one pocket located near the Trp214 residue in subdomain IIA of HSA. The protein tertiary structure was altered during ligand binding and formed an intermediate structure to create stronger ligand binding interactions. The best binding mode of the ligand was initially estimated by docking on an ensemble of HSA crystallographic structures and by molecular dynamics (MD) simulations. Per residue interaction energies were calculated over the MD trajectories as well. Reasonable agreement was found between experimental and theoretical results about the nature of binding, which was dominated by hydrogen bonding and van der Waals contributions.

  19. 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.

  20. 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.

  1. An albumin-derived peptide scaffold capable of binding and catalysis.

    PubMed

    Luisi, Immacolata; Pavan, Silvia; Fontanive, Giampaolo; Tossi, Alessandro; Benedetti, Fabio; Savoini, Adriano; Maurizio, Elisa; Sgarra, Riccardo; Sblattero, Daniele; Berti, Federico

    2013-01-01

    We have identified a 101-amino-acid polypeptide derived from the sequence of the IIA binding site of human albumin. The polypeptide contains residues that make contact with IIA ligands in the parent protein, and eight cysteine residues to form disulfide bridges, that stabilize the polypeptide structure. Seventy-four amino acids are located in six α-helical regions, while the remaining thirty-seven amino acids form six connecting coil/loop regions. A soluble GST fusion protein was expressed in E. coli in yields as high as 4 mg/l. This protein retains the IIA fragment's capacity to bind typical ligands such as warfarin and efavirenz and other albumin's functional properties such as aldolase activity and the ability to direct the stereochemical outcome of a diketone reduction. This newly cloned polypeptide thus represents a valuable starting point for the construction of libraries of binders and catalysts with improved proficiency.

  2. Investigation of the interaction between quercetin and human serum albumin by multiple spectra, electrochemical impedance spectra and molecular modeling.

    PubMed

    Dai, Jie; Zou, Ting; Wang, Li; Zhang, Yezhong; Liu, Yi

    2014-12-01

    Quercetin (Qu), a flavonoid compound, exists widely in the human diet and exhibits a variety of pharmacological activities. This work is aimed at studying the effect of Qu on the bioactive protein, human serum albumin (HSA) under simulated biophysical conditions. Multiple spectroscopic methods (including fluorescence and circular dichroism), electrochemical impedance spectra (EIS) and molecular modeling were employed to investigate the interaction between Qu and HSA. The fluorescence quenching and EIS experimental results showed that the fluorescence quenching of HSA was caused by formation of a Qu-HSA complex in the ground state, which belonged to the static quenching mechanism. Based on the calculated thermodynamic parameters, it concluded that the interaction was a spontaneous process and hydrogen bonds combined with van der Waal's forces played a major role in stabilizing the Qu-HSA complex. Molecular modeling results demonstrated that several amino acids participated in the binding process and the formed Qu-HSA complex was stabilized by H-bonding network at site I in sub-domain IIA, which was further confirmed by the site marker competitive experiments. The evidence from circular dichroism (CD) indicated that the secondary structure and microenvironment of HSA were changed. Alterations in the conformation of HSA were observed with a reduction in the amount of α helix from 59.9% (free HSA) to 56% (Qu-HSA complex), indicating a slight unfolding of the protein polypeptides.

  3. Spectroscopic studies of the interaction of anti-coagulant rodenticide diphacinone with human serum albumin

    NASA Astrophysics Data System (ADS)

    Tang, Jianghong; Qi, Shengda; Chen, Xingguo

    2005-11-01

    The interaction of diphacinone with human serum albumin (HSA) was studied by the methods of fluorescence and Fourier transform infrared (FT-IR) spectroscopy under simulative physiological conditions. Diphacinone can strongly quench the intrinsic fluorescence of HSA by static quenching. The apparent binding constants KT at four different temperatures (291, 300, 310, and 318 K) were obtained according to Scatchard procedure. The Stern-Volmer and Scatchard plots both had an intersection at C diphacinone/C HSA≈1.2, which indicated that diphacinone binded to different binding sites on HSA. The analytical results of fluorescence data showed when C diphacinone/C HSA was lower than 1.2, the numbers of binding sites were near 1.1, and C diphacinone/C HSA was higher than 1.2, the number of binding sites was approximately 1.8. The FT-IR spectra had proved that the secondary structure of HSA changed after interacting with diphacinone in aqueous solution. The thermodynamic parameters were calculated by van't Hoff equation. The enthalpy change (Δ H0) and entropy change (Δ S0) were -14.59 kJ mol -1 and 62.49 J mol -1 K -1, respectively. The results suggested that the hydrophobic interaction might play a main role in the interaction of diphacinone with the HSA. The binding distance ( r) between diphacinone and tryptophan in HSA was obtained according to the Förster energy transfer theory. Furthermore, the study of molecular modeling indicated that diphacinone could bind to the site I of HSA and hydrophobic interaction was the major acting force, which was in agreement with the binding mode study.

  4. Improving the pharmacokinetic properties of biologics by fusion to an anti-HSA shark VNAR domain

    PubMed Central

    Müller, Mischa R.; Saunders, Kenneth; Grace, Christopher; Jin, Macy; Piche-Nicholas, Nicole; Steven, John; O’Dwyer, Ronan; Wu, Leeying; Khetemenee, Lam; Vugmeyster, Yulia; Hickling, Timothy P.; Tchistiakova, Lioudmila; Olland, Stephane; Gill, Davinder; Jensen, Allan; Barelle, Caroline J.

    2012-01-01

    Advances in recombinant antibody technology and protein engineering have provided the opportunity to reduce antibodies to their smallest binding domain components and have concomitantly driven the requirement for devising strategies to increase serum half-life to optimise drug exposure, thereby increasing therapeutic efficacy. In this study, we adopted an immunization route to raise picomolar affinity shark immunoglobulin new antigen receptors (IgNARs) to target human serum albumin (HSA). From our model shark species, Squalus acanthias, a phage display library encompassing the variable binding domain of IgNAR (VNAR) was constructed, screened against target, and positive clones were characterized for affinity and specificity. N-terminal and C-terminal molecular fusions of our lead hit in complex with a naïve VNAR domain were expressed, purified and exhibited the retention of high affinity binding to HSA, but also cross-selectivity to mouse, rat and monkey serum albumin both in vitro and in vivo. Furthermore, the naïve VNAR had enhanced pharmacokinetic (PK) characteristics in both N- and C-terminal orientations and when tested as a three domain construct with naïve VNAR flanking the HSA binding domain at both the N and C termini. Molecules derived from this platform technology also demonstrated the potential for clinical utility by being available via the subcutaneous route of delivery. This study thus demonstrates the first in vivo functional efficacy of a VNAR binding domain with the ability to enhance PK properties and support delivery of multifunctional therapies. PMID:23676205

  5. The complexity of condensed tannin binding to bovine serum albumin--An isothermal titration calorimetry study.

    PubMed

    Kilmister, Rachel L; Faulkner, Peta; Downey, Mark O; Darby, Samuel J; Falconer, Robert J

    2016-01-01

    Isothermal titration calorimetry was applied to study the binding of purified proanthocyanidin oligomers to bovine serum albumin (BSA). The molecular weight of the proanthocyanidin oligomer had a major impact on its binding to BSA. The calculated change in enthalpy (ΔH) and association constant (Ka) became greater as the oligomer size increased then plateaued at the heptameric oligomer. These results support a model for precipitation of proteins by proanthocyanidin where increased oligomer size enhanced the opportunity for cross linkages between proteins ultimately forming sediment-able complexes. The authors suggest tannin binding to proteins is opportunistic and involves multiple sites, each with a different Ka and ΔH of binding. The ΔH of binding comprises both an endothermic hydrophobic interaction and exothermic hydrogen bond component. This suggests the calculated entropy value (ΔS) for tannin-protein interactions is subject to a systematic error and should be interpreted with caution.

  6. Stereoselective bile pigment binding to polypeptides and albumins: a circular dichroism study.

    PubMed

    Goncharova, Iryna; Urbanová, Marie

    2008-12-01

    Stereoselective recognition of bilirubin and biliverdin by poly(L-lysine) (PLL), poly(D-lysine) (PDL), and poly(L-arginine) (PLA) and their micelles with dodecanoate ions (C(12)) at different pH has been studied using a combination of vibrational and electronic circular dichroism. Biliverdin has been found to be more sensitive to pH in its complexes with the polypeptides. In acidic media in the complexes with PLL-C(12) and PDL-C(12) the conformation becomes more closed than the characteristic one found at physiological pH. Partial flattening and chiral self-association of bilirubin molecules takes place at higher concentrations with PLL and PDL. For both pigments, inversions of the ECD signals are observed in the systems with PLA at pH > or = 8.5. This study was carried out in order to clarify the role of Lys and Arg residues in pigment binding to serum albumin. The circular dichroism spectra obtained for bilirubin bound to different mammalian serum albumins have been compared with the homology within the IIA principal ligand-binding structural domains. Analysis suggests that the chiroptical properties of the pigment in the complexes with serum albumins depend on the location of Lys and/or Arg at positions 222 and 199 in the binding site.

  7. Chloride channel ClC-5 binds to aspartyl aminopeptidase to regulate renal albumin endocytosis.

    PubMed

    Lee, Aven; Slattery, Craig; Nikolic-Paterson, David J; Hryciw, Deanne H; Wilk, Sherwin; Wilk, Elizabeth; Zhang, Yuan; Valova, Valentina A; Robinson, Phillip J; Kelly, Darren J; Poronnik, Philip

    2015-04-01

    ClC-5 is a chloride/proton exchanger that plays an obligate role in albumin uptake by the renal proximal tubule. ClC-5 forms an endocytic complex with the albumin receptor megalin/cubilin. We have identified a novel ClC-5 binding partner, cytosolic aspartyl aminopeptidase (DNPEP; EC 3.4.11.21), that catalyzes the release of N-terminal aspartate/glutamate residues. The physiological role of DNPEP remains largely unresolved. Mass spectrometric analysis of proteins binding to the glutathione-S-transferase (GST)-ClC-5 C terminus identified DNPEP as an interacting partner. Coimmunoprecipitation confirmed that DNPEP and ClC-5 also associated in cells. Further experiments using purified GST-ClC-5 and His-DNPEP proteins demonstrated that the two proteins bound directly to each other. In opossum kidney (OK) cells, confocal immunofluorescence studies revealed that DNPEP colocalized with albumin-containing endocytic vesicles. Overexpression of wild-type DNPEP increased cell-surface levels of ClC-5 and albumin uptake. Analysis of DNPEP-immunoprecipitated products from rat kidney lysate identified β-actin and tubulin, suggesting a role for DNPEP in cytoskeletal maintenance. A DNase I inhibition assay showed a significant decrease in the amount of G actin when DNPEP was overexpressed in OK cells, suggesting a role for DNPEP in stabilizing the cytoskeleton. DNPEP was not present in the urine of healthy rats; however, it was readily detected in the urine in rat models of mild and heavy proteinuria (diabetic nephropathy and anti-glomerular basement membrane disease, respectively). Urinary levels of DNPEP were found to correlate with the severity of proteinuria. Therefore, we have identified another key molecular component of the albumin endocytic machinery in the renal proximal tubule and describe a new role for DNPEP in stabilizing the actin cytoskeleton.

  8. Probing the interaction of thionine with human serum albumin by multispectroscopic studies and its in vitro cytotoxic activity toward MCF-7 breast cancer cells.

    PubMed

    Manivel, Perumal; Paulpandi, Manickam; Murugan, Kadarkarai; Benelli, Giovanni; Ilanchelian, Malaichamy

    2016-10-27

    The studies on protein-dye interactions are important in biological process and it is regarded as vital step in rational drug design. The interaction of thionine (TH) with human serum albumin (HSA) was analyzed using isothermal titration calorimetry (ITC), spectroscopic, and molecular docking technique. The emission spectral titration of HSA with TH revealed the formation of HSA-TH complex via static quenching process. The results obtained from absorption, synchronous emission, circular dichroism, and three-dimensional (3D) emission spectral studies demonstrated that TH induces changes in the microenvironment and secondary structure of HSA. Results from ITC experiments suggested that the binding of TH dye was favored by negative enthalpy and a favorable entropy contribution. Site marker competitive binding experiments revealed that the binding site of TH was located in subdomain IIA (Sudlow site I) of HSA. Molecular docking study further substantiates that TH binds to the hydrophobic cavity of subdomain IIA (Sudlow site I) of HSA. Further, we have studied the cytotoxic activity of TH and TH-HSA complex on breast cancer cell lines (MCF-7) by MTT assay and LDH assay. These studies revealed that TH-HSA complex showed the higher level of cytotoxicity in cancer cells than TH dye-treated MCF-7 cells and the significant adverse effect did not found in the normal HBL-100 cells. Fluorescence microscopy analyses of nuclear fragmentation studies validate the significant reduction of viability of TH-HSA-treated human MCF-7 breast cancer cells through activation of apoptotic-mediated pathways.

  9. 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.

  10. The effect of Cu 2+ on the interaction between an antitumor drug-mitoxantrone and human serum albumin

    NASA Astrophysics Data System (ADS)

    Tian, Ming-Yue; Zhang, Xiu-Feng; Xie, Ling; Xiang, Jun-Feng; Tang, Ya-Lin; Zhao, Chang-Qi

    2008-12-01

    The studies on the interaction between HSA and drugs have been an interesting research field in life sciences, chemistry and clinical medicine. There are also many metal ions present in blood plasma, thus the research about the effect of metal ions on the interaction between drugs and plasma proteins is crucial. In this study, we have investigated the effect of a familiar metal ion-Cu 2+ on the interaction between an antitumor drug-mitoxantrone (MTO) and human serum albumin (HSA) by using fluorescence spectroscopy, ultraviolet-visible absorption spectroscopy and circular dichroism spectroscopy, for the first time. The results showed that the quenching efficiency of MTO to HSA is higher with Cu 2+ than that without Cu 2+. In the presence of Cu 2+, the secondary structure of HSA was changed and the α-helix content was increased. The apparent association constant ( KA), the binding sites ( n) and the spatial-distance ( r) between MTO and HSA decreased. These results indicated that Cu 2+ could affect the interaction between MTO and HSA by altering HSA molecular conformation. Further calculation indicated that the binding mode of Cu 2+ in MTO-HSA system was likely to form Cu 2+-HSA complex.

  11. Comparative Studies of Interactions between Fluorodihydroquinazolin Derivatives and Human Serum Albumin with Fluorescence Spectroscopy.

    PubMed

    Wang, Yi; Zhu, Meiqing; Liu, Feng; Wu, Xiangwei; Pan, Dandan; Liu, Jia; Fan, Shisuo; Wang, Zhen; Tang, Jun; Na, Risong; Li, Qing X; Hua, Rimao; Liu, Shangzhong

    2016-10-14

    In the present study, 3-(fluorobenzylideneamino)-6-chloro-1-(3,3-dimethylbutanoyl)-phenyl-2,3-dihydroquinazolin-4(1H)-one (FDQL) derivatives have been designed and synthesized to study the interaction between fluorine substituted dihydroquinazoline derivatives with human serum albumin (HSA) using fluorescence, circular dichroism and Fourier transform infrared spectroscopy. The results indicated that the FDQL could bind to HSA, induce conformation and the secondary structure changes of HSA, and quench the intrinsic fluorescence of HSA through a static quenching mechanism. The thermodynamic parameters, ΔH, ΔS, and ΔG, calculated at different temperatures, revealed that the binding was through spontaneous and hydrophobic forces and thus played major roles in the association. Based on the number of binding sites, it was considered that one molecule of FDQL could bind to a single site of HSA. Site marker competition experiments indicated that the reactive site of HSA to FDQL mainly located in site II (subdomain IIIA). The substitution by fluorine in the benzene ring could increase the interactions between FDQL and HSA to some extent in the proper temperature range through hydrophobic effect, and the substitution at meta-position enhanced the affinity greater than that at para- and ortho-positions.

  12. Extending serum half-life of albumin by engineering neonatal Fc receptor (FcRn) binding.

    PubMed

    Andersen, Jan Terje; Dalhus, Bjørn; Viuff, Dorthe; Ravn, Birgitte Thue; Gunnarsen, Kristin Støen; Plumridge, Andrew; Bunting, Karen; Antunes, Filipa; Williamson, Rebecca; Athwal, Steven; Allan, Elizabeth; Evans, Leslie; Bjørås, Magnar; Kjærulff, Søren; Sleep, Darrell; Sandlie, Inger; Cameron, Jason

    2014-05-09

    A major challenge for the therapeutic use of many peptides and proteins is their short circulatory half-life. Albumin has an extended serum half-life of 3 weeks because of its size and FcRn-mediated recycling that prevents intracellular degradation, properties shared with IgG antibodies. Engineering the strictly pH-dependent IgG-FcRn interaction is known to extend IgG half-life. However, this principle has not been extensively explored for albumin. We have engineered human albumin by introducing single point mutations in the C-terminal end that generated a panel of variants with greatly improved affinities for FcRn. One variant (K573P) with 12-fold improved affinity showed extended serum half-life in normal mice, mice transgenic for human FcRn, and cynomolgus monkeys. Importantly, favorable binding to FcRn was maintained when a single-chain fragment variable antibody was genetically fused to either the N- or the C-terminal end. The engineered albumin variants may be attractive for improving the serum half-life of biopharmaceuticals.

  13. Preferential binding of fisetin to the native state of bovine serum albumin: spectroscopic and docking studies.

    PubMed

    Singha Roy, Atanu; Pandey, Nitin Kumar; Dasgupta, Swagata

    2013-04-01

    We have investigated the binding of the biologically important flavonoid fisetin with the carrier protein bovine serum albumin using multi-spectroscopic and molecular docking methods. The binding constants were found to be in the order of 10(4) M(-1) and the number of binding sites was determined as one. MALDI-TOF analyses showed that one fisetin molecule binds to a single bovine serum albumin (BSA) molecule which is also supported by fluorescence quenching studies. The negative Gibbs free energy change (∆G°) values point to a spontaneous binding process which occurs through the presence of electrostatic forces with hydrophobic association that results in a positive entropy change (+51.69 ± 1.18 J mol(-1) K(-1)). The unfolding and refolding of BSA in urea have been studied in absence and presence of fisetin using steady-state fluorescence and lifetime measurements. Urea denaturation studies indicate that fisetin is gradually released from its binding site on the protein. In the absence of urea, an increase in temperature that causes denaturation of the protein results in the release of fisetin from its bound state indicating that fisetin binds only to the native state of the protein. The circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopic studies showed an increase in % α-helix content of BSA after binding with fisetin. Site marker displacement studies in accordance with the molecular docking results suggested that fisetin binds in close proximity of the hydrophobic cavity in site 1 (subdomain IIA) of the protein. The PEARLS (Program of Energetic Analysis of Receptor Ligand System) has been used to estimate the interaction energy of fisetin with BSA and the results are in good correlation with the experimental findings.

  14. Analysis of binding ability of two tetramethylpyridylporphyrins to albumin and its complex with bilirubin

    NASA Astrophysics Data System (ADS)

    Solomonov, Alexey V.; Shipitsyna, Maria K.; Vashurin, Arthur S.; Rumyantsev, Evgeniy V.; Timin, Alexander S.; Ivanov, Sergey P.

    2016-11-01

    An interaction between 5,10,15,20-tetrakis-(N-methyl-x-pyridyl)porphyrins, x = 2; 4 (TMPyPs) with bovine serum albumin (BSA) and its bilirubin (BR) complex was investigated by UV-Viz and fluorescence spectroscopy under imitated physiological conditions involving molecular docking studies. The parameters of forming intermolecular complexes (binding constants, quenching rate constants, quenching sphere radius etc.) were determined. It was showed that the interaction between proteins and TMPyPs occurs via static quenching of protein fluorescence and has predominantly hydrophobic and electrostatic character. It was revealed that obtained complexes are relatively stable, but in the case of TMPyP4 binding with proteins occurs better than TMPyP2. Nevertheless, both TMPyPs have better binding ability with free protein compared to BRBSA at the same time. The influence of TMPyPs on the conformational changes in protein molecules was studied using synchronous fluorescence spectroscopy. It was found that there is no competition of BR with TMPyPs for binging sites on protein molecule and BR displacement does not occur. Molecular docking calculations have showed that TMPyPs can bind with albumin via tryptophan residue in the hydrophilic binding site of protein molecule but it is not one possible interaction way.

  15. Interactions of thioflavin T with serum albumins: Spectroscopic analyses

    NASA Astrophysics Data System (ADS)

    Sen, Priyankar; Fatima, Sadaf; Ahmad, Basir; Khan, Rizwan Hasan

    2009-09-01

    The interaction of thioflavin T (ThT) with serum albumins from four different mammalian species i.e. human, bovine, porcine and rabbit, has been investigated by circular dichroism (CD), fluorescence spectroscopy and ITC. The binding constant ( K) for HSA was found to be 9.9 × 10 4 M -1, 4.3 × 10 4 M -1 for RSA, 1.07 × 10 4 M -1 for PSA and 0.3 × 10 4 M -1 for BSA and the number of binding sites ( n) were 1.14, 1.06, 0.94 and 0.8, respectively, which is very significant. By using unfolding pathway of HSA in the presence of urea, domain II of HSA has been assigned to possess binding site of ThT. Its binding constant is comparable to many drugs that bind at domain II of HSA, like salicylate, warfarin, digitoxin, etc. Acting force between HSA and ThT is showing that both hydrophobic and electrostatic forces have contributed for the interaction. Δ Gbinding, Δ H and Δ S were calculated to be -28.46 kJ mol -1, -3.50 kJ mol -1 and 81.04 J K -1 mol -1, respectively. The data described here will help to increase our understanding about the interaction of ThT with native proteins. The results also indicate that care must be taken while using ThT as a probe for detecting amyloid fibrils.

  16. Chlorpromazine interactions to sera albumins. A study by the quenching of fluorescence

    NASA Astrophysics Data System (ADS)

    Silva, Dilson; Cortez, Célia M.; Louro, Sônia R. W.

    2004-04-01

    Binding of chlorpromazine (CPZ) and hemin (Hmn) to human (HSA) and bovine (BSA) serum albumin was studied by fluorescence quenching technique. Intrinsic fluorescences of BSA and HSA were measured by selectively exciting their tryptophan residues. Gradual quenching was observed by titration of both proteins with CPZ and Hmn. CPZ is a widely used anti-psychosis drug that causes severe side effects and strongly interacts with biomembranes, both in its lipidic and proteic regions. CPZ also interacts with blood components, influences bioavailability, and affects the function of several biomolecules. Albumin plays an important role in the transport and storage of hormones, ions, fatty acids and others substances, including CPZ, affecting the regulation of their plasmatic concentration. Hmn is an important ferric residue of hemoglobin that binds within the hydrophobic region of albumin with great specificity. Hmn added to HSA and BSA solutions at a molar ratio of 1:1 quenched about half of their fluorescence. Stern-Volmer plots obtained from experiments carried out at 25 and 35 °C showed the quenching of fluorescence of HSA and BSA by CPZ to be a collisional phenomenon. Hmn quenches fluorescence by a static process, which specifically indicates the formation of a complex. Our results suggest the prime binding site for CPZ and Hmn on both HSA and BSA to be near tryptophan residues.

  17. Circular dichroism study of the interaction between mutagens and bilirubin bound to different binding sites of serum albumins

    NASA Astrophysics Data System (ADS)

    Orlov, Sergey; Goncharova, Iryna; Urbanová, Marie

    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.

  18. 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.

  19. Modulation of the reactivity of the thiol of human serum albumin and its sulfenic derivative by fatty acids

    PubMed Central

    Torres, María José; Turell, Lucía; Botti, Horacio; Antmann, Laura; Carballal, Sebastián; Ferrer-Sueta, Gerardo; Radi, Rafael; Alvarez, Beatriz

    2012-01-01

    The single cysteine residue of human serum albumin (HSA-SH) is the most abundant plasma thiol. HSA transports fatty acids (FA), a cargo that increases under conditions of diabetes, exercise or adrenergic stimulation. The stearic acid-HSA (5/1) complex reacted 6-fold faster at pH 7.4 with the disulfide 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and 2-fold faster with hydrogen peroxide and peroxynitrite. The apparent pKa of HSA-SH decreased from 7.9 ± 0.1 to 7.4 ± 0.1. Exposure to H2O2 (2 mM, 5 min, 37 °C) yielded 0.29 ± 0.04 moles of sulfenic acid (HSA-SOH) per mole of FA-bound HSA. The reactivity of HSA-SOH with low molecular weight thiols increased ~3-fold in the presence of FA. The enhanced reactivity of the albumin thiol at neutral pH upon FA binding can be rationalized by considering that the corresponding conformational changes that increase thiol exposure both increase the availability of the thiolate due to a lower apparent pKa and also loosen steric constraints for reactions. Since situations that increase circulating FA are associated with oxidative stress, this increased reactivity of HSA-SH could assist in oxidant removal. PMID:22450170

  20. Neo-epitopes on methylglyoxal modified human serum albumin lead to aggressive autoimmune response in diabetes.

    PubMed

    Jyoti; Mir, Abdul Rouf; Habib, Safia; Siddiqui, Sheelu Shafiq; Ali, Asif; Moinuddin

    2016-05-01

    Glyco-oxidation of proteins has implications in the progression of diabetes type 2. Human serum albumin is prone to glyco-oxidative attack by sugars and methylglyoxal being a strong glycating agent may have severe impact on its structure and consequent role in diabetes. This study has probed the methylglyoxal mediated modifications of HSA, the alterations in its immunological characteristics and possible role in autoantibody induction. We observed an exposure of chromophoric groups, loss in the fluorescence intensity, generation of AGEs, formation of cross-linked products, decrease in α-helical content, increase in hydrophobic clusters, FTIR band shift, attachment of methylglyoxal to HSA and the formation of N(ε)-(carboxyethyl) lysine in the modified HSA, when compared to the native albumin. MG-HSA was found to be highly immunogenic with additional immunogenicity invoking a highly specific immune response than its native counterpart. The binding characteristics of circulating autoantibodies in type 2 diabetes mellitus (DM) patients showed the generation of anti-MG-HSA auto-antibodies in the these patients, that are preferentially recognized by the modified albumin. We propose that MG induced structural perturbations in HSA, result in the generation of neo-epitopes leading to an aggressive auto-immune response and may contribute to the immunopathogenesis of diabetes type 2 associated complications.

  1. Thiolated human serum albumin cross-linked dextran hydrogels as a macroscale delivery system.

    PubMed

    Gao, Yue; Kieltyka, Roxanne E; Jesse, Wim; Norder, Ben; Korobko, Alexander V; Kros, Alexander

    2014-07-21

    Hydrogels play an important role in macroscale delivery systems by enabling the transport of cells and molecules. Here we present a facile and benign method to prepare a dextran-based hydrogel (Dex-sHSA) using human serum albumin (HSA) as a simultaneous drug carrier and covalent cross-linker. Drug binding affinity of the albumin protein was conserved in the thiolation step using 2-iminothiolane and subsequently, in the in situ gelation step. Oscillation rheometry studies confirmed the formation of a three-dimensional viscoelastic network upon reaction of dextran and the HSA protein. The mechanical properties of Dex-sHSA hydrogel can be tuned by the protein concentration, and the degree of thiolation of sHSA. Sustained release of hydrophobic drugs, such as ibuprofen, paclitaxel and dexamethasone, from the Dex-sHSA network was shown over one week. Hence, this albumin-based dextran hydrogel system demonstrates its potential as a macroscale delivery system of hydrophobic therapeutics for a wide range of biomedical applications.

  2. 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

  3. Modeling the accessibility of the interaction of clonazepan to albumins

    NASA Astrophysics Data System (ADS)

    Valdez, Ethel Celene Narvaez; Paulino, Erica Tex; de Morais e Coura, Carla Patrícia; Cortez, Celia Martins; da Silva Fragoso, Viviane Muniz

    2016-12-01

    This paper shows results obtained from the clonazepam (CNZP) interaction with human and bovine serum albumin study, seeking data on the pharmacokinetics and the binding site for the anxiolytic by comparing the responses of these two proteins to this drug. The quenching response of this experiment show a huge interaction between CNZP and the albumins, that confirm the literature information relative to the high affinity of CNZP with the plasma protein, a long plasma half-life and that the single binding site for this drug can be found in or close to subdomain IB of HSA and BSA.

  4. Environment sensitive fluorescent analogue of biologically active oxazoles differentially recognizes human serum albumin and bovine serum albumin: Photophysical and molecular modeling studies.

    PubMed

    Maiti, Jyotirmay; Biswas, Suman; Chaudhuri, Ankur; Chakraborty, Sandipan; Chakraborty, Sibani; Das, Ranjan

    2017-03-15

    An environment sensitive fluorophore, 4-(5-(4-(dimethylamino)phenyl)oxazol-2-yl)benzoic acid (DMOBA), that closely mimics biologically active 2,5-disubstituited oxazoles has been designed to probe two homologous serum proteins, human serum albumin (HSA) and bovine serum albumin (BSA) by means of photophysical and molecular modeling studies. This fluorescent analogue exhibits solvent polarity sensitive fluorescence due to an intramolecular charge transfer in the excited state. In comparison to water, the steady state emission spectra of DMOBA in BSA is characterized by a greater blue shift (~10nm) and smaller Stokes' shift (~5980cm(-1)) in BSA than HSA (Stokes'shift~6600cm(-1)), indicating less polar and more hydrophobic environment of the dye in the former than the latter. The dye-protein binding interactions are remarkably stronger for BSA than HSA which is evident from higher value of the association constant for the DMOBA-BSA complex (Ka~5.2×10(6)M(-1)) than the DMOBA-HSA complex (Ka~1.0×10(6)M(-1)). Fӧrster resonance energy transfer studies revealed remarkably less efficient energy transfer (8%) between the donor tryptophans in BSA and the acceptor DMOBA dye than that (30%) between the single tryptophan moiety in HSA and the dye, which is consistent with a much larger distance between the donor (tryptophan)-acceptor (dye) pair in BSA (34.5Å) than HSA (25.4Å). Site specific competitive binding assays have confirmed on the location of the dye in Sudlow's site II of BSA and in Sudlow's site I of HSA, respectively. Molecular modeling studies have shown that the fluorescent analogue is tightly packed in the binding site of BSA due to strong steric complementarity, where, binding of DMOBA to BSA is primarily dictated by the van der Waals and hydrogen bonding interactions. In contrast, in HSA the steric complementarity is less significant and binding is primarily guided by polar interactions and van der Waals interactions appear to be less significant in the

  5. Environment sensitive fluorescent analogue of biologically active oxazoles differentially recognizes human serum albumin and bovine serum albumin: Photophysical and molecular modeling studies

    NASA Astrophysics Data System (ADS)

    Maiti, Jyotirmay; Biswas, Suman; Chaudhuri, Ankur; Chakraborty, Sandipan; Chakraborty, Sibani; Das, Ranjan

    2017-03-01

    An environment sensitive fluorophore, 4-(5-(4-(dimethylamino)phenyl)oxazol-2-yl)benzoic acid (DMOBA), that closely mimics biologically active 2,5-disubstituited oxazoles has been designed to probe two homologous serum proteins, human serum albumin (HSA) and bovine serum albumin (BSA) by means of photophysical and molecular modeling studies. This fluorescent analogue exhibits solvent polarity sensitive fluorescence due to an intramolecular charge transfer in the excited state. In comparison to water, the steady state emission spectra of DMOBA in BSA is characterized by a greater blue shift ( 10 nm) and smaller Stokes' shift ( 5980 cm- 1) in BSA than HSA (Stokes'shift 6600 cm- 1), indicating less polar and more hydrophobic environment of the dye in the former than the latter. The dye-protein binding interactions are remarkably stronger for BSA than HSA which is evident from higher value of the association constant for the DMOBA-BSA complex (Ka 5.2 × 106 M- 1) than the DMOBA-HSA complex (Ka 1.0 × 106 M- 1). Fӧrster resonance energy transfer studies revealed remarkably less efficient energy transfer (8%) between the donor tryptophans in BSA and the acceptor DMOBA dye than that (30%) between the single tryptophan moiety in HSA and the dye, which is consistent with a much larger distance between the donor (tryptophan)-acceptor (dye) pair in BSA (34.5 Å) than HSA (25.4 Å). Site specific competitive binding assays have confirmed on the location of the dye in Sudlow's site II of BSA and in Sudlow's site I of HSA, respectively. Molecular modeling studies have shown that the fluorescent analogue is tightly packed in the binding site of BSA due to strong steric complementarity, where, binding of DMOBA to BSA is primarily dictated by the van der Waals and hydrogen bonding interactions. In contrast, in HSA the steric complementarity is less significant and binding is primarily guided by polar interactions and van der Waals interactions appear to be less significant in the

  6. Binding of ring-substituted indole-3-acetic acids to human serum albumin.

    PubMed

    Soskić, Milan; Magnus, Volker

    2007-07-01

    The plant hormone, indole-3-acetic acid (IAA), and its ring-substituted derivatives have recently attracted attention as promising pro-drugs in cancer therapy. Here we present relative binding constants to human serum albumin for IAA and 34 of its derivatives, as obtained using the immobilized protein bound to a support suitable for high-performance liquid chromatography. We also report their octanol-water partition coefficients (logK(ow)) computed from retention data on a C(18) coated silica gel column. A four-parameter QSPR (quantitative structure-property relationships) model, based on physico-chemical properties, is put forward, which accounts for more than 96% of the variations in the binding affinities of these compounds. The model confirms the importance of lipophilicity as a global parameter governing interaction with serum albumin, but also assigns significant roles to parameters specifically related to the molecular topology of ring-substituted IAAs. Bulky substituents at ring-position 6 increase affinity, those at position 2 obstruct binding, while no steric effects were noted at other ring-positions. Electron-withdrawing substituents at position 5 enhance binding, but have no obvious effect at other ring positions.

  7. 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.

  8. Molecular interaction investigation between three CdTe:Zn(2+) quantum dots and human serum albumin: A comparative study.

    PubMed

    Huang, Shan; Qiu, Hangna; Liu, Yi; Huang, Chusheng; Sheng, Jiarong; Su, Wei; Xiao, Qi

    2015-12-01

    Water-soluble Zn-doped CdTe quantum dots (CdTe:Zn(2+) QDs) have attracted great attention in biological and biomedical applications. In particular, for any potential in vivo application, the interaction of CdTe:Zn(2+) QDs with human serum albumin (HSA) is of greatest importance. As a step toward the elucidation of the fate of CdTe:Zn(2+) QDs introduced to organism, the molecular interactions between CdTe:Zn(2+) QDs with three different sizes and HSA were systematically investigated by spectroscopic techniques. Three CdTe:Zn(2+) QDs with maximum emission of 514 nm (green QDs, GQDs), 578 nm (yellow QDs, YQDs), and 640 nm (red QDs, RQDs) were tested. The binding of CdTe:Zn(2+) QDs with HSA was a result of the formation of HSA-QDs complex and electrostatic interactions played major roles in stabilizing the complex. The Stern-Volmer quenching constant, associative binding constant, and corresponding thermodynamic parameters were calculated. The site-specific probe competitive experiments revealed that the binding location of CdTe:Zn(2+) QDs with HSA was around site I. The microenvironmental and conformational changes of HSA induced by CdTe:Zn(2+) QDs were analyzed. These results suggested that the conformational change of HSA was dramatically at secondary structure level and the biological activity of HSA was weakened in the present of CdTe:Zn(2+) QDs with bigger size.

  9. Toxic effects of chrysoidine on human serum albumin: isothermal titration calorimetry and spectroscopic investigations.

    PubMed

    Sun, Haoyu; Liu, Yingxue; Li, Meng; Han, Songlin; Yang, Xudan; Liu, Rutao

    2016-03-01

    Chrysoidine is widely used in industry as a type of azo dye, and is sometimes used illegally as a food additive despite its potential toxicity. Human serum albumin (HSA) is one of the most important proteins in blood plasma and possesses major physiological functions. In the present study, the conformational and functional effects of chrysoidine on HSA were investigated by isothermal titration calorimetry (ITC), multiple spectroscopic methods, a molecular docking study and an esterase activity assay. Based on the ITC results, the binding stoichiometry of chrysoidine to HSA was estimated to be 1.5:1, and was a spontaneous process via a single hydrogen bond. The binding of chrysoidine to HSA induced dynamic quenching in fluorescence, and changes in secondary structure and in the microenvironment of the Trp-214 residue. In addition, the hydrogen bond (1.80 Å) formed between the chrysoidine molecule and the Gln-211 residue. The esterase activity of HSA decreased following the addition chrysoidine due to the change in protein structure. This study details the direct interaction between chrysoidine and HSA at the molecular level and the mechanism for toxicity as a result of the functional changes induced by HSA structural variation upon binding to chrysoidine in vitro. This study provides useful information towards detailing the transportation mechanism and toxicity of chrysoidine in vivo.

  10. New Perspectives in the Renin-Angiotensin-Aldosterone System (RAAS) II: Albumin Suppresses Angiotensin Converting Enzyme (ACE) Activity in Human

    PubMed Central

    Fagyas, Miklós; Úri, Katalin; Siket, Ivetta M.; Fülöp, Gábor Á.; Csató, Viktória; Daragó, Andrea; Boczán, Judit; Bányai, Emese; Szentkirályi, István Elek; Maros, Tamás Miklós; Szerafin, Tamás; Édes, István; Papp, Zoltán; Tóth, Attila

    2014-01-01

    About 8% of the adult population is taking angiotensin-converting enzyme (ACE) inhibitors to treat cardiovascular disease including hypertension, myocardial infarction and heart failure. These drugs decrease mortality by up to one-fifth in these patients. We and others have reported previously that endogenous inhibitory substances suppress serum ACE activity, in vivo, similarly to the ACE inhibitor drugs. Here we have made an effort to identify this endogenous ACE inhibitor substance. ACE was crosslinked with interacting proteins in human sera. The crosslinked products were immunoprecipitated and subjected to Western blot. One of the crosslinked products was recognized by both anti-ACE and anti-HSA (human serum albumin) antibodies. Direct ACE-HSA interaction was confirmed by binding assays using purified ACE and HSA. HSA inhibited human purified (circulating) and human recombinant ACE with potencies (IC50) of 5.7±0.7 and 9.5±1.1 mg/mL, respectively. Effects of HSA on the tissue bound native ACE were tested on human saphenous vein samples. Angiotensin I evoked vasoconstriction was inhibited by HSA in this vascular tissue (maximal force with HSA: 6.14±1.34 mN, without HSA: 13.54±2.63 mN), while HSA was without effects on angiotensin II mediated constrictions (maximal force with HSA: 18.73±2.17 mN, without HSA: 19.22±3.50 mN). The main finding of this study is that HSA was identified as a potent physiological inhibitor of the ACE. The enzymatic activity of ACE appears to be almost completely suppressed by HSA when it is present in its physiological concentration. These data suggest that angiotensin I conversion is limited by low physiological ACE activities, in vivo. PMID:24691203

  11. Molecular hybridization approach of bio-potent Cu(II)/Zn(II) complexes derived from N, O donor bidentate imine scaffolds: Synthesis, spectral, human serum albumin binding, antioxidant and antibacterial studies.

    PubMed

    Shakir, Mohammad; Hanif, Summaiya; Alam, Md Fazle; Younus, Hina

    2016-12-01

    Novel bio-relevant monometallic Schiff base complexes of the type, [Cu(L(1))2] (1), [Zn(L(1))2]·2H2O (2), [Cu(L(2))2]·2H2O (3) and [Zn(L(2))2]·H2O (4) [L(1)(E)-3-(((3-chloro-4-hydroxyphenyl)imino)methyl)naphthalen-2-ol and L(2)(E)-2-chloro-4-((1-(5-chloro-2-hydroxyphenyl)ethylidene)amino)phenol] were synthesized and characterized. A comparative account of analytical, spectroscopic (FT-IR, (1)H and (13)C NMR, Mass, UV-vis and EPR), thermal (TGA/DTA), XRD and SEM studies revealed a correlation between the structure and function of these biologically active molecular entities. HSA (Human serum albumin) binding profiles of the metal complexes (1-4) were monitored using biophysical techniques viz., absorbance, fluorescence, circular dichromism (CD) and foster resonance energy transfer (FRET). The intrinsic binding constant (Kb) demonstrated substantial binding propensity of L(1) linked complexes (1 and 2) in comparison to L(2) complexes (3 and 4) suggesting L(1) to be more bio-active pharmacophore due to higher planarity and conjugation as compared to L(2) ligand. The outcome of fluorescence study revealed static quenching mechanism on the basis of the quenching of HSA by the complexes (1-4). However, modifications in the secondary structure of HSA by complexes (1-4) inferred via CD measurements which revealed the enhancement of α-helicity (67.47% to 69.20%) with the preference order of 1>2>3>4. Furthermore, in-vitro antibacterial study against different bacteria and antioxidant activities against DPPH and superoxide radical (O2(-)) at variable concentrations outspread discernible bio-potencies of the metal complexes as compared to free ligand scaffolds due to the chelation effect.

  12. Investigations on the interactions between naphthalimide-based anti-tumor drugs and human serum albumin by spectroscopic and molecular modeling methods.

    PubMed

    Cheng, Huiyuan; Zou, Ting; Xu, Yongliang; Wang, Ying; Wu, Aibin; Dai, Jie; Zhang, Yezhong; Liu, Yi

    2016-02-01

    The interactions between the three kinds of naphthalimide-based anti-tumor drugs (NADA, NADB, NADC) and human serum albumin (HSA) under simulated physiological conditions were investigated by fluorescence spectroscopy, circular dichroism spectroscopy and molecular modeling. The results of the fluorescence quenching spectroscopy showed that the quenching mechanisms for different drugs were static and their affinity was in a descending order of NADA > NADB > NADC. The relative thermodynamic parameters indicated that hydrophobic force was the predominant intermolecular force in the binding of NAD to HSA, while van der Waals interactions and hydrogen bonds could not be ignored. The results of site marker competitive experiment confirmed that the binding site of HSA primarily took place in site I. Furthermore, the molecular modeling study was consistent with these results. The study of circular dichroism spectra demonstrated that the presence of NADs decreased the α-helical content of HSA and induced the change of the secondary structure of HSA.

  13. New insight into the binding interaction of hydroxylated carbon nanotubes with bovine serum albumin.

    PubMed

    Guan, Yonghui; Zhang, Hongmei; Wang, Yanqing

    2014-04-24

    In order to understand the effects of carbon nanotubes on the structural stability of proteins, the ligand-binding ability, fibrillation, and chemical denaturation of bovine serum albumin in the presence of a multi-walled hydroxylated carbon nanotubes (HO-MWCNTs) was characterized by UV-vis, circular dichroism, fluorescence spectroscopy and molecule modeling methods at the molecular level. The experiment results indicated that the fluorescence intensity of BSA was decreased obviously in presence of HO-MWCNTs. The binding interaction of HO-MWCNTs with BSA led to the secondary structure changes of BSA. This interaction could not only affect the ligand-binding ability of BSA, but also change the rate of fibrillation and denaturation of BSA. This work gave us some important information about the structures and properties of protein induced by carbon nanotubes.

  14. Assessment of the europium(III) binding sites on albumin using fluorescence spectroscopy.

    PubMed

    Tikhonova, Tatiana N; Shirshin, Evgeny A; Budylin, Gleb S; Fadeev, Victor V; Petrova, Galina P

    2014-06-19

    Intrinsic fluorescence quenching of bovine serum albumin (BSA) and europium(III) luminescence in BSA complexes were investigated. The number of BSA binding sites (n) and equilibrium constant (Keq) values were determined from both measurements provided qualitatively different results. While the modified Stern-Volmer relation for BSA fluorescence quenching gave n = 1 at pH 4.5 and pH 6, two sets of binding sites were determined from Eu(3+) luminescence with n1 = 2, n2 = 4 at pH 6 and n1 = 1, n2 = 2 at pH 4.5. The model explaining the discrepancy between the results obtained by these fluorescent approaches was suggested, and the limitations in application of the "log-log" Stern-Volmer plots in analysis of binding processes were discussed.

  15. Interaction of 5-fluoro-5'-deoxyuridine with human serum albumin under physiological and non-physiological condition: a biophysical investigation.

    PubMed

    Ishtikhar, Mohd; Rabbani, Gulam; Khan, Rizwan Hasan

    2014-11-01

    Uridine analogs 5'dFUrd (a cytotoxic metabolite of a prodrug capecitabine that enzymatically converted into 5'dFUrd) commonly used in the treatment of advanced human cancers, especially gastrointestinal tract, ovary, colorectal, breast cancers etc. Drugs/metabolites are transported in the blood by transporter proteins like human serum albumin (HSA). Here we investigate the interaction of 5'dFUrd to HSA by spectroscopic and calorimetric techniques at physiological (pH 7.4) and non-physiological (pH 9.0) conditions. The binding constant (Kb), enthalpy change (ΔH°), entropy change (ΔS°) and Gibbs free energy change (ΔG°) were also calculated under both conditions. The secondary structure of HSA showed greater alteration in helicity at physiological pH. ITC measurement reveals that HSA have high binding affinity at physiological pH as compares to non-physiological conditions. The thermostability of HSA alone as well of the HSA-drug complex was found to be higher at physiological pH. The binding study was also explored through molecular docking studies which revealed that 5'dFUrd was bound to subdomain IIA of Sudlow's site I through multiple mode of interaction. These results suggest that 5'dFUrd have high binding affinity at physiological condition or "N" isoform so lower drug concentrations are required in compare to non-physiological or "B" isoform of HSA to completely occupied the binding site of the protein.

  16. A spectroscopic investigations of anticancer drugs binding to bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Bakkialakshmi, S.; Chandrakala, D.

    2012-03-01

    The binding of anticancer drugs (i) Uracil (U), (ii) 5-Fluorouracil (5FU) and (iii) 5-Chlorouracil (5ClU), to bovine serum albumin (BSA) at two levels of temperature was studied by the fluorescence of quenching method. UV/Vis, time-resolved fluorescence, Fourier transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance (1H NMR) and scanning electron microscope (SEM) analyses were also made. Binding constants (Ka) and binding sites (n) at various levels of temperature were calculated. The obtained binding sites were found to be equal to one for all the three quenchers (U, 5FU and 5ClU) at two different temperature levels. Thermodynamic parameters ΔH, ΔG and ΔS have been calculated and were presented in tables. Change in FTIR absorption intensity shows strong binding of anticancer drugs to BSA. Changes in chemical shifts of NMR and fluorescence lifetimes of the drugs indicate the presence of interaction and binding of BSA to anticancer drugs. 1H NMR spectra and SEM photographs also conform this binding.

  17. Relations between high-affinity binding sites for L-tryptophan, diazepam, salicylate and Phenol Red on human serum albumin.

    PubMed Central

    Kragh-Hansen, U

    1983-01-01

    Binding of L-tryptophan, diazepam, salicylate and Phenol Red to defatted human serum albumin was studied by ultrafiltration at pH 7.0. All ligands bind to one high-affinity binding site with association constants of the order of 10(4)-10(5)M-1. The number of secondary binding sites was found to vary from zero to five, with association constants about 10(3)M-1. Competitive binding studies with different pairs of the ligands were performed. Binding of both ligands was determined simultaneously. L-Tryptophan and diazepam were found to compete for a common high-affinity binding site on albumin. The following combinations of ligands do not bind competitively to albumin: L-tryptophan-Phenol Red, L-tryptophan-salicylate and Phenol Red-salicylate. On the other hand, high-affinity bindings of the three ligands do not take place independently but in such a way that binding of one of the ligands results in a decrease in binding of the other ligands. The decreases in binding are reciprocal and can be accounted for by introducing a coupling constant. The magnitude of the constant is dependent on the ligands being bound. In the present study, the mutual decrease in binding was more pronounced with L-tryptophan-salicylate and Phenol Red-salicylate than with L-tryptophan-Phenol Red. PMID:6847607

  18. Reversible binding of tolmetin, zomepirac, and their glucuronide conjugates to human serum albumin and plasma.

    PubMed

    Ojingwa, J C; Spahn-Langguth, H; Benet, L Z

    1994-02-01

    Acyl glucuronides of drugs and bilirubin have been shown in the past decade to be reactive metabolites undergoing acyl migration and irreversible binding. The latter reaction has been hypothesized to be facilitated by or to proceed through the formation of a reversible complex. Furthermore, it has been suggested that the decreased binding seen in patients with compromised excretory function may be due to competition by elevated plasma concentrations of the glucuronides. In these reversible binding studies, we characterized the extent and the "site" of binding of tolmetin, zomepirac, their glucuronides and isomeric conjugates. We also examined the displacement between the parent drugs and their glucuronide conjugates using a rapid ultrafiltration method. Tolmetin exhibited three classes of binding sites with a primary association constant of 1.7 x 10(6) M-1 (Kd1 = 0.60 microM). The primary association constant of zomepirac (1.16 x 10(6) M-1, Kd1 = 0.86 microM) is similar to that of tolmetin. The beta 1 and alpha/beta 3 glucuronides of both compounds bind to a lesser extent than their parent aglycones. The isomeric glucuronide conjugates of both compounds showed much stronger binding than the beta/1 conjugates. Of the four glucuronides investigated, tolmetin glucuronide-alpha/beta 3 isomer was bound by fatty acid free human serum albumin with the highest affinity (4.6 x 10(5) M-1, Kd = 2.22 microM). Protein binding of the parent drugs and conjugates were decreased significantly at pH 5.0. In displacement studies, except for salicylate and acetylsalicylate, drugs known to bind to Sites I and II as well as the digitoxin and tamoxifen binding sites had little inhibitory effect on the binding of tolmetin, zomepirac, and their glucuronide conjugates.

  19. Investigation of the interaction of deltamethrin (DM) with human serum albumin by multi-spectroscopic method

    NASA Astrophysics Data System (ADS)

    Wang, Jiaman; Ma, Liang; Zhang, Yuhao; Jiang, Tao

    2017-02-01

    The interaction of Deltamethrin (DM) with human serum albumin (HSA) under the condition of simulating human blood pH environment (pH = 7.4) was investigated by fluorescence, UV-Vis absorbance and circular dichroism (CD) spectroscopy. It was shown that DM was a static quencher of HSA. The binding constants (Ka) are 3.598 × 104 L mol-1 (25 °C); the thermodynamic parameters (ΔH = -3.269 × 104 kJ mol-1, ΔS = -22.81 kJ mol-1 k-1, ΔG = -25889.8 kJ mol-1) obtained with the thermodynamic equation. The hydrogen bond and Vander Waals were the main driving force. The effect of DM on the conformation of HSA was observed by three-dimensional (3D) fluorescence and circular dichroism spectra, indicating that the interaction between DM and HSA was achieved through the binding of DM with the tryptophan and tyrosine residues of HSA. The study on the interaction of DM and Bovine Serum Albumin (BSA) was researched and compared. Difference exists in the interactions of with each of the serum albumins. We will verify and supplement that DM residue in animals and human metabolism, toxicology and other mechanisms are different.

  20. Spectroscopic investigation of the effect of salt on binding of tartrazine with two homologous serum albumins: quantification by use of the Debye-Hückel limiting law and observation of enthalpy-entropy compensation.

    PubMed

    Bolel, Priyanka; Datta, Shubhashis; Mahapatra, Niharendu; Halder, Mintu

    2012-08-30

    Formation of ion pair between charged molecule and protein can lead to interesting biochemical phenomena. We report the evolution of thermodynamics of the binding of tartrazine, a negatively charged azo colorant, and serum albumins with salt. The dye binds predominantly electrostatically in low buffer strengths; however, on increasing salt concentration, affinity decreases considerably. The calculated thermodynamic parameters in high salt indicate manifestation of nonelectrostatic interactions, namely, van der Waals force and hydrogen bonding. Site-marker competitive binding studies and docking simulations indicate that the dye binds with HSA in the warfarin site and with BSA at the interface of warfarin and ibuprofen binding sites. The docked poses indicate nearby amino acid positive side chains, which are possibly responsible for electrostatic interaction. Using the Debye-Hückel interionic attraction theory for binding equilibria, it is shown that, for electrostatic binding the calculated free energy change increases linearly with square root of ionic strength. Also UV-vis, fluorescence, CD data indicate a decrease of interaction with salt concentration. This study quantitatively relates how ionic strength modulates the strength of the protein-ligand electrostatic interaction. The binding enthalpy and entropy have been found to compensate one another. The enthalpy-entropy compensation (EEC), general property of weak intermolecular interactions, has been discussed.

  1. Investigations on the binding of mercury ions to albumins employing differential pulse voltammetry.

    PubMed

    Castro, Clarissa Silva Pires de; SouzaDe, Jurandir Rodrigues; Bloch, Carlos

    2003-04-01

    Binding of mercury to BSA and Ovalbumin was investigated by Differential Pulse Voltammetry. The method relies on the direct monitoring of peak current variation due to mercury oxidation in the presence of these two albumins. Linear calibration graphs were obtained for both BSA and Ovalbumin in concentrations ranging from 2.49 x 10(-9) to 19.6 x 10(-9) mol L(-1). The acquired data was used to quantify these two proteins independently and to calculate the dissociation constants of Hg-BSA and Hg-Ovalbumin complexes.

  2. Spectroscopic Studies on Binding of Lotus Seedpod Oligomeric Procyanidins to Bovine Serum Albumin

    NASA Astrophysics Data System (ADS)

    Wu, Q.; Li, Sh.; Fu, X.; Yang, T.; Chen, H.; Guan, Y.; Xie, B.; Sun, Zh.

    2014-01-01

    The binding of lotus seedpod oligomeric procyanidins (LSOPC) and catechin (a major constituent unit of LSOPC) to bovine serum albumin (BSA) was studied by a fluorescence quenching technique. The results revealed that LSOPC could strongly quench the intrinsic fluorescence of BSA through a static quenching procedure, but catechin could not. The Stern-Volmer quenching constant, K SV, and corresponding thermodynamic parameters, Δ G 0, Δ H 0 and Δ S 0, were calculated. The results of synchronous fluorescence and circular dichroism studies showed that LSOPC could cause a conformational change in BSA. In addition, glucose and metal ions could affect the interaction between LSOPC and BSA.

  3. The mouse albumin enhancer contains a negative regulatory element that interacts with a novel DNA-binding protein.

    PubMed Central

    Herbst, R S; Boczko, E M; Darnell, J E; Babiss, L E

    1990-01-01

    The far-upstream mouse albumin enhancer (-10.5 to -8.43 kilobases) has both positive and negative regulatory domains which contribute to the rate and tissue specificity of albumin gene transcription. (R. S. Herbst, N. Friedman, J. E. Darnell, Jr., and L. E. Babiss, Proc. Natl. Acad. Sci. USA 86:1553-1557). In this work, the negative regulatory region has been functionally localized to sequences -8.7 to -8.43 kilobases upstream of the albumin gene cap site. In the absence of the albumin-modulating region (in which there are binding sites for the transcription factor C/EBP), the negative region can suppress a neighboring positive-acting element, thereby interfering with albumin enhancer function. The negative region is also capable of negating the positive action of the heterologous transthyretin enhancer in an orientation-independent fashion. Within this negative-acting region we can detect two DNA-binding sites, both of which are recognized by a protein present in all cell types tested. This DNA-binding activity is not competed for by any of a series of known DNA-binding sites, and hence this new protein is a candidate for a role in suppressing the albumin gene in nonhepatic cells. Images PMID:2370857

  4. Glucose and Fluoxetine Induce Fine Structural Change in Human Serum Albumin

    PubMed Central

    Shahani, Minoo; Daneshi-Mehr, Fatemeh; Tadayon, Roya; Hoseinzade Salavati, Behrooz; Akbar Zadeh-Baghban, Ali-Reza; Zamanian, Abbas; Rezaei-Tavirani, Mostafa

    2013-01-01

    Human serum albumin has been used as a model protein for protein folding and ligand binding studies over many decades. Due to its long life period and high concentration in plasma, HSA is highly sensitive to glycation. It is reported that 175 mg/dL glucose concentration is a threshold of kidney activity for the beginning of excretion of glucose. pH denaturation of HSA in absence and presence of different concentrations of glucose is studied and based on the Pace two-state model, the findings are analyzed. In addition, florescence emission data of albumin range in the period of 300-500 nm was depicted. The amounts of free energy change and [D]1/2 parameters of unfolding in correspond to florescence date indicate that glucose induces fine structural change in human serum albumin. Results showed that 175 mg/dL glucose concentration is a critical point for albumin structural and functional alteration. PMID:24250587

  5. 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.

  6. Cordycepin and N6-(2-Hydroxyethyl)-Adenosine from Cordyceps pruinosa and Their Interaction with Human Serum Albumin

    PubMed Central

    Meng, Zebin; Kang, Jichuan; Wen, Tingchi; Lei, Bangxing; Hyde, Kevin David

    2015-01-01

    Cordyceps pruinosa (CP) is often used as Traditional Chinese Medicine, but the substance basis of its medicinal properties is unclear. In this study, two compounds were isolated from CP cultures by column chromatography, and identified as cordycepin and N6-(2-hydroxyethyl)-adenosine (HEA) by Nuclear Magnetic Resonance. In order to understand the efficacy of these two substances as potential therapeutic agents, it is necessary to explore their binding with proteins. The molecular mechanisms of interaction between cordycepin, HEA and human serum albumin (HSA) were studied using UV and fluorescence spectroscopy. The bingding constants between HSA and cordycepin were 4.227, 3.573 and 3.076 × 103·at 17, 27 and 37°C respectively, and that of HSA and HEA were 27.102, 19.409 and 13.002 × 103·at the three tempretures respectively. Both cordycepin and HEA can quench the intrinsic fluorescence of HSA via static quenching, and they can bind with HSA to form complexes with a single binding site. The interaction forces between cordycepin and HSA were determined as electrostatic and hydrophobic, and those of HEA and HSA were hydrogen bonding and van der Waals forces. Using Foster's equation, the distance between fluorophores of cordycepin and HSA, and HEA and HSA are estimated to be 5.31 nm and 4.98 nm, respectively. In this study, cordycepin was isolated for the first time from CP, and will provide a new source of cordycepin and expand the use of this taxon. The interaction mechanisms between cordycepin and HSA was studied for the first time, which will provide a useful guide for the clinical application of cordycepin. The pharmacological importance of this study is to understand the interaction of HSA with cordycepin and HEA, which will be essential for the future designing of drugs based on the two compounds. PMID:25811172

  7. Mechanism and conformational studies of farrerol binding to bovine serum albumin by spectroscopic methods

    NASA Astrophysics Data System (ADS)

    Zhang, Guowen; Wang, Lin; Fu, Peng; Hu, Mingming

    2011-11-01

    The mechanism and conformational changes of farrerol binding to bovine serum albumin (BSA) were studied by spectroscopic methods including fluorescence quenching technique, UV-vis absorption, circular dichroism (CD) spectroscopy and Fourier transform infrared (FT-IR) spectroscopy under simulative physiological conditions. The results of fluorescence titration revealed that farrerol could strongly quench the intrinsic fluorescence of BSA through a static quenching procedure. The thermodynamic parameters enthalpy change and entropy change for the binding were calculated to be -29.92 kJ mol -1 and 5.06 J mol -1 K -1 according to the van't Hoff equation, which suggested that the both hydrophobic interactions and hydrogen bonds play major role in the binding of farrerol to BSA. The binding distance r deduced from the efficiency of energy transfer was 3.11 nm for farrerol-BSA system. The displacement experiments of site markers and the results of fluorescence anisotropy showed that warfarin and farrerol shared a common binding site I corresponding to the subdomain IIA of BSA. Furthermore, the studies of synchronous fluorescence, CD and FT-IR spectroscopy showed that the binding of farrerol to BSA induced conformational changes in BSA.

  8. Binding of chloroquine-conjugated gold nanoparticles with bovine serum albumin.

    PubMed

    Joshi, Prachi; Chakraborty, Soumyananda; Dey, Sucharita; Shanker, Virendra; Ansari, Z A; Singh, Surinder P; Chakrabarti, Pinak

    2011-03-15

    We have conjugated chloroquine, an anti-malarial, antiviral and anti-tumor drug, with thiol-functionalized gold nanoparticles and studied their binding interaction with bovine serum albumin (BSA) protein. Gold nanoparticles have been synthesized using sodium borohydride as reducing agent and 11-mercaptoundecanoic acid as thiol functionalizing ligand in aqueous medium. The formation of gold nanoparticles was confirmed from the characteristic surface plasmon absorption band at 522 nm and transmission electron microscopy revealed the average particle size to be ~7 nm. Chloroquine was conjugated to thiolated gold nanoparticles by using EDC/NHS chemistry and the binding was analyzed using optical density measurement and Fourier transform infrared spectroscopy. The chloroquine-conjugated gold nanoparticles (GNP-Chl) were found to interact efficiently with BSA. Thermodynamic parameters suggest that the binding is driven by both enthalpy and entropy, accompanied with only a minor alteration in protein's structure. Competitive drug binding assay revealed that the GNP-Chl bind at warfarin binding site I in subdomain IIA of BSA and was further supported by Trp212 fluorescence quenching measurements. Unraveling the nature of interactions of GNP-Chl with BSA would pave the way for the design of nanotherapeutic agents with improved functionality, enriching the field of nanomedicine.

  9. Enantioselective binding interaction of the metolachlor pesticide enatiomers with bovine serum albumin - A spectroscopic analysis study

    NASA Astrophysics Data System (ADS)

    Ni, Yongnian; Zhang, Fangyuan; Kokot, Serge

    2012-11-01

    Enantioselective binding interaction of the pesticides, metolachlor (RAC-metolachlor) and its S-enantiomer (S-metolachlor), with bovine serum albumin (BSA) was investigated by fluorescence and UV-vis absorption spectroscopy. Both RAC- and S-metolachlors quenched the intrinsic fluorescence of BSA via a static mechanism, and various binding parameters indicated that electrostatic forces were involved in the binding of both of these compounds. Site marker competitive experiments demonstrated that S-metolachlor bound to site I of BSA, while R-metolachlor bound to site II, indicating the importance of enantiomeric factors for binding site selection. Further experiments showed that S-metolachlor had a higher binding affinity to BSA than R-metolachlor. The obtained spectral data were resolved with use of the multivariate curve resolution-alternating least squares method (MCR-ALS), and the extracted concentration profiles of the reacting species in the interaction were obtained. These profiles indicated that S-metolachlor was the main active constituent of RAC-metolachlor for binding with BSA, and these findings have significant implications in providing an explanation why S-metolachlor is the preferred herbicide in practice than RAC-metolachlor.

  10. Binding interaction of sorafenib with bovine serum albumin: Spectroscopic methodologies and molecular docking.

    PubMed

    Shi, Jie-Hua; Chen, Jun; Wang, Jing; Zhu, Ying-Yao; Wang, Qi

    2015-01-01

    The binding interaction of sorafenib with bovine serum albumin (BSA) was studied using fluorescence, circular dichrosim (CD) and molecular docking methods. The results revealed that there was a static quenching of BSA induced by sorafenib due to the formation of sorafenib-BSA complex. The binding constant and number of binding site of sorafenib with BSA under simulated physiological condition (pH=7.4) were 6.8×10(4) M(-1) and 1 at 310 K, respectively. Base on the sign and magnitude of the enthalpy and entropy changes (ΔH(0)=-72.2 kJ mol(-1) and ΔS(0)=-140.4J mol(-1) K(-1)) and the results of molecular docking, it could be suggested that the binding process of sorafenib and BSA was spontaneous and the main interaction forces of sorafenib with BSA were van der Waals force and hydrogen bonding interaction. From the results of site marker competitive experiments and molecular docking, it could be deduced that sorafenib was inserted into the subdomain IIA (site I) of BSA and leads to a slight change of the conformation of BSA. And, the significant change of conformation of sorafenib occurred in the binding process with BSA to increase the stability of the sorafenib-BSA system, implying that the flexibility of sorafenib played an important role in the binding process.

  11. Investigation of Cu(II) Binding to Bovine Serum Albumin by Potentiometry with an Ion Selective Electrode

    ERIC Educational Resources Information Center

    Jie Liu

    2004-01-01

    A laboratory project that investigates Cu(II) bind to bovine serum albumin (BSA) in an aqueous solution is developed to assist undergraduate students in gaining better understanding of the interaction of ligands with biological macromolecule. Thus, students are introduced to investigation of Cu(II) binding to BSA by potentiometry with the Cu(II)…

  12. 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.

  13. The effect of methylamine on the solution structures of human and bovine serum albumins

    NASA Astrophysics Data System (ADS)

    Hamdani, S.; Joly, D.; Carpentier, R.; Tajmir-Riahi, H. A.

    2009-11-01

    Serum albumins are the major soluble protein constituents of the circulatory system and have many physiological functions including transporting a variety of compounds. Methylamine, a monoamine with one positive charge complexes with protein and alters protein secondary structure. The aim of this study was to examine the interactions of human serum albumin (HSA) and bovine serum albumin (BSA) with methylamine at physiological conditions, using constant protein concentration and various monoamine concentrations. FTIR, UV-vis, CD and fluorescence spectroscopic methods were used to analyse methylamine binding mode, the binding constant and the effects of monoamine on HSA and BSA stability and conformations. Structural analysis showed that methylamine binds HSA and BSA via hydrophilic (polypeptide and amine polar groups) and hydrophobic interactions with overall binding constants of Kmet-HSA = 2.42 (±0.5) × 10 2 M -1 and Kmet-BSA = 1.34 (±0.3) × 10 3 M -1 with the number of bound methylamine around one molecule per protein. Methylamine complexation alters protein conformation by major reduction of α-helix and increase in random coil and turn structures indicating a partial protein unfolding.

  14. 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.

  15. Mechanism of singlet oxygen chemiluminescence enhancement by human serum albumin

    NASA Astrophysics Data System (ADS)

    Zhou, Jing; Xing, Da; Chen, Qun

    2006-02-01

    Fluoresceinyl Cypridina Luciferin Analog (FCLA) is a chemiluminescence (CL) probe for detecting reactive oxygen species (ROS). Its detection efficiency of singlet oxygen can be significantly enhanced in the presence of human serum albumin (HSA). In the current study, the mechanism of the FCLA-HSA CL system is studied by means of direct CL measurement and spectroscopy techniques. Our results show that FCLA can combine with HSA via a single binding site to form a complex. The CL efficiency of the system is largely governed by an inter-system energy transfer between the two components upon interaction with singlet oxygen. The CL production reaches maximum in a synergetic manner when equal amount of FCLA and HSA are present simultaneously, but the production is less efficient at other ratios. This suggests that the FCLA-HSA system maybe used as a singlet oxygen detecting technique with higher sensitivity compared with that of conventional CL techniques. It may also provide a potential new technique for quantitatively analyze the presence of HSA in a sample.

  16. Characterization of the binding of 2-mercaptobenzimidazole to bovine serum albumin.

    PubMed

    Teng, Yue; Zou, Luyi; Huang, Ming; Zong, Wansong

    2015-04-01

    2-Mercaptobenzimidazole (MBI) is widely utilized as a corrosion inhibitor, copper-plating brightener and rubber accelerator. The residue of MBI in the environment is potentially harmful to human health. In this article, the interaction of MBI with bovine serum albumin (BSA) was explored using spectroscopic and molecular docking methods under physiological conditions. The positively charged MBI can spontaneously bind with the negatively charged BSA through electrostatic forces with one binding site. The site marker competition experiments and the molecular docking study revealed that MBI bound into site II (subdomain IIIA) of BSA, which further led to some secondary structure and microenvironmental changes of BSA. This work provides useful information on understanding the toxicological actions of MBI at the molecular level.

  17. Interaction between phillygenin and human serum albumin based on spectroscopic and molecular docking

    NASA Astrophysics Data System (ADS)

    Song, W.; Ao, M. Z.; Shi, Y.; Yuan, L. F.; Yuan, X. X.; Yu, L. J.

    2012-01-01

    In this paper, the interaction of human serum albumin (HSA) with phillygenin was investigated by fluorescence, circular dichroism (CD), UV-vis spectroscopic and molecular docking methods under physiological conditions. The Stern-Volmer analysis indicated that the fluorescence quenching of HSA by phillygenin resulted from static mechanism, and the binding constants were 1.71 × 10 5, 1.61 × 10 5 and 1.47 × 10 4 at 300, 305 and 310 K, respectively. The results of UV-vis spectra show that the secondary structure of the protein has been changed in the presence of phillygenin. The CD spectra showed that HSA conformation was altered by phillygenin with a major reduction of α-helix and an increase in β-sheet and random coil structures, indicating a partial protein unfolding. The distance between donor (HSA) and acceptor (phillygenin) was calculated to be 3.52 nm and the results of synchronous fluorescence spectra showed that binding of phillygenin to HSA can induce conformational changes in HSA. Molecular docking experiments found that phillygenin binds with HSA at IIIA domain of hydrophobic pocket with hydrogen bond interactions. The ionic bonds were formed with the O (4), O (5) and O (6) of phillygenin with nitrogen of ASN109, ARG186 and LEU115, respectively. The hydrogen bonds are formed between O (2) of phillygenin and SER419. In the presence of copper (II), iron (III) and alcohol, the apparent association constant KA and the number of binding sites of phillygenin on HSA were both decreased in the range of 88.84-91.97% and 16.09-18.85%, respectively. In view of the evidence presented, it is expected to enrich our knowledge of the interaction dynamics of phillygenin to the important plasma protein HSA, and it is also expected to provide important information of designs of new inspired drugs.

  18. Probing the binding sites and the effect of berbamine on the structure of bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Cheng, Xiao-Xia; Lui, Yi; Zhou, Bo; Xiao, Xiao-He; Liu, Yi

    2009-06-01

    Berbamine, a naturally occurring isoquinoline alkaloid extracted from Berberis sp., is the active constituent of some Chinese herbal medicines and exhibits a variety of pharmacological activities. The effects of berbamine on the structure of bovine serum albumin (BSA) were investigated by circular dichroism, fluorescence and absorption spectroscopy under physiological conditions. Berbamine caused a static quenching of the intrinsic fluorescence of BSA, and the quenching data were analyzed by application of the Stern-Volmer equation. There was a single primary berbamine-binding site on BSA with a binding constant of 2.577 × 10 4 L mol -1 at 298 K. The thermodynamic parameters, enthalpy change (Δ H0) and entropy change (Δ S0) for the reaction were -76.5 kJ mol -1 and -173.4 J mol -1 K -1 according to the van't Hoff equation. The results showed that the hydrogen bond and van der Waals interaction were the predominant forces in the binding process. Competitive experiments revealed a displacement of warfarin by berbamine, indicating that the binding site was located at Drug sites I. The distance r between the donor (BSA) and the acceptor (berbamine) was obtained according to the Förster non-radiation energy transfer theory. The results of three-dimensional fluorescence spectra, UV-vis absorption difference spectra and circular dichroism of BSA in the presence of berbamine showed that the conformation of BSA was changed. The results provide a quantitative understanding of the effect of berbamine on the structure of bovine serum albumin, providing a useful guideline for further drug design.

  19. Site-selective conjugation of an anticoagulant aptamer to recombinant albumins and maintenance of neonatal Fc receptor binding.

    PubMed

    Schmøkel, Julie; Voldum, Anders; Tsakiridou, Georgia; Kuhlmann, Matthias; Cameron, Jason; Sørensen, Esben; Wengel, Jesper; Howard, Kenneth A

    2017-03-31

    Aptamers are an attractive molecular medicine that offers high target specificity. Nucleic acid-based aptamers however, are prone to nuclease degradation and rapid renal excretion that require blood circulatory half-life extension enabling technologies. The long circulatory half-life, predominately facilitated by engagement with the cellular recycling neonatal Fc receptor (FcRn), and ligand transport properties of albumin promote it as an attractive candidate to improve the pharmacokinetic profile of aptamers. This study investigates the effect of Cys34 site-selective covalent attachment of a factor IXa anticoagulant aptamer on aptamer functionality and FcRn engagement using recombinant human albumin (rHA) of either a wild type (WT) or an engineered human FcRn high binding variant (HB). Aptamer-albumin conjugates, connected covalently through a heterobifunctional succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate linker, were successfully prepared and purified by high performance liquid chromatography as confirmed by gel electrophoresis band-shift analysis and matrix-assisted laser desorption/ionization time of flight. Minimal reduction (~ 25%) in activity of WT-linked aptamer to that of aptamer alone was found using an anticoagulant activity assay measuring temporal levels of activated partial thrombin. Covalent aptamer-albumin conjugation, however, substantially compromised binding to FcRn, to 10% affinity of that of non-conjugated WT, determined by biolayer interferometry. Binding could be rescued by aptamer conjugation to recombinant albumin engineered for higher FcRn affinity (HB) that exhibited an 8-fold affinity compared to WT alone. This work describes a novel albumin-based aptamer delivery system whose FcRn binding can be increased using a high binding engineered albumin.

  20. Binding of anti-inflammatory drug cromolyn sodium to bovine serum albumin.

    PubMed

    Hu, Yan-Jun; Liu, Yi; Sun, Ting-Quan; Bai, Ai-Min; Lü, Jian-Quan; Pi, Zhen-Bang

    2006-11-15

    Fluorescence spectroscopy in combination with circular dichroism (CD) and UV-vis absorption spectroscopy were employed to investigate the binding of anti-inflammatory drug cromolyn sodium (Intal) to bovine serum albumin (BSA) under the physiological conditions with Intal concentrations of 0-6.4 x 10(-5)mol L(-1). In the mechanism discussion, it was proved that the fluorescence quenching of BSA by Intal is a result of the formation of Intal-BSA complex. Quenching constants were determined using the Stern-Volmer equation to provide a measure of the binding affinity between Intal and BSA. The thermodynamic parameters Delta G, Delta H, Delta S at different temperatures (298, 304, and 310 K) were calculated and the results indicate the electrostatic interactions play a major role in Intal-BSA association. Binding studies concerning the number of binding sites (n=1) and apparent binding constant K(b) were performed by fluorescence quenching method. Utilizing fluorescence resonant energy transfer (FRET) the distance R between the donor (BSA) and acceptor (Intal) has been obtained. Furthermore, CD and synchronous fluorescence spectrum were used to investigate the structural change of BSA molecules with addition of Intal, the results indicate that the secondary structure of BSA molecules was changed in the presence of Intal.

  1. Structural Insights into the Competitive Binding of Diclofenac and Naproxen by Equine Serum Albumin.

    PubMed

    Sekula, Bartosz; Bujacz, Anna

    2016-01-14

    The binding modes to equine serum albumin (ESA) of two nonsteroidal anti-inflammatory drugs (NSAIDs), diclofenac (Dic) and naproxen (Nps), were studied by X-ray crystallography and isothermal titration calorimetry. On the basis of the crystal structure of ESA/Dic determined to a resolution of 1.92 Å and the structure of the previously described ESA/Nps complex (2.42 Å), it was found that both NSAIDs bind within drug site 2 (DS2) of ESA and both occupy secondary binding sites in separate cavities of domain II (Nps) and domain III (Dic). The two structures of the ternary complex ESA/Dic/Nps, obtained by competitive cocrystallization (2.19 Å) and through a displacement experiment (2.35 Å), were determined to investigate possible competition of these widely used pharmaceutical drugs in binding to ESA. In these complexes Nps occupies the DS2 pocket common for both drugs, whereas the other distinct binding sites of Dic and Nps remain unaffected. These results suggest that combined application of both drugs may result in increased concentration of free diclofenac in plasma.

  2. Investigation of Interaction Between Ozagrel and Human Serum Albumin by Spectroscopic and Electrochemical Methods

    NASA Astrophysics Data System (ADS)

    Li, S.; Wang, Li; Hao, J.; Wang, L.; Tong, Y.-J.; Fu, Z.-Q.; Zhang, A.-P.

    2017-01-01

    The interaction between ozagrel and human serum albumin (HSA) was investigated by fl uorescence spectroscopy, UV-Vis absorption spectroscopy, cyclic voltammetry (CV), differential pulse voltammetry (DPV), and Fourier transform infrared spectroscopy (FTIR) under simulative physiological conditions. The results of CV, DPV and fl uorescence titration revealed that ozagrel bound to HSA. The enthalpy change (ΔH) and the entropy change (ΔS) were derived to be positive values, indicating that the hydrophobic force played the main role in the binding of ozagrel with HSA. The binding distance between ozagrel and HSA was 1.75 nm. Upon binding with ozagrel, the conformation and the secondary structure of HSA molecules were changed. The percentage of α-helix and β-sheet structures decreased by 7.25% and 4.58%, respectively, while the percentage of a β-turn structure increased by 2.67%. The effect of common ions on the binding of ozagrel with HSA was also examined. This study will give an insight into the evaluation of the drug's stabi-lity during transport and its releasing effi ciency at the target site under simulative physiological conditions.

  3. Mode of encapsulation of linezolid by β-cyclodextrin and its role in bovine serum albumin binding.

    PubMed

    Natesan, Sudha; Sowrirajan, Chandrasekaran; Yousuf, Sameena; Enoch, Israel V M V

    2015-01-22

    We describe, in this article, the associative interaction between Linezolid and β-Cyclodextrin, and the influence of β-Cyclodextrin on Linezolid's binding to Bovine serum albumin. β-Cyclodextrin forms a 1:1 inclusion complex with Linezolid, with a binding constant value of 3.51×10(2)M(-1). The binding is studied using ultraviolet-visible absorption, fluorescence, nuclear magnetic resonance, and rotating-frame overhauser effect spectroscopic techniques. The amide substituent on the oxazolidinone ring of Linezolid is involved in its binding to β-Cyclodextrin. The binding of the Linezolid to bovine serum albumin, in the absence and the presence of β-Cyclodextrin, is studied by analyzing the fluorescence quenching and Förster resonance energy transfer. The Stern-Volmer quenching constant, the binding constant, and energy transfer occurring on the interaction of the Linezolid with BSA are found to be smaller in the presence of β-Cyclodextrin than in water.

  4. Synthesis, comparative photosensitizing efficacy, human serum albumin (site II) binding ability, and intracellular localization characteristics of novel benzobacteriochlorins derived from vic-dihydroxybacteriochlorins.

    PubMed

    Li, Guolin; Graham, Andrew; Chen, Yihui; Dobhal, Mahabeer P; Morgan, Janet; Zheng, Gang; Kozyrev, Andrei; Oseroff, Allan; Dougherty, Thomas J; Pandey, Ravindra K

    2003-12-04

    In a sequence of reactions, methyl mesopyropheophorbide a, mesochlorin e(6) trimethyl ester, mesochlorin p(6) trimethyl ester, mesopurpurin-18-N-hexylimide methyl ester, and mesopurpurin-18-N-3,5-bis(trifluoromethyl)benzylimide methyl ester were synthesized from chlorophyll-a. These chlorins on reacting with osmium tetraoxide produced the corresponding vic-dihydroxybacteriochlorins. The 8-vinylchlorins obtained by refluxing the related vic-dihydroxybacteriochlorins in o-dichlorobenzene were individually treated with dimethylacetylenedicarboxylate (DMAD) under Diels-Alder reaction conditions. The intermediate adducts on 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) treatment rearranged to the corresponding stable benzobacteriochlorins, exhibiting the longest wavelength absorption in the range of 737 to 805 nm. In preliminary in vitro (RIF tumor cells) and in vivo screening (C3H/HeJ mice bearing RIF tumors), some of these compounds were found to be quite effective. Under similar treatment conditions (drug dose: 5.0 micromol/kg; light dose: 135 J/cm(2), tumors were exposed to light for 30 min at 24 h postinjection), the benzobacteriochlorins containing N-substituted-imide ring system produced enhanced photosensitizing efficacy with limited skin phototoxicity. These compounds were also found to bind to site II of human serum albumin (HSA). However, no correlation between the binding constant values and photosensitizing efficacy was observed. A competitive intracellular localization study of these novel structures with Rhodamine-123 (a mitochondrial probe) indicated their preferential localization in mitochondria, without producing any specific displacement of (3)H-PK11195 (PBR probe, (3)H-labeled 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide). These results suggest that the mitochondrial peripheral benzodiazepine receptor (PBR) is not the cellular binding site for this class of compounds.

  5. Interaction of phenylbutazone and colchicine in binding to serum albumin in rheumatoid therapy: 1H NMR study

    NASA Astrophysics Data System (ADS)

    Maciążek-Jurczyk, M.; Sułkowska, A.; Bojko, B.; Równicka-Zubik, J.; Sułkowski, W. W.

    2009-09-01

    The monitoring of drug concentration in blood serum is necessary in multi-drug therapy. Mechanism of drug binding with serum albumin (SA) is one of the most important factors which determine drug concentration and its transport to the destination tissues. In rheumatoid diseases drugs which can induce various adverse effects are commonly used in combination therapy. Such proceeding may result in the enhancement of those side effects due to drug interaction. Interaction of phenylbutazone and colchicine in binding to serum albumin and competition between them in gout has been studied by proton nuclear magnetic resonance ( 1H NMR) technique. The aim of the study was to determine the low affinity binding sites, the strength and kind of interaction between serum albumin and drugs used in combination therapy. The study of competition between phenylbutazone and colchicine in binding to serum albumin points to the change of their affinity to serum albumin in the ternary systems. This should be taken into account in multi-drug therapy. This work is a subsequent part of the spectroscopic study on Phe-COL-SA interactions [A. Sułkowska, et al., J. Mol. Struct. 881 (2008) 97-106].

  6. DEER in biological multispin-systems: A case study on the fatty acid binding to human serum albumin

    NASA Astrophysics Data System (ADS)

    Junk, Matthias J. N.; Spiess, Hans W.; Hinderberger, Dariush

    2011-06-01

    In this study, self-assembled systems of human serum albumin (HSA) and spin-labeled fatty acids are characterized by double electron-electron resonance (DEER). HSA, being the most important transport protein of the human blood, is capable to host up to seven paramagnetic fatty acid derivatives. DEER measurements of these self-assembled multispin clusters are strongly affected by correlations of more than two spins, the evaluation of the latter constituting the central topic of this paper. While the DEER modulation depth can be used to obtain qualitative information of the number of coupled spins, the quantitative analysis is hampered by the occurrence of cluster mixtures with different numbers of coupled spins and contributions from unbound spin-labeled material. Applying flip angle dependent DEER measurements, unwanted multispin correlations were found to lead not only to a broadening of the distance peaks but also to cause small distances to be overestimated and large distances to be suppressed. It is thus favorable to use spin-diluted systems with an average of two paramagnetic molecules per spin cluster when a quantitative analysis of the distance distribution is sought.

  7. Design and development of in situ albumin binding surfaces: Evaluation in the paradigm of blood-biomaterial compatibility

    NASA Astrophysics Data System (ADS)

    Guha Thakurta, Sanjukta

    Biocompatibility of natural and synthetic implant materials as blood contacting devices is crucial to host response. Implantation often raises complications from thrombotic and thromboembolic events. The aspect of hemocompatibility concentrates on minimizing thrombotic and thromboembolic response of foreign materials in contact with blood. The initial layer of surface adsorbed proteins plays a pivotal role in the adhesion and subsequent aggregation of platelets and in the activation of the coagulation cascade. Therefore, an improved surface architecture is required to gain control over the initial protein adsorption events, thereby extending the sustainability of an implantable device. In general, surfaces with an ability to bind endogenous albumin has been known to minimize platelet adhesion and activation. While the scope of applicability is broad, in this study silicon-based surfaces were selected as model surfaces. A densely packed uniformly distributed silane monolayer was achieved on silicon based surfaces with -- NH2 functionality, upon a careful optimization of hydroxylation and the subsequent silanization with 2 vol% of 3-Aminopropyltriethoxy Silane (APTES). Two linear peptides with affinity for albumin over other serum proteins were selected to create affinity surfaces. Silanized surfaces covalently immobilized with albumin binding peptides were evaluated in the paradigm of blood-biomaterial compatibility. When compared to control surfaces, albumin binding surfaces prepared in this study: (a) possessed 2.0 to 3.0 mug/cm2 of surface bound albumin with minimal surface adsorbed fibrinogen, (b) depicted low levels of adhered platelets and supported a rounded platelet morphology, (c) displayed delayed clotting, (d) showed reduced platelet adhesion and activation under shearing, and (f) exhibited faster adsorption kinetics. Conclusively, in-situ albumin binding surfaces selectively and specifically interacted with albumin without being severely displaced by

  8. Sulfadiazine binds and unfolds bovine serum albumin: an in vitro study.

    PubMed

    Al-Lohedan, Hamad A; Sajih Ali, Mohd

    2013-11-01

    Sulfonamide derivatives, such as sulfadiazine (SD) are used as antibiotics and, very recently, anti-amyloid properties of these have been reported. We have evaluated binding of SD with bovine serum albumin (BSA) followed by unfolding of protein. Studies were accomplished at physiological conditions of temperature (37 °C) and pH (7.4), employing UV, fluorescence, circular dichroism (CD) and Fourier transform infra-red (FTIR) spectroscopies. In presence of drug, UV spectrum of BSA was altered from the spectrum of native BSA due to the interaction between albumin and drug. Excitation of protein at 295 nm showed that fluorescence quenching of BSA by SD is a result of the formation of SD–BSA complex. The data were analyzed using Stern–Volmer and Lineweaver–Burk methods. From both methods it was evaluated that the quenching involved in BSA–SD binding was static. BSA had only one binding site for SD. Synchronous fluorescence spectra have shown a red shift and advocated that hydrophobicity around both Trp and Tyr residues was decreased. CD results revealed that the conformation of macromolecule remain undisturbed at low concentrations (up to 20 μM of the SD) and there was small perturbation in the secondary structure from 20 to 50 μM of SD followed by a large change and consequent unfolding on further increase in the drug concentration. Both synchronous and CD measurements were consistent to each other. FTIR spectra revealed the shifting of amide I band which is also an indication of conformational change of the protein.

  9. Effects of gene carrier polyethyleneimines on the structure and binding capability of bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Guo, Zhiyong; Kong, Zhijie; Wei, Yanshan; Li, Hua; Wang, Yajing; Huang, Aimin; Ma, Lin

    2017-02-01

    Polyethyleneimine (PEI), one of the most effective non-viral gene carriers, is also cytotoxic, however the molecular basis is poorly understood. Little is known about the effects of PEI on the structure and functions of the biomacromolecules. In this work, fluorescence, UV-vis absorption, circular dichroism (CD) spectroscopy and zeta-potential measurement were conducted to reveal the interaction between PEIs (average molecular weight 25, 10 and 1.8 kDa) and bovine serum albumin (BSA), and to evaluate the effects on the conformation of BSA as long as its binding capability to the model compounds, 8-anilino-1-naphthalenesulfonic acid (ANS) and quercetin. PEIs were found to complex with BSA and induced a conformational change of the protein by a major reduction of α-helix at PEI concentration < 0.2 mg·mL- 1 and an increase at higher PEI concentration. The binding efficacy of ANS and quercetin to BSA was greatly reduced by the competitive binding by PEI and influenced by the conformational change of BSA, which was found to display a similar trend to the change of the α-helix content of the protein. The polymer size played an important role in PEI-BSA interaction. PEI of higher molecular weight was more favorable to interact with BSA and more efficient to perturb the conformation and binding capability of the protein.

  10. Spectroscopic study on binding of gentisic acid to bovine serum albumin.

    PubMed

    Garzón, Andrés; Bravo, Iván; Carrión-Jiménez, M Rosario; Rubio-Moraga, Ángela; Albaladejo, José

    2015-01-01

    The interaction of (gentisic acid) GA with (bovine serum albumin) BSA has been studied by different spectroscopic techniques. GA is a monoanionic specie at the working pH of 7.4, it was determined by combining UV-Vis absorption spectroscopy and theoretical calculations. A set of fluorescence quenching experiments at different temperatures was carried out employing the native fluorescence of BSA. A Stern-Volmer constant (KSV) of (2.07±0.12)×10(4) mol(-1) L and a binding constant (Ka) of (8.47±4.39)×10(3) were determined at 310 K. The static quenching caused by the BSA-GA complex formation seems to play a significant role in the overall quenching process. A single binding site on BSA for GA was observed. ΔH=-55.6±0.2 kJ mol(-1) and ΔS=-104.3±0.6 J mol(-1) K(-1) were determined in a set of experiments on the dependence of Ka with the temperature. The binding process is, therefore, spontaneous and enthalpy-driven. Van der Waals forces and hydrogen bonds could also play the major role in the binding mode. The secondary structure changes of BSA in the absence and presence of GA were studied by FTIR and UV-Vis absorption spectroscopy.

  11. Characterization of the binding of nevadensin to bovine serum albumin by optical spectroscopic technique

    NASA Astrophysics Data System (ADS)

    Yu, Zhaolian; Li, Daojin; Ji, Baoming; Chen, Jianjun

    2008-10-01

    Binding of nevadensin to bovine serum albumin (BSA) has been studied in detail at 298 and 310 K using spectrophotometric technique. The intrinsic fluorescence of BSA was strongly quenched by the addition of nevadensin and spectroscopic observations are mainly rationalized in terms of a static quenching process at lower concentration of nevadensin ( Cdrug/ CBSA < 1) and a combined quenching process at higher concentration of nevadensin ( Cdrug/ CBSA > 1). The binding parameters for the reaction at a pH above (7.40) or below (3.40) the isoelectric point have been calculated according to the double logarithm regression curve. The thermodynamic parameters Δ H0, Δ G0, Δ S0 at different temperatures and binding mechanism of nevadensin to BSA at pH 7.40 and 3.40 were evaluated. The binding ability of nevadensin to BSA at pH 7.40 was stronger than that at pH 3.40. Steady fluorescence, synchronous fluorescence and circular dichroism (CD) were applied to investigate protein conformation. A value of 2.15 nm for the average distance r between nevadensin (acceptor) and tryptophan residues (Trp) of BSA (donor) was derived from the fluorescence resonance energy transfer. Moreover, influence of pH on the interaction nevadensin with BSA was investigated.

  12. A comparison study on the binding of hesperetin and luteolin to bovine serum albumin by spectroscopy

    NASA Astrophysics Data System (ADS)

    Tang, Lin; Jia, Wanteng

    2013-02-01

    Binding mechanism of luteolin (LUT) and hesperetin (HES) to bovine serum albumin (BSA) was investigated at 288,298,310 K and pH = 7.40 by UV absorption spectroscopy, fluorescence quenching and synchronous fluorescence spectroscopy. Under simulated physiological conditions, the fluorescence data indicated that hesperetin binding to BSA mainly occurs through a static mechanism. In contrast, binding of luteolin to BSA is a combined quenching process while static quenching is prevailing. Linear interval of the Stern-Volmer plot of LUT-BSA for the concentration ratio of LUT to BSA ranged from 0.5 to 1.25 was obtained. The thermodynamic parameters obtained from the Van't Hoff equation indicated that electrostatic force was the predominant force in the LUT-BSA and HES-BSA complex. The inner filter effect was eliminated to get accurate data. The conformational changes of BSA caused by LUT and HES were observed in the UV absorption. Results of fluorescence quenching and synchronous fluorescence showed that degree of luteolin-BSA quenching was higher than hesperetin-BSA quenching, which indicated that the 4'-hydroxide radical was more helpful to the ligand binding to proteins than 4'-methoxyl group for flavones.

  13. Binding interaction of atorvastatin with bovine serum albumin: Spectroscopic methods and molecular docking.

    PubMed

    Wang, Qi; Huang, Chuan-ren; Jiang, Min; Zhu, Ying-yao; Wang, Jing; Chen, Jun; Shi, Jie-hua

    2016-03-05

    The interaction of atorvastatin with bovine serum albumin (BSA) was investigated using multi-spectroscopic methods and molecular docking technique for providing important insight into further elucidating the store and transport process of atorvastatin in the body and the mechanism of action and pharmacokinetics. The experimental results revealed that the fluorescence quenching mechanism of BSA induced atorvastatin was a combined dynamic and static quenching. The binding constant and number of binding site of atorvastatin with BSA under simulated physiological conditions (pH=7.4) were 1.41 × 10(5) M(-1) and about 1 at 310K, respectively. The values of the enthalpic change (ΔH(0)), entropic change (ΔS(0)) and Gibbs free energy (ΔG(0)) in the binding process of atorvastatin with BSA at 310K were negative, suggesting that the binding process of atorvastatin and BSA was spontaneous and the main interaction forces were van der Waals force and hydrogen bonding interaction. Moreover, atorvastatin was bound into the subdomain IIA (site I) of BSA, resulting in a slight change of the conformation of BSA.

  14. Binding interaction of atorvastatin with bovine serum albumin: Spectroscopic methods and molecular docking

    NASA Astrophysics Data System (ADS)

    Wang, Qi; Huang, Chuan-ren; Jiang, Min; Zhu, Ying-yao; Wang, Jing; Chen, Jun; Shi, Jie-hua

    2016-03-01

    The interaction of atorvastatin with bovine serum albumin (BSA) was investigated using multi-spectroscopic methods and molecular docking technique for providing important insight into further elucidating the store and transport process of atorvastatin in the body and the mechanism of action and pharmacokinetics. The experimental results revealed that the fluorescence quenching mechanism of BSA induced atorvastatin was a combined dynamic and static quenching. The binding constant and number of binding site of atorvastatin with BSA under simulated physiological conditions (pH = 7.4) were 1.41 × 105 M- 1 and about 1 at 310 K, respectively. The values of the enthalpic change (ΔH0), entropic change (ΔS0) and Gibbs free energy (ΔG0) in the binding process of atorvastatin with BSA at 310 K were negative, suggesting that the binding process of atorvastatin and BSA was spontaneous and the main interaction forces were van der Waals force and hydrogen bonding interaction. Moreover, atorvastatin was bound into the subdomain IIA (site I) of BSA, resulting in a slight change of the conformation of BSA.

  15. Effects of gene carrier polyethyleneimines on the structure and binding capability of bovine serum albumin.

    PubMed

    Guo, Zhiyong; Kong, Zhijie; Wei, Yanshan; Li, Hua; Wang, Yajing; Huang, Aimin; Ma, Lin

    2017-02-15

    Polyethyleneimine (PEI), one of the most effective non-viral gene carriers, is also cytotoxic, however the molecular basis is poorly understood. Little is known about the effects of PEI on the structure and functions of the biomacromolecules. In this work, fluorescence, UV-vis absorption, circular dichroism (CD) spectroscopy and zeta-potential measurement were conducted to reveal the interaction between PEIs (average molecular weight 25, 10 and 1.8kDa) and bovine serum albumin (BSA), and to evaluate the effects on the conformation of BSA as long as its binding capability to the model compounds, 8-anilino-1-naphthalenesulfonic acid (ANS) and quercetin. PEIs were found to complex with BSA and induced a conformational change of the protein by a major reduction of α-helix at PEI concentration <0.2mg·mL(-1) and an increase at higher PEI concentration. The binding efficacy of ANS and quercetin to BSA was greatly reduced by the competitive binding by PEI and influenced by the conformational change of BSA, which was found to display a similar trend to the change of the α-helix content of the protein. The polymer size played an important role in PEI-BSA interaction. PEI of higher molecular weight was more favorable to interact with BSA and more efficient to perturb the conformation and binding capability of the protein.

  16. Controlling the taste receptor accessible structure of rebaudioside A via binding to bovine serum albumin.

    PubMed

    Mudgal, Samriddh; Keresztes, Ivan; Feigenson, Gerald W; Rizvi, S S H

    2016-04-15

    We illustrate a method that uses bovine serum albumin (BSA) to control the receptor-accessible part of rebaudioside A (Reb A). The critical micelle concentration (CMC) of Reb A was found to be 4.5 mM and 5 mM at pH 3 and 6.7 respectively. NMR studies show that below its CMC, Reb A binds weakly to BSA to generate a Reb A-protein complex ("RPC"), which is only modestly stable under varying conditions of pH (3.0-6.7) and temperature (4-40°C) with its binding affinities determined to be in the range of 5-280 mM. Furthermore, saturation transfer difference (STD) NMR experiments confirm that the RPC has fast exchange of the bitterness-instigating diterpene of Reb A into the binding sites of BSA. Our method can be used to alter the strength of Reb A-receptor interaction, as a result of binding of Reb A to BSA, which may ultimately lead to moderation of its taste.

  17. Why mammals more susceptible to the hepatotoxic microcystins than fish: evidences from plasma and albumin protein binding through equilibrium dialysis.

    PubMed

    Zhang, Wei; Liang, Gaodao; Wu, Laiyan; Tuo, Xun; Wang, Wenjing; Chen, Jun; Xie, Ping

    2013-08-01

    To elucidate the interspecies variation of susceptibility to microcystins (MCs), fresh plasma and purified albumin from six kinds of mammals and fish were used in toxins-substances binding test. Protein contents in the test plasma were analyzed and the binding characteristics to MCs were compared. Two kinds of widely observed MCs, microcystin-LR (MC-LR) and microcystin-RR (MC-RR) were tested and data were collected through the method of equilibrium dialysis. It was found that total plasma protein and albumin content in mammals were nearly two times and four times higher than that in fish, respectively. In the test range of 0-100 μg/mL, binding rates of fish plasma to MCs were considered significant lower (p < 0.01) than that of mammals. And human plasma demonstrated the highest binding rate in mammals. In all the test species, plasma protein binding rates of MC-RR were significantly higher than MC-LR (p < 0.01). Besides, binding profiles of albumin were acquired under the protein content of 0.67 mg/mL. Human serum albumin demonstrated the highest affinity to MCs throughout the six species and differences among the other five species were considered not significant (p > 0.05). From the view of protein binding, it is concluded that both the variation of plasma protein composition and albumin binding characteristic could influence the existing form of MCs in circulation, change MCs utilization, alter MCs half-life and further contribute to the difference of susceptibility between mammals and fish.

  18. New approach to measure protein binding based on a parallel artificial membrane assay and human serum albumin.

    PubMed

    Lázaro, Elisabet; Lowe, Philip J; Briand, Xavier; Faller, Bernard

    2008-04-10

    We report here a new, label-free approach to measure serum protein binding constants. The assay is able to measure HSA K d values in the milli-molar to micromolar range. The protein is not immobilized on any surface and the assay self-corrects for nonspecific adsorption. No mass balance is required to get accurate binding constants and it is not necessary to wait for equilibrium to extract the binding constant. The assay runs in a 96-well format using commercially available parts and is, therefore, relatively easy to implement and automate. As the chemical membranes used are not water permeable, there is no volume change due to the osmotic pressure and pretreatment (soaking) is not necessary. The concept can potentially be extended to other proteins and could thus serve as a label-free technique for general binding constant measurements.

  19. Water-in-silicone oil emulsion stabilizing surfactants formed from native albumin and alpha,omega-triethoxysilylpropyl-polydimethylsiloxane.

    PubMed

    Zelisko, Paul M; Flora, Kulwinder K; Brennan, John D; Brook, Michael A

    2008-08-01

    Contact with hydrophobic silicones frequently leads to protein denaturation. However, it is demonstrated that albumin in water-in-silicone oil emulsions retains its native structure in the presence of a functional, triethoxysilyl-terminated silicone polymer, TES-PDMS. Both HSA and TES-PDMS were essential for the formation of stable water-in-silicone oil emulsions: attempts to generate stable emulsions using independently either the protein or the functionalized silicone as a surfactant failed. Confocal microscopy indicated that the human serum albumin (HSA) preferentially adsorbed at the oil/water interface, even in the presence of another protein (glucose oxidase). A variety of experiments demonstrated that the hydrolysis of the Si-OEt groups on the functional silicone occurred only to a limited extent, consistent with the absence of a covalent linkage between the silicone and protein, or of cross-linked silicones at the interface. The fluorescence spectra of HSA extracted from the emulsions, front-faced fluorescence experiments on the HSA/silicone emulsion itself, and HSA/salicylate binding studies all demonstrated that the stability of the water/oil interface decreased as the protein began to unfold: unfolding of the protein in the emulsion was slower than in aqueous solution. The experimental evidence indicated that the interaction between HSA and TES-PDMS is not associated with either homomolecular (HSA/HSA; TES-PDMS/TES-PDMS) interactions or with covalent linkage between two the polymers. Rather, the data is consistent with the direct binding of unhydrolyzed Si(OEt) 3 groups to native HSA. The nature of these interactions is discussed.

  20. Investigation of interaction of antibacterial drug sulfamethoxazole with human serum albumin by molecular modeling and multi-spectroscopic method.

    PubMed

    Wang, Qin; Zhang, Sheng-Rui; Ji, Xiaohui

    2014-04-24

    Interaction of sulfamethoxazole (SMX) with human serum albumin (HSA) was investigated by molecular modeling and multi-spectroscopic methods under physiological conditions. The interaction mechanism was firstly predicted through molecular modeling that confirmed the interaction between SMX and HSA. The binding parameters and the thermodynamic parameters at different temperatures for the reaction had been calculated according to the Stern-Volmer, Hill, Scatchard and the Van't Hoff equations, respectively. One independent class of binding site existed during the interaction between HSA and SMX. The binding constants decreased with the increasing temperatures, which meant that the quenching mechanism was a static quenching. The thermodynamic parameters of the reaction, namely standard enthalpy ΔH(0) and entropy ΔS(0), had been calculated to be -16.40 kJ mol(-1) and 32.33 J mol(-1) K(-1), respectively, which suggested that the binding process was exothermic, enthalpy driven and spontaneous. SMX bound to HSA was mainly based on electrostatic interaction, but hydrophobic interactions and hydrogen bonds could not be excluded from the binding. The conformational changes of HSA in the presence of SMX were confirmed by the three-dimensional fluorescence spectroscopy, UV-vis absorption spectroscopy and circular dichroism (CD) spectroscopy. CD data suggested that the protein conformation was altered with the reduction of α-helices from 55.37% to 41.97% at molar ratio of SMX/HSA of 4:1.

  1. Characterization of erythrosine B binding to bovine serum albumin and bilirubin displacement.

    PubMed

    Mathavan, Vinodaran M K; Boh, Boon Kim; Tayyab, Saad

    2009-08-01

    The interaction of crythrosine B (ErB), a commonly used dye for coloring foods and drinks, with bovine scrum albumin (BSA) was investigated both in the absence and presence of bilirubin (BR) using absorption and absorption difference spectroscopy. ErB binding to BSA was reflected from a significant red shift of 11 nm in the absorption maximum of ErB (527 nm) with the change in absorbance at lamdamax. Analysis of absorption difference spectroscopic titration results of BSA with increasing concentrations of ErB3 using Benesi-Hildebrand equation gave the association constant, K as 6.9 x 10(4) M(-1). BR displacing action of ErB was revealed by a significant blue shift in the absorption maximum, accompanied by a decrease in absorbance difference at lamdamax in the difference spectrum of BR-BSA complex upon addition of increasing concentrations of ErB. This was further substantiated by fluorescence spectroscopy, as addition of increasing concentrations of ErB to BR-BSA complex caused a significant decrease in fluoresccnce at 510 nm. The results suggest that ErB binds to a site in the vicinity of BR binding site on BSA. Therefore, intake of ErB may increase the risk of hyperbilirubinemia in the healthy subjects.

  2. Urea-induced binding between diclofenac sodium and bovine serum albumin: a spectroscopic insight.

    PubMed

    Dohare, Neeraj; Khan, Abbul Bashar; Athar, Fareeda; Thakur, Sonu Chand; Patel, Rajan

    2016-06-01

    We investigated the interaction of diclofenac sodium (Dic.Na) with bovine serum albumin (BSA) in the absence and presence of urea using different spectroscopic techniques. A fluorescence quenching study revealed that the Stern-Volmer quenching constant decreases in the presence of urea, decreasing further at higher urea concentrations. The binding constant and number of binding sites were also evaluated for the BSA-Dic.Na interaction system in the absence and presence of urea using a modified Stern-Volmer equation. The binding constant is greater at high urea concentrations, as shown by the fluorescence results. In addition, for the BSA-Dic.Na interaction system, a static quenching mechanism was observed, which was further confirmed using time-resolved fluorescence spectroscopy. UV-vis spectroscopy provided information about the formation of a complex between BSA and Dic.Na. Circular dichroism was carried out to explain the conformational changes in BSA induced by Dic.Na in the absence and presence of urea. The presence of urea reduced the α-helical content of BSA as the Dic.Na concentration varied. The distance r between the donor (BSA) and acceptor (Dic.Na) was also obtained in the absence and presence of urea, using fluorescence resonance energy transfer. Copyright © 2015 John Wiley & Sons, Ltd.

  3. Elucidation of the binding sites of two novel Ru(II) complexes on bovine serum albumin.

    PubMed

    Nišavić, Marija; Masnikosa, Romana; Butorac, Ana; Perica, Kristina; Rilak, Ana; Korićanac, Lela; Hozić, Amela; Petković, Marijana; Cindrić, Mario

    2016-06-01

    Hyphenated mass spectrometry (MS) techniques have attained an important position in analysis of covalent and non-covalent interactions of metal complexes with peptides and proteins. The aim of the present study was to qualitatively and quantitatively determine ruthenium binding sites on a protein using tandem mass spectrometry and allied techniques, i.e. liquid chromatography (LC) and inductively coupled plasma optical emission spectrometry (ICP-OES). For that purpose, two newly synthesized Ru(II) complexes of a meridional geometry, namely mer-[Ru(4' Cl-tpy)(en)Cl](+) (1) and mer-[Ru(4' Cl-tpy)(dach)Cl](+) (2) (where 4' Cl-tpy=4'-chloro-2,2':6',2″-terpyridine, en=1,2-diaminoethane and dach=1,2-diaminocyclohexane), and bovine serum albumin were used. The binding of the complexes to the protein was investigated by means of size exclusion- and reversed phase-LC, ICP OES, matrix-assisted laser desorption ionization MS and MS/MS. Ruthenated peptide sequence and a binding target amino acid were revealed through accurate elucidation of MS/MS spectra. The results obtained in this study suggest a high binding capacity of the protein towards both complexes, with up to 5.77±0.14 and 6.95±0.43mol of 1 and 2 bound per mol of protein, respectively. The proposed binding mechanism for the selected complexes includes the release of Cl ligand, its replacement with water molecule and further coordination to electron donor histidine residue.

  4. Insight into the interaction of antitubercular and anticancer compound clofazimine with human serum albumin: spectroscopy and molecular modelling.

    PubMed

    Ajmal, Mohammad Rehan; Zaidi, Nida; Alam, Parvez; Nusrat, Saima; Siddiqi, Mohd Khursheed; Badr, Gamal; Mahmoud, Mohamed H; Khan, Rizwan Hasan

    2017-01-01

    The binding of clofazimine to human serum albumin (HSA) was investigated by applying optical spectroscopy and molecular docking methods. Fluorescence quenching data revealed that clofazimine binds to protein with binding constant in the order of 10(4) M(-1), and with the increase in temperature, Stern-Volmer quenching constants gradually decreased indicating quenching mode to be static. The UV-visible spectra showed increase in absorbance upon interaction of HSA with clofazimine which further reveals formation of the drug-albumin complex. Thermodynamic parameters obtained from fluorescence data indicate that the process is exothermic and spontaneous. Forster distance (Ro) obtained from fluorescence resonance energy transfer is found to be 2.05 nm. Clofazimine impelled rise in α-helical structure in HSA as observed from far-UV CD spectra while there are minor alterations in tertiary structure of the protein. Clofazimine interacts strongly with HSA inducing secondary structure in the protein and slight alterations in protein topology as suggested by dynamic light scattering results. Moreover, docking results indicate that clofazimine binds to hydrophobic pocket near to the drug site II in HSA.

  5. Studies on the interactions of 3,6-diaminoacridine derivatives with human serum albumin by fluorescence spectroscopy.

    PubMed

    Gökoğlu, Elmas; Kıpçak, Fulya; Seferoğlu, Zeynel

    2014-11-01

    This study reports the preparation and investigation of the modes of binding of the two symmetric 3,6-diaminoacridine derivatives obtained from proflavine, which are 3,6-diphenoxycarbonyl aminoacridine and 3,6-diethoxycarbonyl aminoacridine to human serum albumin (HSA). The interaction of HSA with the derivatives was investigated using fluorescence quenching and ultraviolet-visible absorption spectra at pH 7.2 and different temperatures. The results suggest that the derivatives used can interact strongly with HSA and are the formation of HSA-derivative complexes and hydrophobic interactions as the predominant intermolecular forces in stabilizing for each complex. The Stern-Volmer quenching constants, binding constants, binding sites and corresponding thermodynamic parameters ΔH, ΔS and ΔG were calculated at different temperatures. The binding distance (r) ~ 3 nm between the donor (HSA) and acceptors (3,6-diethoxycarbonyl aminoacridine, 3,6-diphenoxycarbonyl aminoacridine and proflavine) was obtained according to Förster's non-radiative energy transfer theory. Moreover, the limit of detection and limit of quantification of derivatives were calculated in the presence of albumin.

  6. Binding studies of lophirone B with bovine serum albumin (BSA): Combination of spectroscopic and molecular docking techniques

    NASA Astrophysics Data System (ADS)

    Chaves, Otávio Augusto; da Silva, Veridiana A.; Sant'Anna, Carlos Maurício R.; Ferreira, Aurélio B. B.; Ribeiro, Tereza Auxiliadora N.; de Carvalho, Mário G.; Cesarin-Sobrinho, Dari; Netto-Ferreira, José Carlos

    2017-01-01

    The interaction between the transport protein bovine serum albumin (BSA) and the natural product lophirone B, was investigated by spectroscopic techniques combined with a computational method (molecular docking). From the KSV and kq values it was concluded that lophirone B quenches the fluorescence of BSA by dynamic and static mechanisms. The Ka values, of the order of 104 M-1, and the number of binding sites (n ≈ 1), indicate that the binding is moderate and there is just one main binding site in BSA for lophirone B. The negative ΔG° values are in accordance with the spontaneity of the process and the positive ΔH° and ΔS° values indicate that the binding is entropically driven; the main binding forces for the association BSA:lophirone B are probably lipophilic interactions. Circular dichroism (CD) studies show there is not a significant perturbation on the secondary structure of the albumin upon the binding process. In order to better understand the spectroscopic results, a computational method was applied: molecular docking suggests Trp-213 site, as the main binding site for the ligand. Lophirone B seems to be exposed to the aqueous media as well as accommodated inside the protein cavity, resulting in a moderate affinity for the albumin. The Arg-198, His-287, Lys-294 and Lys-439 residues are interacting via hydrogen bonding with lophirone B, whereas the interaction with Trp-213 residue occurs through a lipophilic interaction.

  7. A Comprehensive Spectroscopic and Computational Investigation to Probe the Interaction of Antineoplastic Drug Nordihydroguaiaretic Acid with Serum Albumins.

    PubMed

    Nusrat, Saima; Siddiqi, Mohammad Khursheed; Zaman, Masihuz; Zaidi, Nida; Ajmal, Mohammad Rehan; Alam, Parvez; Qadeer, Atiyatul; Abdelhameed, Ali Saber; Khan, Rizwan Hasan

    2016-01-01

    Exogenous drugs that are used as antidote against chemotheray, inflammation or viral infection, gets absorbed and interacts reversibly to the major serum transport protein i.e. albumins, upon entering the circulatory system. To have a structural guideline in the rational drug designing and in the synthesis of drugs with greater efficacy, the binding mechanism of an antineoplastic and anti-inflammatory drug Nordihydroguaiaretic acid (NDGA) with human and bovine serum albumins (HSA & BSA) were examined by spectroscopic and computational methods. NDGA binds to site II of HSA with binding constant (Kb) ~105 M-1 and free energy (ΔG) ~ -7.5 kcal.mol-1. It also binds at site II of BSA but with lesser binding affinity (Kb) ~105 M-1 and ΔG ~ -6.5 kcal.mol-1. The negative value of ΔG, ΔH and ΔS for both the albumins at three different temperatures confirmed that the complex formation process between albumins and NDGA is spontaneous and exothermic. Furthermore, hydrogen bonds and hydrophobic interactions are the main forces involved in complex formation of NDGA with both the albumins as evaluated from fluorescence and molecular docking results. Binding of NDGA to both the albumins alter the conformation and causes minor change in the secondary structure of proteins as indicated by the CD spectra.

  8. A Comprehensive Spectroscopic and Computational Investigation to Probe the Interaction of Antineoplastic Drug Nordihydroguaiaretic Acid with Serum Albumins

    PubMed Central

    Nusrat, Saima; Siddiqi, Mohammad Khursheed; Zaman, Masihuz; Zaidi, Nida; Ajmal, Mohammad Rehan; Alam, Parvez; Qadeer, Atiyatul; Abdelhameed, Ali Saber

    2016-01-01

    Exogenous drugs that are used as antidote against chemotheray, inflammation or viral infection, gets absorbed and interacts reversibly to the major serum transport protein i.e. albumins, upon entering the circulatory system. To have a structural guideline in the rational drug designing and in the synthesis of drugs with greater efficacy, the binding mechanism of an antineoplastic and anti-inflammatory drug Nordihydroguaiaretic acid (NDGA) with human and bovine serum albumins (HSA & BSA) were examined by spectroscopic and computational methods. NDGA binds to site II of HSA with binding constant (Kb) ~105 M-1 and free energy (ΔG) ~ -7.5 kcal.mol-1. It also binds at site II of BSA but with lesser binding affinity (Kb) ~105 M-1 and ΔG ~ -6.5 kcal.mol-1. The negative value of ΔG, ΔH and ΔS for both the albumins at three different temperatures confirmed that the complex formation process between albumins and NDGA is spontaneous and exothermic. Furthermore, hydrogen bonds and hydrophobic interactions are the main forces involved in complex formation of NDGA with both the albumins as evaluated from fluorescence and molecular docking results. Binding of NDGA to both the albumins alter the conformation and causes minor change in the secondary structure of proteins as indicated by the CD spectra. PMID:27391941

  9. Development of an affinity silica monolith containing human serum albumin for chiral separations.

    PubMed

    Mallik, Rangan; Hage, David S

    2008-04-14

    An affinity monolith based on silica and containing immobilized human serum albumin (HSA) was developed and evaluated in terms of its binding, efficiency and selectivity in chiral separations. The results were compared with data obtained for the same protein when used as a chiral stationary phase with HPLC-grade silica particles or a monolith based on a copolymer of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA). The surface coverage of HSA in the silica monolith was similar to values obtained with silica particles and a GMA/EDMA monolith. However, the higher surface area of the silica monolith gave a material that contained 1.3-2.2-times more immobilized HSA per unit volume when compared to silica particles or a GMA/EDMA monolith. The retention, efficiency and resolving power of the HSA silica monolith were evaluated using two chiral analytes: d/l-tryptophan and R/S-warfarin. The separation of R- and S-ibuprofen was also considered. The HSA silica monolith gave higher retention and higher or comparable resolution and efficiency when compared with HSA columns that contained silica particles or a GMA/EDMA monolith. The silica monolith also gave lower back pressures and separation impedances than these other materials. It was concluded that silica monoliths can be valuable alternatives to silica particles or GMA/EDMA monoliths when used with immobilized HSA as a chiral stationary phase.

  10. A diclofenac suppository-nabumetone combination therapy for arthritic pain relief and a monitoring method for the diclofenac binding capacity of HSA site II in rheumatoid arthritis.

    PubMed

    Setoguchi, Nao; Takamura, Norito; Fujita, Ken-ichi; Ogata, Kenji; Tokunaga, Jin; Nishio, Toyotaka; Chosa, Etsuo; Arimori, Kazuhiko; Kawai, Keiichi; Yamamoto, Ryuichi

    2013-03-01

    Diclofenac suppository, a non-steroidal anti-inflammatory drug (NSAID), is used widely in rheumatoid arthritis (RA) patients with severe arthritic pain. As the binding percentage of diclofenac to serum proteins is high, its free (unbound) concentration after rectal administration is low. To increase temporarily the free concentration of diclofenac and to enhance its analgesic effect by inhibiting the protein binding of diclofenac, the analgesic effect of diclofenac was examined before and after the start of an inhibitor administration to RA patients with insufficient control of arthritic pain, and the protein binding capacity of diclofenac was evaluated. Binding experiments were performed by ultrafiltration, and arthritic pain was recorded by the face scale. Free fractions of diazepam and diclofenac were augmented by increasing 6-methoxy-2-naphthylacetic acid (6-MNA; the active metabolite of the NSAID nabumetone) concentrations. The free fraction of diazepam increased after the start of nabumetone administration to RA patients, and arthritic pain relief was observed. These results suggest that 6-MNA has an inhibitory effect on the protein binding of diclofenac and the free fraction of diazepam can be used to evaluate the binding capacity of diclofenac. It is considered that diclofenac suppository-nabumetone combination therapy and the method for protein binding monitoring by diazepam can positively benefit RA patients with insufficient control of arthritic pain.

  11. Structural consistency analysis of recombinant and wild-type human serum albumin

    NASA Astrophysics Data System (ADS)

    Cao, Hui-Ling; Sun, Li-Hua; Liu, Li; Li, Jian; Tang, Lin; Guo, Yun-Zhu; Mei, Qi-Bing; He, Jian-Hua; Yin, Da-Chuan

    2017-01-01

    Recombinant human serum albumin (rHSA) is potential alternatives for human serum albumin (HSA) which may ease severe shortage of HSA worldwide. In theory, rHSA and HSA are the same. Structure decides function. Therefore, the 3D structural consistency analysis of rHSA and HSA is outmost importance, which is the base of their function consistency. In this paper, the crystal structures of rHSA at resolution limit of 2.22 Å and HSA at 2.30 Å were determined by X-ray diffraction (XRD), which were deposited in the Protein Data Bank (PDB) with accession codes 4G03 (rHSA) and 4G04 (HSA). The differences between rHSA and HSA were systematically analyzed from the crystallization behavior, diffraction data and three-dimensional (3D) structure. The superimposed contrasted analysis indicated that rHSA and HSA achieved a structural similarity of 99% with an r.m.s. deviation of 0.397 Å for the corresponding overall Cα atoms. In addition, the number of α-helices in the rHSA or HSA molecule was verified to be 30. As a result, rHSA can potentially replace HSA. The study provides a theoretical and experimental basis for the clinical and additional applications of rHSA. Meanwhile, it is also a good example for applications of genetic engineering.

  12. Development and characterization of small bispecific albumin-binding domains with high affinity for ErbB3.

    PubMed

    Nilvebrant, Johan; Astrand, Mikael; Löfblom, John; Hober, Sophia

    2013-10-01

    Affinity proteins based on small scaffolds are currently emerging as alternatives to antibodies for therapy. Similarly to antibodies, they can be engineered to have high affinity for specific proteins. A potential problem with small proteins and peptides is their short in vivo circulation time, which might limit the therapeutic efficacy. To circumvent this issue, we have engineered bispecificity into an albumin-binding domain (ABD) derived from streptococcal Protein G. The inherent albumin binding was preserved while the opposite side of the molecule was randomized for selection of high-affinity binders. Here we present novel ABD variants with the ability to bind to the epidermal growth factor receptor 3 (ErbB3). Isolated candidates were shown to have an extraordinary thermal stability and affinity for ErbB3 in the nanomolar range. Importantly, they were also shown to retain their affinity to albumin, hence demonstrating that the intended strategy to engineer bispecific single-domain proteins against a tumor-associated receptor was successful. Moreover, competition assays revealed that the new binders could block the natural ligand Neuregulin-1 from binding to ErbB3, indicating a potential anti-proliferative effect. These new binders thus represent promising candidates for further development into ErbB3-signaling inhibitors, where the albumin interaction could result in prolonged in vivo half-life.

  13. Evaluation of the binding interaction between bovine serum albumin and dimethyl fumarate, an anti-inflammatory drug by multispectroscopic methods

    NASA Astrophysics Data System (ADS)

    Jattinagoudar, Laxmi; Meti, Manjunath; Nandibewoor, Sharanappa; Chimatadar, Shivamurti

    2016-03-01

    The information of the quenching reaction of bovine serum albumin with dimethyl fumarate is obtained by multi-spectroscopic methods. The number of binding sites, n and binding constants, KA were determined at different temperatures. The effect of increasing temperature on Stern-Volmer quenching constants (KD) indicates that a dynamic quenching mechanism is involved in the interaction. The analysis of thermodynamic quantities namely, ∆H° and ∆S° suggested hydrophobic forces playing a major role in the interaction between dimethyl fumarate and bovine serum albumin. The binding site of dimethyl fumarate on bovine serum albumin was determined by displacement studies, using the site probes viz., warfarin, ibuprofen and digitoxin. The determination of magnitude of the distance of approach for molecular interactions between dimethyl fumarate and bovine serum albumin is calculated according to the theory of Förster energy transfer. The CD, 3D fluorescence spectra, synchronous fluorescence measurements and FT-IR spectral results were indicative of the change in secondary structure of the protein. The influence of some of the metal ions on the binding interaction was also studied.

  14. 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.

  15. NMR metabolomics profiling of blood plasma mimics shows that medium- and long-chain fatty acids differently release metabolites from human serum albumin.

    PubMed

    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.

  16. Combined albumin and bicarbonate induces head-to-head sperm agglutination which physically prevents equine sperm-oviduct binding.

    PubMed

    Leemans, Bart; Gadella, Bart M; Stout, Tom A E; Sostaric, Edita; De Schauwer, Catharina; Nelis, Hilde; Hoogewijs, Maarten; Van Soom, Ann

    2016-04-01

    In many species, sperm binding to oviduct epithelium is believed to be an essential step in generating a highly fertile capacitated sperm population primed for fertilization. In several mammalian species, this interaction is based on carbohydrate-lectin recognition. D-galactose has previously been characterized as a key molecule that facilitates sperm-oviduct binding in the horse. We used oviduct explant and oviduct apical plasma membrane (APM) assays to investigate the effects of various carbohydrates; glycosaminoglycans; lectins; S-S reductants; and the capacitating factors albumin, Ca(2+) and HCO3(-) on sperm-oviduct binding in the horse. Carbohydrate-specific lectin staining indicated that N-acetylgalactosamine, N-acetylneuraminic acid (sialic acid) and D-mannose or D-glucose were the most abundant carbohydrates on equine oviduct epithelia, whereas D-galactose moieties were not detected. However, in a competitive binding assay, sperm-oviduct binding density was not influenced by any tested carbohydrates, glycosaminoglycans, lectins or D-penicillamine, nor did the glycosaminoglycans induce sperm tail-associated protein tyrosine phosphorylation. Furthermore, N-glycosidase F (PNGase) pretreatment of oviduct explants and APM did not alter sperm-oviduct binding density. By contrast, a combination of the sperm-capacitating factors albumin and HCO3(-) severely reduced (>10-fold) equine sperm-oviduct binding density by inducing rapid head-to-head agglutination, both of which events were independent of Ca(2+) and an elevated pH (7.9). Conversely, neither albumin and HCO3(-) nor any other capacitating factor could induce release of oviduct-bound sperm. In conclusion, a combination of albumin and HCO3(-) markedly induced sperm head-to-head agglutination which physically prevented stallion sperm to bind to oviduct epithelium.

  17. Comparative serum albumin interactions and antitumor effects of Au(III) and Ga(III) ions.

    PubMed

    Sarioglu, Omer Faruk; Ozdemir, Ayse; Karaboduk, Kuddusi; Tekinay, Turgay

    2015-01-01

    In the present study, interactions of Au(III) and Ga(III) ions on human serum albumin (HSA) were studied comparatively via spectroscopic and thermal analysis methods: UV-vis absorbance spectroscopy, fluorescence spectroscopy, Fourier transform infrared (FT-IR) spectroscopy and isothermal titration calorimetry (ITC). The potential antitumor effects of these ions were studied on MCF-7 cells via Alamar blue assay. It was found that both Au(III) and Ga(III) ions can interact with HSA, however; Au(III) ions interact with HSA more favorably and with a higher affinity. FT-IR second derivative analysis results demonstrated that, high concentrations of both metal ions led to a considerable decrease in the α-helix content of HSA; while Au(III) led to around 5% of decrease in the α-helix content at 200μM, it was around 1% for Ga(III) at the same concentration. Calorimetric analysis gave the binding kinetics of metal-HSA interactions; while the binding affinity (Ka) of Au(III)-HSA binding was around 3.87×10(5)M(-1), it was around 9.68×10(3)M(-1) for Ga(III)-HSA binding. Spectroscopy studies overall suggest that both metal ions have significant effects on the chemical structure of HSA, including the secondary structure alterations. Antitumor activity studies on MCF7 tumor cell line with both metal ions revealed that, Au(III) ions have a higher antiproliferative activity compared to Ga(III) ions.

  18. 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.

  19. Structural basis of albumin-thyroxine interactions and familial dysalbuminemic hyperthyroxinemia.

    PubMed

    Petitpas, Isabelle; Petersen, Charles E; Ha, Chung-Eun; Bhattacharya, Ananyo A; Zunszain, Patricia A; Ghuman, Jamie; Bhagavan, Nadhipuram V; Curry, Stephen

    2003-05-27

    Human serum albumin (HSA) is the major protein component of blood plasma and serves as a transporter for thyroxine and other hydrophobic compounds such as fatty acids and bilirubin. We report here a structural characterization of HSA-thyroxine interactions. Using crystallographic analyses we have identified four binding sites for thyroxine on HSA distributed in subdomains IIA, IIIA, and IIIB. Mutation of residue R218 within subdomain IIA greatly enhances the affinity for thyroxine and causes the elevated serum thyroxine levels associated with familial dysalbuminemic hyperthyroxinemia (FDH). Structural analysis of two FDH mutants of HSA (R218H and R218P) shows that this effect arises because substitution of R218, which contacts the hormone bound in subdomain IIA, produces localized conformational changes to relax steric restrictions on thyroxine binding at this site. We have also found that, although fatty acid binding competes with thyroxine at all four sites, it induces conformational changes that create a fifth hormone-binding site in the cleft between domains I and III, at least 9 A from R218. These structural observations are consistent with binding data showing that HSA retains a high-affinity site for thyroxine in the presence of excess fatty acid that is insensitive to FDH mutations.

  20. Binding of PFOS to serum albumin and DNA: insight into the molecular toxicity of perfluorochemicals

    PubMed Central

    Zhang, Xian; Chen, Ling; Fei, Xun-Chang; Ma, Yin-Sheng; Gao, Hong-Wen

    2009-01-01

    Background Health risk from exposure of perfluorochemicals (PFCs) to wildlife and human has been a subject of great interest for understanding their molecular mechanism of toxicity. Although much work has been done, the toxigenicity of PFCs remains largely unknown. In this work, the non-covalent interactions between perfluorooctane sulfonate (PFOS) and serum albumin (SA) and DNA were investigated under normal physiological conditions, aiming to elucidate the toxigenicity of PFCs. Results In equilibrium dialysis assay, the bindings of PFOS to SA correspond to the Langmuir isothermal model with two-step sequence model. The saturation binding number of PFOS was 45 per molecule of SA and 1 per three base-pairs of DNA, respectively. ITC results showed that all the interactions were spontaneous driven by entropy change. Static quenching of the fluorescence of SA was observed when interacting with PFOS, indicating PFOS bound Trp residue of SA. CD spectra of SA and DNA changed obviously in the presence of PFOS. At normal physiological conditions, 1.2 mmol/l PFOS reduces the binding ratio of Vitamin B2 to SA by more than 30%. Conclusion The ion bond, van der Waals force and hydrophobic interaction contributed to PFOS binding to peptide chain of SA and to the groove bases of DNA duplex. The non-covalent interactions of PFOS with SA and DNA alter their secondary conformations, with the physiological function of SA to transport Vitamin B2 being inhibited consequently. This work provides a useful experimental method for further studying the toxigenicity of PFCs. PMID:19239717

  1. The binding of analogs of porphyrins and chlorins with elongated side chains to albumin

    PubMed Central

    Ben Dror, Shimshon; Bronshtein, Irena; Weitman, Hana; Smith, Kevin M.; O’Neal, William G.; Jacobi, Peter A.; Ehrenberg, Benjamin

    2012-01-01

    In previous studies, we demonstrated that elongation of side chains of several sensitizers endowed them with higher affinity for artificial and natural membranes and caused their deeper localization in membranes. In the present study, we employed eight hematoporphyrin and protoporphyrin analogs and four groups containing three chlorin analogs each, all synthesized with variable numbers of methylenes in their alkyl carboxylic chains. We show that these tetrapyrroles’ affinity for bovine serum albumin (BSA) and their localization in the binding site are also modulated by chain lengths. The binding constants of the hematoporphyrins and protoporphyrins to BSA increased as the number of methylenes was increased. The binding of the chlorins depended on the substitution at the meso position opposite to the chains. The quenching of the sensitizers’ florescence by external iodide ions decreased as the side chains became longer, indicating to deeper insertion of the molecules into the BSA binding pocket. To corroborate this conclusion, we studied the efficiency of photodamage caused to tryptophan in BSA upon illumination of the bound sensitizers. The efficiency was found to depend on the side-chain lengths of the photosensitizer. We conclude that the protein site that hosts these sensitizers accommodates different analogs at positions that differ slightly from each other. These differences are manifested in the ease of access of iodide from the external aqueous phase, and in the proximity of the photosensitizers to the tryptophan. In the course of this study, we developed the kinetic equations that have to be employed when the sensitizer itself is being destroyed. PMID:19330323

  2. Loss of albumin and megalin binding to renal cubilin in rats results in albuminuria after total body irradiation.

    PubMed

    Yammani, Raghunatha R; Sharma, Mukut; Seetharam, Shakuntla; Moulder, John E; Dahms, Nancy M; Seetharam, Bellur

    2002-08-01

    The role of the renal apical brush-border membrane (BBM) endocytic receptors cubilin and megalin in the onset of albuminuria in rats exposed to a single dose of total body irradiation (TBI) has been investigated. Albuminuria was evident as immunoblot (IB) analysis of the urine samples from TBI rats revealed excretion of large amounts of albumin. IB analysis of the BBM proteins did not reveal any significant changes in cubilin or megalin levels, but (125)I-albumin binding to BBM from TBI rats declined by 80% with a fivefold decrease (from 0.5 to 2.5 microM) in the affinity for albumin. IB analysis of cubilin from the BBM demonstrated a 75% loss when purified using albumin, but not intrinsic factor (IF)-cobalamin (Cbl) ligand affinity chromatography. Immunoprecipitation (IP) of Triton X-100 extract of the BBM with antiserum to cubilin followed by IB of the immune complex with an antiserum to megalin revealed a 75% loss of association between megalin and cubilin. IP studies with antiserum to cubilin or megalin and IB with antiserum to the cation-independent mannose 6-phosphate/insulin-like growth factor II-receptor (CIMPR) revealed that CIMPR interacted with both cubilin and megalin. In addition, TBI did not disrupt the association of CIMPR with either cubilin or megalin in BBM. These results suggest that albuminuria noted in TBI rats is due to selective loss of albumin and megalin, but not CIMPR or IF-Cbl binding by cubilin. Furthermore, these results also suggest that albumin and IF-Cbl binding to cubilin occur at distinct sites and that in the rat renal BBM, CIMPR interacts with both cubilin and megalin.

  3. Binding thermodynamics of synthetic dye Allura Red with bovine serum albumin.

    PubMed

    Lelis, Carini Aparecida; Hudson, Eliara Acipreste; Ferreira, Guilherme Max Dias; Ferreira, Gabriel Max Dias; da Silva, Luis Henrique Mendes; da Silva, Maria do Carmo Hespanhol; Pinto, Maximiliano Soares; Pires, Ana Clarissa Dos Santos

    2017-02-15

    The interaction between Allura Red and bovine serum albumin (BSA) was studied in vitro at pH 7.4. The fluorescence quenching was classified as static quenching due to the formation of AR-BSA complex, with binding constant (K) ranging from 3.26±0.09 to 8.08±0.0610(4)L.mol(-1), at the warfarin binding site of BSA. This complex formation was driven by increasing entropy. Isothermal titration calorimetric measurements also showed an enthalpic contribution. The Allura Red diffusion coefficient determined by the Taylor-Aris technique corroborated these results because it reduced with increasing BSA concentration. Interfacial tension measurements showed that the AR-BSA complex presented surface activity, since interfacial tension of the water-air interface decreased as the colorant concentration increased. This technique also provided a complexation stoichiometry similar to those obtained by fluorimetric experiments. This work contributes to the knowledge of interactions between BSA and azo colorants under physiological conditions.

  4. Binding interaction of quinclorac with bovine serum albumin: A biophysical study

    NASA Astrophysics Data System (ADS)

    Han, Xiao-Le; Mei, Ping; Liu, Yi; Xiao, Qi; Jiang, Feng-Lei; Li, Ran

    2009-10-01

    Quinclorac (QUC) is a new class of highly selective auxin herbicides. The interaction between QUC and bovine serum albumin (BSA) was investigated by fluorescence spectroscopy, synchronous fluorescence, three-dimensional fluorescence, CD spectroscopy and UV-vis absorption spectroscopy under simulative physiological condition. It was proved that the probable quenching mechanism of BSA by quinclorac was dynamic quenching. The Stern-Volmer quenching model has been successfully applied and the activation energy of the interaction as much as 8.03 kJ mol -1, corresponding thermodynamic parameters Δ Hθ, Δ Sθ and Δ Gθ were calculated. The results indicated that the acting forces between QUC and BSA were mainly hydrogen bonding and van der Waals forces. According to the Förster non-radiation energy transfer theory, the average binding distance between donor (BSA) and acceptor (QUC) was obtained ( r = 3.12 nm). The alterations of protein secondary structure in the presence of QUC were confirmed by the evidences from three-dimensional fluorescence, synchronous fluorescence and CD spectroscopy. Furthermore, the site marker competitive experiments indicated that the binding of QUC to BSA primarily took place in Sudlow site I.

  5. Ellipsometric studies of synthetic albumin-binding chitosan-derivatives and selected blood plasma proteins

    NASA Astrophysics Data System (ADS)

    Sarkar, Sabyasachi

    This dissertation summarizes work on the synthesis of chitosan-derivatives and the development of ellipsometric methods to characterize materials of biological origin. Albumin-binding chitosan-derivatives were synthesized via addition reactions that involve amine groups naturally present in chitosan. These surfaces were shown to have an affinity towards human serum albumin via ELISA, UV spectroscopy and SDS PAGE. Modified surfaces were characterized with IR ellipsometry at various stages of their synthesis using appropriate optical models. It was found that spin cast chitosan films were anisotropic in nature. All optical models used for characterizing chitosan-derivatives were thus anisotropic. Chemical signal dependence on molecular structure and composition was illustrated via IR spectroscopic ellipsometry (IRSE). An anisotropic optical model of an ensemble of Lorentz oscillators were used to approximate material behavior. The presence of acetic acid in spin-cast non-neutralized chitosan samples was thus shown. IRSE application to biomaterials was also demonstrated by performing a step-wise chemical characterizations during synthesis stages. Protein adsorbed from single protein solutions on these modified surfaces was monitored by visible in-situ variable wavelength ellipsometry. Based on adsorption profiles obtained from single protein adsorption onto silicon surfaces, lumped parameter kinetic models were developed. These models were used to fit experimental data of immunoglobulin-G of different concentrations and approximate conformational changes in fibrinogen adsorption. Biomaterial characterization by ellipsometry was further extended to include characterization of individual protein solutions in the IR range. Proteins in an aqueous environment were characterized by attenuated total internal reflection (ATR) IR ellipsometry using a ZnSe prism. Parameterized dielectric functions were created for individual proteins using Lorentz oscillators. These

  6. Multi-spectroscopic and molecular modelling approach to investigate the interaction of riboflavin with human serum albumin.

    PubMed

    Alam, Md Maroof; Abul Qais, Faizan; Ahmad, Iqbal; Alam, Parvez; Hasan Khan, Rizwan; Naseem, Imrana

    2017-03-09

    Riboflavin (RF) plays an important role in various metabolic redox reactions in the form of flavin adenine dinucleotide and flavin mononucleotide. Human serum albumin (HSA) is an important protein involved in the transportation of drugs, hormones, fatty acid and other molecules which determine the biodistribution and physiological fate of these molecules. In this study, we have investigated the interaction of riboflavin RF with HSA under simulative physiological conditions using various biophysical, calorimetric and molecular docking techniques. Results demonstrate the formation of riboflavin-HSA complex with binding constant in the order of 10(4) M(-1). Fluorescence spectroscopy confirms intermediate strength having a static mode of quenching with stoichiometry of 1:1. Experimental results suggest that the binding site of riboflavin mainly resides in sub-domain IIA of HSA and that ligand interaction increases the α-helical content of HSA. These parameters were further verified by isothermal titration calorimetry ITC which confirms the thermodynamic parameters obtained by fluorescence spectroscopy. Molecular docking was employed to suggest a binding model. Based on thermodynamic, spectroscopic and computational observations it can be concluded that HSA-riboflavin complex is mainly stabilized by various non-covalent forces with binding energy of -7.2 kcal mol(-1).

  7. New insights into non-enzymatic glycation of human serum albumin biopolymer: A study to unveil its impaired structure and function.

    PubMed

    Raghav, Alok; Ahmad, Jamal; Alam, Khursheed; Khan, Asad U

    2017-03-18

    Albumin glycation and subsequent formation of advanced glycation end products (AGEs) correlate with diabetes and associated complications. Human Serum Albumin (HSA) was modified with d-glucose for a 40day period under sterile conditions at 37°C. Modified samples along with native HSA (unmodified) were analyzed for impairment in biochemical characteristics ((fructosamine, carbonyl, thiol, lysine, arginine, hydroxymethyl furfural [HMF] content), electrochemical (electrical conductance), simulated gastric fluid assay, simulated intestinal fluid assay, spectroscopic properties (UV, fluorescence), optical (surface contact angle) and fluid dynamics (viscosity and Stokes radius). Impairment in drug binding capacity of glycosylated-HSA was assessed with molecular docking experiment using metformin. UV-absorbance and fluorescence measurement were performed for drug bound glycated and native-HSA. Extensively modified HSA has been used to study its relevance in diabetes mellitus. Glucose modified-HSA resulted in AGEs formation. It suggests deleterious impairment in biochemical, electrochemical, spectroscopic, optical and fluidity properties of HSA at high concentrations of glucose. The results of the present study can be useful to understand the phenomenon of proteins damage in hyperglycemic conditions.

  8. Multi-spectroscopic and molecular modeling studies of interaction between two different angiotensin I converting enzyme inhibitory peptides from gluten hydrolysate and human serum albumin.

    PubMed

    Assaran Darban, Reza; Shareghi, Behzad; Asoodeh, Ahmad; Chamani, Jamshidkhan

    2016-12-26

    The present study was carried out to characterize Angiotensin-converting enzyme (ACE) inhibitory peptides which are released from the trypsin hydrolysate of wheat gluten protein. The binding of two inhibitory peptide (P4 and P6) to human serum albumin (HSA) under physiological conditions has been investigated by multi-spectroscopic in combination with molecular modeling techniques. Time-resolved and quenching fluorescence spectroscopies results revealed that the quenching of HSA fluorescence by P4 and P6 in the binary and ternary systems caused HSA-peptides complexes formation. The results indicated that both peptides quenched the fluorescence intensity of HSA through a static mechanism. The binding affinities and number of binding sites were obtained for the HSA-peptides complexes. The circular dichroism (CD) data revealed that the presence of both peptides increased the α-helix content of HSA and induced the remarkable folding of the polypeptide of the protein. Therefore, the CD data determined that the protein structure has been stabilized in the percent of ACE inhibitory peptides in binary and ternary systems. The binding distances between HSA and both peptides were estimated by the Forster theory, and it was revealed that nonradiative energy transfer from HSA to peptides occurred with a high probability. ITC experiments reveal that, in the absence and presence of P6, the dominant forces are electrostatic in binary and ternary systems. Furthermore, molecular modeling studies confirmed the experimental results. Molecular modeling investigation suggested that P4 bound to the site IA and IIA of HSA in binary and ternary systems, respectively. This study on the interaction of peptides with HSA should prove helpful for realizing the distribution and transportation of food compliments and drugs in vivo, elucidating the action mechanism and dynamics of food compliments and drugs at the molecular level. It should moreover be of great use for understanding the

  9. Differential modulation in binding of ketoprofen to bovine serum albumin in the presence and absence of surfactants: spectroscopic and calorimetric insights.

    PubMed

    Misra, Pinaki P; Kishore, Nand

    2013-07-01

    Surfactants have long been implicated in the unique static and dynamic effect on the structure and function of serum albumins. However, there is very little information on the mode of interactions of drugs to serum albumins in presence of surfactants. The importance of such studies lay in the fact that apart from binding to serum albumins, surfactants are known to radically influence the solvents' micro environment and protein structure. Thus, we have studied the binding of the racemic form of ketoprofen with bovine serum albumin at pH 7.4 in the presence and absence of hexadecyl trimethyl ammonium bromide, sodium dodecyl sulfate, Triton X-100, and NaCl. The structural studies of ketoprofen with bovine serum albumin as investigated by circular dichroism spectroscopy revealed a significant stabilization of bovine serum albumin. However, the combined presence of the surfactants, NaCl and ketoprofen, demonstrated an extremely erratic behavior in terms of stabilization. Further the values of Stern-Volmer and dynamic quenching constant suggested the binding site of ketoprofen to be scattered in the region of domain I B and II A, close to Trp 134. The results of differential scanning calorimetry revealed that the binding of ketoprofen to bovine serum albumin leads to its temperature-dependent separation into two units. The binding parameters of bovine serum albumin obtained from isothermal titration calorimetry in the combined presence of ketoprofen and surfactants/NaCl correlate well with the differential scanning calorimetry studies further confirming the localization of ketoprofen in domain I B and II A. In the combined presence of surfactants, NaCl and ketoprofen, the binding of ketoprofen to bovine serum albumin exhibited altered binding parameters far different from the binding of ketoprofen alone. Overall, the experimental findings strongly indicated positive as well as negative modulation in the binding of ketoprofen to bovine serum albumin in the presence of

  10. Spectroscopic studies on the interaction of cinnamic acid and its hydroxyl derivatives with human serum albumin

    NASA Astrophysics Data System (ADS)

    Min, Jiang; Meng-Xia, Xie; Dong, Zheng; Yuan, Liu; Xiao-Yu, Li; Xing, Chen

    2004-04-01

    Cinnamic acid and its derivatives possess various biological effects in remedy of many diseases. Interaction of cinnamic acid and its hydroxyl derivatives, p-coumaric acid and caffeic acid, with human serum albumin (HSA), and concomitant changes in its conformation were studied using fluorescence and Fourier transform infrared spectroscopic methods. Fluorescence data revealed the presence of one binding site on HSA for cinnamic acid and its hydroxyl derivatives, and their binding constants ( KA) are caffeic acid> p-coumaric acid> cinnamic acid when Cdrug/ CHSA ranging from 1 to 10. The changes of the secondary structure of HSA after interacting with the three drugs are estimated, respectively by combining the curve-fitting results of amid I and amid III bands. The α-helix structure has a decrease of ≈9, 5 and 3% after HSA interacted with caffeic acid, p-coumaric acid and cinnamic acid, respectively. It was found that the hydroxyls substituted on aromatic ring of the drugs play an important role in the changes of protein's secondary structure. Combining the result of fluorescence quenching and the changes of secondary structure of HSA after interaction with the three drugs, the drug-HSA interaction mode was discussed.

  11. Recombinant albumin adsorption on mica studied by AFM and streaming potential measurements.

    PubMed

    Kujda, Marta; Adamczyk, Zbigniew; Morga, Maria; Sofińska, Kamila

    2015-03-01

    Recombinant human serum albumin (rHSA) in monomeric state is widely used in pharmaceutical industry as a drug excipient and for preparing coatings for medical devices. In this work the adsorption process of rHSA on model mica surface at pH 3.5 was studied using the atomic force microscopy (AFM) and in situ streaming potential measurements. The kinetics of albumin adsorption was determined by a direct enumeration of single molecules over various substrate areas. These results were consistent with streaming potential measurements carried out for the parallel-plate channel flow and with theoretical predictions derived from the random sequential adsorption (RSA) model. Desorption kinetics of albumin under flow conditions was also evaluated via the streaming potential measurements. In this way, the amount of irreversibly bound albumin was quantitatively evaluated to be 0.64 and 1.2 mg m(-2) for ionic strength of 0.01 and 0.15 M, respectively. This agrees with previous results obtained for HSA and theoretical calculations derived from the RSA model. Additionally, it was demonstrated that there existed a fraction of reversibly bound albumin that can be fully eluted within a few hours. The binding energy of these fraction of molecules was -18 kT that is consistent with the electrostatic controlled adsorption mechanism of albumin at this pH. It was concluded that the rHSA monolayers of well-defined coverage can find applications for quantitatively analyzing ligand binding and for performing efficient biomaterials and immunological tests.

  12. Regulation of inflammation-primed activation of macrophages by two serum factors, vitamin D3-binding protein and albumin.

    PubMed Central

    Yamamoto, N; Kumashiro, R; Yamamoto, M; Willett, N P; Lindsay, D D

    1993-01-01

    A very small amount (0.0005 to 0.001%) of an ammonium sulfate [50% saturated (NH4)2SO4]-precipitable protein fraction of alpha 2-globulin efficiently supported inflammation-primed activation of macrophages. This fraction contains vitamin D3-binding protein essential for macrophage activation. Comparative macrophage activation studies with fetal calf serum, alpha 2-globulin fraction, 50% (NH4)2SO4 precipitate, and purified bovine vitamin D3-binding protein revealed that fetal calf serum and alpha 2-globulin fraction appear to contain an inhibitor for macrophage activation while ammonium sulfate precipitate contains no inhibitor. This inhibitor was found to be serum albumin. When bovine serum albumin (25 micrograms/ml) was added to a medium supplemented with 0.0005 to 0.05% (NH4)2SO4 precipitate or 1 to 10 ng of vitamin D3-binding protein per ml, activation of macrophages was inhibited. PMID:8225612

  13. Surfactant-cobalt(III) complexes: The impact of hydrophobicity on interaction with HSA and DNA - insights from experimental and theoretical approach.

    PubMed

    Veeralakshmi, Selvakumar; Sabapathi, Gopal; Nehru, Selvan; Venuvanalingam, Ponnambalam; Arunachalam, Sankaralingam

    2017-02-16

    To develop surfactant-based metallodrugs, it is very important to know about their hydrophobicity, micelle forming capacity, their interaction with biomacromolecules such as proteins and nucleic acids, and biological activities. Here, diethylenetriamine (dien) and tetradecylamine ligand (TA) based surfactant-cobalt(III) complexes with single chain domain, [Co(dien)(TA)Cl2]ClO4 (1) and double chain domain [Co(dien)(TA)2Cl](ClO4)2 (2) were chosen to study the effect of hydrophobicity on the interaction with human serum albumin and calf thymus DNA. The obtained results showed that (i) single chain surfactant-cobalt(III) complex (1) interact with HSA and DNA via electrostatic interaction and groove binding, respectively; (ii) double chain surfactant-cobalt(III) complex (2) interact with HSA and DNA via hydrophobic interaction and partial intercalation, respectively, due to the play of hydrophobicity by single and double chain domains. Further it is noted that, double chain surfactant-cobalt(III) complex interact strongly with HSA and DNA, compared single chain surfactant-cobalt(III) complex due to their more hydrophobicity nature. DFT and molecular docking studies offer insights into the mechanism and mode of binding towards the molecular target CT-DNA and HSA. Hence, the present findings will create new avenue towards the use of hydrophobic metallodrugs for various therapeutic applications.

  14. Interactions of human serum albumin with doxorubicin in different media

    NASA Astrophysics Data System (ADS)

    Gun'ko, Vladimir M.; Turov, Vladimir V.; Krupska, Tetyana V.; Tsapko, Magdalina D.

    2017-02-01

    Interactions of human serum albumin (10 wt% H2O and 0.3 wt% sodium caprylate) with doxorubicin hydrochloride (1 wt%) were studied alone or with addition of HCl (3.6 wt% HCl) using 1H NMR spectroscopy. A model of hydrated HSA/12DOX was calculated using PM7 method with COSMO showing large variations in the binding constant depending on structural features of DOX/HSA complexes. DOX molecules/ions displace bound water from narrow intramolecular voids in HSA that leads to diminution of freezing-melting point depression of strongly bound water (SBW). Structure of weakly bound water (WBW) depends much weaker on the presence of DOX than SBW because a major fraction of DOX is bound to adsorption sites of HSA. Addition of HCl results in strong changes in structure of macromolecules and organization of water in hydration shells of HSA (i.e., mainly SBW) and in the solution (i.e., WBW + non-bound bulk water).

  15. Chemical Conjugation of Evans Blue Derivative: A Strategy to Develop Long-Acting Therapeutics through Albumin Binding

    PubMed Central

    Chen, Haojun; Wang, Guohao; Lang, Lixin; Jacobson, Orit; Kiesewetter, Dale O.; Liu, Yi; Ma, Ying; Zhang, Xianzhong; Wu, Hua; Zhu, Lei; Niu, Gang; Chen, Xiaoyuan

    2016-01-01

    The efficacy of therapeutic drugs is highly dependent on their optimal in vivo pharmacokinetics. Albumin conjugation is considered to be one of the most effective means of protracting the short lifespan of peptides and proteins. In this study, we proposed a novel platform for developing long lasting therapeutics by conjugating a small molecular albumin binding moiety, truncated Evans blue, to either peptides or proteins. Using the anti-diabetic peptide drug Exendin-4 as a model peptide, we synthesized a new long-acting Exendin-4 derivative (denoted as Abextide). Through complexation with albumin in situ, the biological half-life of Abextide was significantly extended. The hypoglycemic effect of Abextide was also improved remarkably over Exendin-4. Thus, Abextide has considerable potential to treat type 2 diabetes. This strategy as a general technology platform can be applied to other small molecules and biologics for the development of long-acting therapeutic drugs. PMID:26877782

  16. Photoreactions of macrocyclic dyes bound to human serum albumin.

    PubMed

    Davila, J; Harriman, A

    1990-01-01

    The photophysical properties of tetrakis(4-sulfonatophenyl)porphyrin (H2TSPP), its tin (IV) complex (SnTSPP), aluminium(III) trisulfonatophthalocyanine (AIPCS), and the corresponding zinc(II) complex (ZnPCS), have been measured in H2O, D2O, and upon binding to human serum albumin (HSA). The triplet excited states of the various macrocyclic dyes generate singlet molecular oxygen, O2(1 delta g) in high quantum yield upon illumination in O2-saturated solution, even in the presence of HSA. The triplet states also abstract an electron from 4-aminophenol, forming the radical anion of the macrocycle. Quenching rate constants and quantum yields have been measured for the various processes in the presence and absence of HSA. It is found that HSA binds all the dyes at nonspecific sites close to the interface in such a manner that the dyes remain accessible to species residing in the solution phase. Dyes that do not possess axial ligands complexed to the central cation (e.g. H2TSPP, ZnPCS) are able to bind also at a deeper, more specific site on the protein where they are protected from species in solution. Under such conditions, triplet quenching by 4-aminophenol is restricted to long-distance electron tunnelling, for which the rate is relatively slow.

  17. Structural basis and anticancer properties of ruthenium-based drug complexed with human serum albumin.

    PubMed

    Zhang, Yao; Ho, Andy; Yue, Jiping; Kong, Linlin; Zhou, Zuping; Wu, Xiaoyang; Yang, Feng; Liang, Hong

    2014-10-30

    Ruthenium-based anticancer complexes have become increasingly popular for study over the last two decades. Although ruthenium complexes are currently being investigated in clinical trials, there are still some difficulties with their delivery and associated side effects. Human serum albumin (HSA)-based delivery systems are promising for improving anticancer drug targeting and reducing negative side effects. However, there have been few studies regarding the HSA delivery system for metal-based anticancer compounds and no mention of its structural mechanism. Therefore, we studied the structure and anticancer properties of the ruthenium-based compound [RuCl5(ind)](2-) in complex with HSA. The structure revealed that [RuCl5(ind)](2-) has two binding sites in HSA. In the IB subdomain, [RuCl5(ind)](2-) binds to a new sub-site by coordinating with His-146. In the IIA subdomain, ruthenium (III) of [RuCl5(ind)](2-) binds to the hydrophobic cavity and forms coordination bonds by replacing chlorine atoms with the His-242 and Lys-199 residues of HSA. Interestingly, [RuCl5(ind)](2-), together with HSA, can enhance cytotoxicity by two to five times in cancer cells but has no effect on normal cells in vitro. Compared with unbound drug, the HSA-[RuCl5(ind)](2-) complex promotes MGC-803 cell apoptosis and also has a stronger capacity for cell cycle arrest at the G2 phase in MGC-803. In conclusion, this study will guide the rational design and development of ruthenium-containing or ruthenium-centered drugs and an HSA delivery system for ruthenium-based drugs.

  18. Molecular interaction study of flavonoid derivative 3d with human serum albumin using multispectroscopic and molecular modeling approach.

    PubMed

    Wei, Juntong; Jin, Feng; Wu, Qin; Jiang, Yuyang; Gao, Dan; Liu, Hongxia

    2014-08-01

    Human serum albumin (HSA) has been developed as a model protein to study drug-protein interaction. In the present work, the interaction between our synthesized flavonoid derivative 3d (possessing potent antitumor activity against HepG2 cells) and HSA was investigated using fluorescence spectroscopy, circular dichroism spectroscopy, UV-vis spectroscopy and molecular modeling approach. Fluorescence spectroscopy showed that the fluorescence of HSA can be quenched remarkably by 3d under physiological condition with a slight shift of maximum fluorescence emission bands from 360nm to 363nm. Calculated results from Stern-Volmer equation and modified Stern-Volmer equation indicated that the fluorescence was quenched by static quenching processing with association constant 5.26±0.04×10(4)L mol(-1) at 298K. After comprehensive consideration of the free energy change ΔG, enthalpy change ΔH and entropy change ΔS, electrostatic interactions were confirmed as the main factor that participate in stabilizing the 3d-HSA complex. Both dichroism spectroscopy and UV-vis spectroscopy indicated conformational change of HSA after binding to 3d. Moreover, the structure of HSA was loosened and the percentage of α-helix decreased with increasing concentration of 3d. Molecular modeling results demonstrated that 3d could bind to HSA well into subdomain IIA, which is related to its capability of deposition and delivery. Three cation-π interactions and three hydrogen bonds occurred between 3d and amino acid residuals ARG218, ARG222 and LYS199. In conclusion, flavonoid derivative 3d can bind to HSA with noncovalent bond in a relatively stable way, so it can be delivered by HSA in a circulatory system.

  19. Effect of serum albumin presence on the binding constant of metronidazole to the phospholipid membranes fluorescence study

    NASA Astrophysics Data System (ADS)

    Sułkowska, A.

    1999-05-01

    The experiment was designed to test the possibility of entrapping the drug metronidazole into liposome vesicles in order to use liposome vesicles as a drug carrier. We estimated the effect of size of the liposome and the presence of serum albumin on the leakage of the drug. Some interactions between the lipid membrane surface and serum albumin were demonstrated. As the effects of lipid composition and physical states in membranes on protein adsorption and binding are poorly understood, it is difficult to estimate the protein interaction with the lipid membrane surface. The fluorescence quenching technique was used in this investigation. The fluorescence of serum albumin tryptophanyl residues is reduced by the presence of metronidazole. When incorporated into liposomes, metronidazole reduces the fluorescence of tryptophanyl residues of BSA to a lesser extent. The least effect of metronidazole on the fluorescence of albumin is shown when the drug is incorporated into large liposomes (450 nm). Larger liposomes are less susceptible to the presence of serum albumin than the smaller ones. Large size of the liposomes is necessary to retain their stability in plasma.

  20. Asymmetric dimethylarginine (ADMA) in human blood: effects of extended haemodialysis in the critically ill patient with acute kidney injury, protein binding to human serum albumin and proteolysis by thermolysin.

    PubMed

    Sitar, Mustafa Erinc; Kayacelebi, Arslan Arinc; Beckmann, Bibiana; Kielstein, Jan T; Tsikas, Dimitrios

    2015-09-01

    Free, non-protein bound asymmetrically guanidine-dimethylated arginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthesis. Human erythrocytic membrane comprises considerable amounts of large (>50 kDa) ADMA-containing proteins. Location in the erythrocyte membrane and identity and physiological functions of ADMA-containing proteins are unrevealed. In healthy subjects, the concentration of free ADMA in heparinised plasma is almost identical to that of serum. We hypothesised that the robustness of free ADMA concentration in human blood is due to a remarkable resistance of erythrocytic ADMA-containing proteins against proteases. In vivo, we investigated the course of the concentration of ADMA in serum and EDTA plasma of a critically ill patient with acute kidney injury during extended haemodialysis. In vitro, we studied the effects of thermolysin, a useful experimental proteolytic enzyme of erythrocyte membrane proteins, on erythrocytic ADMA. The protein binding (PB) of ADMA to human serum albumin (HSA) was also determined. In these studies, ADMA was measured by a previously reported, fully validated GC-MS/MS method. We measured almost identical ADMA concentrations in plasma and serum samples of the patient. During dialysis, the circulating ADMA concentration decreased slowly and moderately indicating removal of this substance, which was however much less than expected from its low molecular weight (202 Da) and high water solubility. After dialysis, circulating ADMA concentration increased again, a phenomenon called rebound, and ADMA reached higher levels compared to the baseline. The PB value of ADMA to HSA was about 30 %. This surprisingly high PB value of ADMA to HSA may be an explanation for the rather poor dialysance of ADMA. Washed human erythrocytes suspended in phosphate-buffered physiological saline were found not to release appreciable amounts of free and ADMA-containing proteins. The lack of effect of coagulation or anticoagulation on the

  1. Excited triplet state photophysics of the sulphonated aluminium phthalocyanines bound to human serum albumin.

    PubMed

    Foley, M S; Beeby, A; Parker, A W; Bishop, S M; Phillips, D

    1997-03-01

    The binding of the sulphonated aluminum phthalocyanines to human serum albumin (HSA) in aqueous phosphate buffer solution at 25 degrees C has been studied by measuring the properties of the triplet excited states of these dyes. The triplet lifetimes were measured by triplet-triplet absorption flash photolysis. The triplet lifetime of the disulphonated AlS2Pc (2.5 microM) varies from 500 +/- 30 microseconds in the absence of protein to 1.100 microseconds and longer with HSA concentrations above 100 microM. Under identical conditions, the maximum triplet lifetimes of the mono-, tri- and tetrasulphonated compounds bound to HSA are shorter than those for the disulphonated species. The increase in the triplet state lifetimes is attributed to the ability of the bulk aqueous phase to interact with the sensitizer at the site of binding; the site of binding being dependent on the degree of sulphonation. For AlS2Pc and AlS3Pc at all HSA concentrations, and regardless of the degree of sulphonation, all the triplet state decay profiles follow simple pseudo-first-order kinetics. The exponential decay of the triplet phthalocyanine at all HSA concentrations is ascribed to the rapid association and dissociation of the phthalocyanine-HSA complex on the time-scales of the triplet state lifetimes. A simplified one-step binding model is utilized to describe the results. The association of AlS1Pc with HSA results in substantial quenching of the triplet state quantum yield, and a more complex model is required to analyze the results. The tetrasulphonated compound (AlS4Pc) binds to the protein at a site where it experiences some protection from the aqueous phase.

  2. Interaction of VO2+ ion with human serum transferrin and albumin.

    PubMed

    Sanna, Daniele; Garribba, Eugenio; Micera, Giovanni

    2009-04-01

    The complexation of VO(2+) ion with the high molecular mass components of the blood serum, human serum transferrin (hTf) and albumin (HSA), has been re-examined using EPR spectroscopy. In the case of transferrin, the results confirm those previously obtained, showing that VO(2+) ion occupies three different binding sites, A, B(1) and B(2), distinguishable in the X-band anisotropic spectrum recorded in D(2)O. With albumin the results show that a dinuclear complex (VO)(2)(d)HSA is formed in equimolar aqueous solutions or with an excess of protein; in the presence of an excess of VO(2+), the multinuclear complex (VO)(x)(m)HSA is the prevalent species, where x=5-6 indicates the equivalents of metal ion coordinated by HSA. The structure of the dinuclear species is discussed and the donor atoms involved in the metal coordination are proposed on the basis of the measured EPR parameters. Two different binding modes of albumin can be distinguished varying the pH, with only one species being present at the physiological value. The results show that the previously named "strong" site is not the N-terminal copper binding site, and some hypothesis on the metal coordination is discussed, with the (51)V A(z) values for the proposed donor sets obtained by DFT (density functional theory) calculations. Finally, preliminary results obtained in the ternary system VO(2+)/hTf/HSA are shown in order to determine the different binding strength of the two proteins. Due to the low VO(2+) concentration used, the recording of the EPR spectra through the repeated acquisition of the weak signals is essential to obtain a good signal to noise ratio in these systems.

  3. Fluorescence quenching of serum albumin by rifamycin antibiotics and their analytical application.

    PubMed

    Yang, Ji-Dong; Deng, Shi-Xing; Liu, Zhong-Fang; Kong, Ling; Liu, Shao-Pu

    2007-01-01

    In neutral medium, rifamycin antibiotics such as rifapentin (RFPT), rifampicin (RFP), rifandin (RFD) and rifamycin SV (RFSV) can bind with human serum albumin (HSA) and bovine serum albumin (BSA) to form complexes, resulting in the quenching of the intrinsic fluorescence (lambda(ex)/lambda(em) = 285/355 nm) of the BSA and HSA. The quenching intensity (DeltaF) is directly proportional to the concentration of the rifamycin antibiotics. Therefore, a new analytical method was established to determine trace rifamycin antibiotics. The method had fairly high sensitivity and the detecting limits (3sigma) for RFPT, RFP, RFD and RFSV were 0.85, 0.98, 1.83, 1.89 ng/mL, respectively, for the HSA system and 0.76, 0.89, 1.55, 1.77 ng/mL, respectively, for the BSA system. All relative standard deviations (RSDs) were <3.8%. In this work, the characteristics of the fluorescence spectra were studied and the optimum reaction conditions and influencing factors were investigated. The influence of coexisting substances was tested and the results showed that the method had good selectivity and could be applied to determine trace rifamycin antibiotics in medicine capsules and urine samples. Taking the RFSV-serum albumin system as an example, the reaction mechanisms, such as binding constants, binding sites, binding distance and the type of fluorescence quenching, were investigated.

  4. Albumin administration prevents neurological damage and death in a mouse model of severe neonatal hyperbilirubinemia

    PubMed Central

    Vodret, Simone; Bortolussi, Giulia; Schreuder, Andrea B.; Jašprová, Jana; Vitek, Libor; Verkade, Henkjan J.; Muro, Andrés F.

    2015-01-01

    Therapies to prevent severe neonatal unconjugated hyperbilirubinemia and kernicterus are phototherapy and, in unresponsive cases, exchange transfusion, which has significant morbidity and mortality risks. Neurotoxicity is caused by the fraction of unconjugated bilirubin not bound to albumin (free bilirubin, Bf). Human serum albumin (HSA) administration was suggested to increase plasma bilirubin-binding capacity. However, its clinical use is infrequent due to difficulties to address its potential preventive and curative benefits, and to the absence of reliable markers to monitor bilirubin neurotoxicity risk. We used a genetic mouse model of unconjugated hyperbilirubinemia showing severe neurological impairment and neonatal lethality. We treated mutant pups with repeated HSA administration since birth, without phototherapy application. Daily intraperitoneal HSA administration completely rescued neurological damage and lethality, depending on dosage and administration frequency. Albumin infusion increased plasma bilirubin-binding capacity, mobilizing bilirubin from tissues to plasma. This resulted in reduced plasma Bf, forebrain and cerebellum bilirubin levels. We showed that, in our experimental model, Bf is the best marker to determine the risk of developing neurological damage. These results support the potential use of albumin administration in severe acute hyperbilirubinemia conditions to prevent or treat bilirubin neurotoxicity in situations in which exchange transfusion may be required. PMID:26541892

  5. Evaluation of the interaction between naringenin and human serum albumin: Insights from fluorescence spectroscopy, electrochemical measurement and molecular docking.

    PubMed

    Tu, Bao; Wang, Yang; Mi, Ran; Ouyang, Yu; Hu, Yan-Jun

    2015-01-01

    Naringenin (Nar) is a flavanone compound found in grapefruits that is endowed with diverse pharmacological and biological activities. Here, the interaction between Nar and human serum albumin (HSA) was investigated via various methods, including fluorescence spectroscopy, electrochemical methods and molecular docking. The Stern-Volmer quenching constants inversely correlated with temperature, demonstrating that the fluorescence quenching about HSA-Nar system is initiated by the formation of a compound, which has confirmed by electrochemical measurements. Three-dimensional fluorescence demonstrated that Nar induces the slight unfolding of the polypeptides of HSA. The calculated thermodynamic parameters suggesting that the binding of Nar to HSA is spontaneous, and the mainly force is electrostatic interactions. In addition, site marker competitive experiments indicated that Nar binds to HSA both on site I (subdomain IIA) and site II (subdomain IIIA), with higher affinity to the latter one, consistence with molecular docking. Furthermore, the fluorescence resonance energy transfer (FRET) experiment showed the binding distance (r) is 2.65 nm. And the effects of metal ions on the HSA-Nar system are also discussed.

  6. Interaction of albumin with perylene-diimides with aromatic substituents

    NASA Astrophysics Data System (ADS)

    Farooqi, Mohammed; Penick, Mark; Burch, Jessica; Negrete, George; Brancaleon, Lorenzo

    2015-03-01

    Polyaromatic hydrocarbons (PAH) binding to proteins remains one of the fundamental aspects of research in biophysics. Ligand binding can regulate the function of proteins. Binding to small ligands remains a very important aspect in the study of the function of many proteins. Perylene diimide or PDI derivatives have attracted initial interest as industrial dyes and pigments. Recently, much attention has been focused on their strong π - π stacks resulting from the large PDI aromatic core. These PDI stacks have distinct optical properties, and provide informative models that mimic the light-harvesting system and initial charge separation and charge transfer in the photosynthetic system. The absorption property of PDI derivatives may be largely tuned from visible to near-infrared region by chemical modifications at the bay-positions. We are currently studying a new class of PDI derivatives with substituents made of the side chains of aromatic amino acids (Tyrosine, Tryptophan and Phenylalanine). We have looked at the fluorescence absorption and emission of these PDIs in water and other organic solvents. PDIs show evidence of dimerization and possible aggregation. We also present binding studies of these PDIs with Human Serum Albumin (HSA). The binding was studied using fluorescence emission quenching of the HSA Tryptophan residue. Stern-Volmer equation is used to derive the quenching constants. PDI binding to HSA also has an effect on the fluorescence emission of the PDIs themselves by red shifting the spectra. Funded by RCMI grant.

  7. Drug-protein binding of Danhong injection and the potential influence of drug combination with aspirin: Insight by ultrafiltration LC-MS and molecular modeling.

    PubMed

    Zhu, Junfeng; Yi, Xiaojiao; Huang, Peng; Chen, Shuqing; Wu, Yongjiang

    2017-02-05

    Danhong injection (DHI) is a widely used Chinese medicine injection (CMI) for the clinical treatment of cardiovascular and cerebrovascular diseases. In this study, a simple and efficient in vitro method based on ultrafiltration LC-MS and molecular modeling has been developed to study the human serum albumin (HSA) binding of the compounds in DHI. Seven major components including protocatechuic aldehyde, p-coumaric acid, salvianolic acid D, rosmarinic acid, salvianolic acid E, lithospermic acid and salvianolic acid B were identified as HSA ligands and their binding degrees in the proposed non-saturated model were 26.17, 37.69, 99.77, 91.78, 96.91, 99.42 and 98.10%, respectively. Considering the drug-HSA binding property of the compounds in DHI may change during drug combination therapy, competitive binding assay was carried out to evaluate the influence of aspirin on the DHI-HSA binding. Experimental results revealed that the salvianolic acids in DHI had stronger binding ability to HSA than sodium salicylate. To further verify the results above, molecular modeling and probe displacement assay were conducted to investigate the optimum binding site and binding affinity of the ligands on HSA. Our findings suggested that the established method could be a powerful tool to study the drug-HSA binding property of CMIs.

  8. Maximizing the relaxivity of Gd-complex by synergistic effect of HSA and carboxylfullerene.

    PubMed

    Zhen, Mingming; Zheng, Junpeng; Ye, Lei; Li, Shumu; Jin, Chan; Li, Kai; Qiu, Dong; Han, Hongbin; Shu, Chunying; Yang, Yongji; Wang, Chunru

    2012-07-25

    Macromolecular magnetic resonance imaging (MRI) contrast agent Gd-DTPA-HSA (DTPA, diethylene triamine pentacetate acid; HSA, human serum albumin) as a model has been successfully conjugated with trimalonic acid modified C60 for contrast enhancement at clinically used magnetic field strength. The Gd-DTPA-HSA-C60 conjugate exhibit maximal relaxivity (r1 = 86 mM(-1) s(-1) at 0.5 T, 300 K) reported so far, which is much superior to that of the control Gd-DTPA-HSA (r1 = 38 mM(-1 )s(-1)) under the same condition and comparable to the theoretical maximum (r1 = 80-120 mM(-1) s(-1), at 20 MHz and 298 K), indicating the synergistic effect of HSA and carboxylfullerene on the increased contrast enhancement. TEM characterization reveals that both Gd-DTPA-HSA-C60 and Gd-DTPA-HSA can penetrate the cells via endocytosis and trans-membrane, respectively, suggesting the potential to sensitively image the events at the cellular and subcellular levels. In addition, the fusion of fullerene with Gd-DTPA-HSA will further endow the resulting complex with photodynamic therapy (PDT) property and thus combine the modalities of therapy (PDT) and diagnostic imaging (MRI) into one entity. More importantly, the payloaded Gd-DTPA may substitute for other more stable Gd-DOTA and HSA as a theranostic package can further work as a drug delivery carrier and effectively control drug release through proteolysis.

  9. Developing an anticancer copper(II) pro-drug based on the nature of cancer cell and human serum albumin carrier IIA subdomain: mouse model of breast cancer

    PubMed Central

    Qi, Jinxu; Chen, Shifang; Zhou, Zuping; Wu, Xiaoyang; Liang, Hong; Yang, Feng

    2016-01-01

    Human serum albumin (HSA)-based drug delivery systems are promising for improving delivery efficiency, anticancer activity and selectivity of anticancer agents. To rationally guide to design HSA carrier for anticancer metal agent, we built a breast mouse model on developing anti-cancer copper (Cu) pro-drug based on the nature of IIA subdomain of HSA carrier and cancer cells. Thus, we first synthesized a new Cu(II) compound derived from tridentate (E)-N'-(5-bromo-2-hydroxybenzylidene)benzohydrazide Schiff base ligand (HL) containing 2 potential leaving groups [indazole (Ind) and NO3−], namely, [Cu(L)(Ind)NO3]. Structural analysis of the HSA complex showed that Cu(L)(Ind)(NO3) could bind to the hydrophobic pocket of the HSA IIA subdomain. Lys199 and His242 coordinate with Cu2+ by replacing the indazole and NO3 ligands of [Cu(L)(Ind)NO3]. The release behavior of the Cu compound from the HSA complex is different at different pH levels. [Cu(L)(Ind)NO3] can enhance cytotoxicity by 2 times together with HSA specifically in cancer cells but has no such effect on normal cells in vitro. Importantly, our in vivo results showed that the HSA complex displayed increased selectivity and capacity to inhibit tumor growth and was less toxic than [Cu(L)(Ind)NO3] alone. PMID:27564255

  10. Dual-targeting peptide probe for sequence- and structure-sensitive sensing of serum albumin.

    PubMed

    Yu, Yang; Huang, Yanyan; Jin, Yulong; Zhao, Rui

    2017-04-02

    Peptide-protein interactions mediate numerous biologic processes and provide great opportunity for developing peptide probes and analytical approaches for detecting and interfering with recognition events. Molecular interactions usually take place on the heterogeneous surface of proteins, and the spatial distribution and arrangement of probes are therefore crucial for achieving high specificity and sensitivity in the bioassays. In this study, small linear peptides, homogenous peptide dimers and hetero bivalent peptides were designed for site-specific recognition of human serum albumin (HSA). Three hydrophilic regions located at different subdomains of HSA were chosen as targets for the molecular design. The binding affinity, selectivity and kinetics of the candidates were screened with surface plasmon resonance imaging (SPRi) and fluoroimmuno assays. Benefiting from the synergistic effect from the surface-targeted peptide binders and the flexible spacer, a heterogenetic dimer peptide (heter-7) with fast binding and slow dissociation behavior was identified as the optimized probe. Heter-7 specifically recognizes the target protein HSA, and effectively blocks the binding of antibody to HSA. Its inhibitory activity was estimated as 83nM. It is noteworthy that heter-7 can distinguish serum albumins from different species despite high similarities in sequence and structure of these proteins. This hetero bivalent peptide shows promise for use in serum proteomics, disease detection and drug transport, and provides an effective approach for promoting the affinity and selectivity of ligands to achieve desirable chemical and biological outcomes.

  11. Interactions of aptamers with sera albumins

    NASA Astrophysics Data System (ADS)

    Cortez, Célia Martins; Silva, Dilson; Silva, Camila M. C.; Missailidis, Sotiris

    2012-09-01

    The interactions of two short aptamers to human and bovine serum albumins were studied by fluorescence spectroscopic techniques. Intrinsic fluorescence of BSA and