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Sample records for recombinant human albumin

  1. Preparation of Tc-99m-macroaggregated albumin from recombinant human albumin for lung perfusion imaging.

    PubMed

    Hunt, A P; Frier, M; Johnson, R A; Berezenko, S; Perkins, A C

    2006-01-01

    Human serum albumin (HSA) extracted from pooled blood taken from human donors is used in the production of (99m)Tc-labelled macroaggregated albumin (MAA) for lung perfusion imaging. However, concerns for the safety of blood-derived products due to potential contamination by infective agents (e.g. new variant CJD), make alternative production methods necessary. Recombinant DNA technology is a promising method of albumin production avoiding problems associated with human-derived HSA. This paper presents results comparing MAA prepared from recombinant human albumin (rHA, Recombumin) (rMAA) with in-house produced HSA MAA (hMAA) and commercially available MAA (cMAA). (99m)Tc-MAA was prepared using previously published production methods by heating a mixture of albumin and stannous chloride in acetate buffer (pH 5.4) at 70 degrees C for 20 min. Parameters investigated include aggregate size, radiolabelling efficiency, radiochemical and aggregate stability at 4 degrees C and in vitro (in whole human blood) at 37 degrees C and biodistribution studies. Results showed that rMAA could be produced with similar morphology, labelling efficiency and stability to hMAA and cMAA. Our findings confirm that rHA shows significant potential as a direct replacement for HSA in commercially available MAA.

  2. Stabilization of a hydrophobic recombinant cytokine by human serum albumin.

    PubMed

    Hawe, Andrea; Friess, Wolfgang

    2007-11-01

    The objective was to evaluate the impact of pH and NaCl content on aggregation, particle formation, and solubility of a hydrophobic recombinant human cytokine in formulations with human serum albumin (HSA) as stabilizing excipient. While cytokine-HSA formulations were stable at physiological pH, a tremendous increase in turbidity at pH 5.0, close to the isoelectric point of HSA was caused by a partially irreversible precipitation. By dynamic light scattering (DLS), disc centrifugation, atomic force microscopy (AFM), and light obscuration it could be shown that the turbidity was mainly caused by particles larger than 120 nm. SDS-PAGE provided evidence that the precipitation at pH 5.0 was mainly caused by the cytokine. The HSA-stabilizers Na-octanoate and Na-N-acetyltryptophante were less effective in preventing the turbidity increase of unstabilized-HSA compared to NaCl. The interactions between HSA and cytokine were weakened by NaCl, as determined by fluorescence spectroscopy. The positive effect of NaCl on the formulation could be attributed to a direct stabilization of HSA and weaker interactions between HSA and the cytokine, which in consequence provided an overall stabilization of the cytokine. Copyright 2007 Wiley-Liss, Inc.

  3. Strategy to tether organometallic ruthenium-arene anticancer compounds to recombinant human serum albumin.

    PubMed

    Ang, Wee Han; Daldini, Elisa; Juillerat-Jeanneret, Lucienne; Dyson, Paul J

    2007-10-29

    In order to utilize macromolecules for drug targeting and delivery, a strategy to tether organometallic ruthenium-arene drugs to carrier protein molecules was developed. The approach involves the design of a drug fragment capable of conjugating to linker molecules on a modified carrier protein via hydrazone bond formation. The proof-of-concept using recombinant human serum albumin is described.

  4. 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.

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

    PubMed

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

    2014-04-01

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

  6. 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.

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

    PubMed Central

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

    2013-01-01

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

  8. Recombinant human albumin supports single cell cloning of CHO cells in chemically defined media.

    PubMed

    Zhu, Jiang; Wooh, Jong Wei; Hou, Jeff Jia Cheng; Hughes, Benjamin S; Gray, Peter P; Munro, Trent P

    2012-01-01

    Biologic drugs, such as monoclonal antibodies, are commonly made using mammalian cells in culture. The cell lines used for manufacturing should ideally be clonal, meaning derived from a single cell, which represents a technically challenging process. Fetal bovine serum is often used to support low cell density cultures, however, from a regulatory perspective, it is preferable to avoid animal-derived components to increase process consistency and reduce the risk of contamination from adventitious agents. Chinese hamster ovary (CHO) cells are the most widely used cell line in industry and a large number of serum-free, protein-free, and fully chemically defined growth media are commercially available, although these media alone do not readily support efficient single cell cloning. In this work, we have developed a simple, fully defined, single-cell cloning media, specifically for CHO cells, using commercially available reagents. Our results show that a 1:1 mixture of CD-CHO™ and DMEM/F12 supplemented with 1.5 g/L of recombinant albumin (Albucult®) supports single cell cloning. This formulation can support recovery of single cells in 43% of cultures compared to 62% in the presence of serum.

  9. Unfolding properties of recombinant human serum albumin products are due to bioprocessing steps.

    PubMed

    Lang, Brian E; Cole, Kenneth D

    2015-01-01

    We have used differential scanning calorimetry (DSC) to determine the unfolding properties of commercial products of human serum albumin (HSA) prepared from pooled human blood, transgenic yeast, and transgenic rice. The initial melting temperatures (Tm1 ) for the unfolding transitions of the HSA products varied from 62°C to 75°C. We characterized the samples for purity, fatty acid content, and molecular weight. The effects of adding fatty acids, heat pasteurization, and a low pH defatting technique on the transition temperatures were measured. Defatted HSA has a structure with the lowest stability (Tm of ∼62°C). When fatty acids are bound to HSA, the structure is stabilized (Tm of ∼64-72°C), and prolonged heating (pasteurization at 60°C) results in a heat-stabilized structural form containing fatty acids (Tm of ∼75-80°C). This process was shown to be reversible by a low pH defatting step. This study shows that the fatty acid composition and bioprocessing history of the HSA commercial products results in the large differences in the thermal stability.

  10. Human albumin: old, new, and emerging applications.

    PubMed

    Rozga, Jacek; Piątek, Tomasz; Małkowski, Piotr

    2013-05-10

    Human serum albumin has been widely used in an array of clinical settings for nearly 7 decades. Although there is no evidence to support the use of albumin rather than crystalloid in acute volume resuscitation, many clinicians continue to use albumin because it has other important physiologic effects besides the oncotic function. In keeping with the improved understanding of albumin physiology and pathophysiology of many acute and chronic diseases, use of albumin for medical applications has increased in recent years. This, along with increased costs of manufacturing and lower production volume of medical-grade albumin, has lead to an ongoing shortage and rapid increase in albumin prices. This review is based on the analysis of major publications, related to albumin chemistry, physiology, and medical uses including guidelines developed by professional and governmental organizations. Results reflect current knowledge about the role of albumin in health and disease and relevance of albumin therapy in specific clinical settings. Albumin therapy is currently recommended in spontaneous bacterial peritonitis with ascites, refractory ascites not responsive to diuretics, large-volume paracentesis, post-paracentesis syndrome, and the treatment of hepatorenal syndrome as an adjunct to vasoconstrictors. New indications for albumin therapy are linked to the antioxidant activity of albumin and its effects on capillary integrity. In recent years, large-pore hemofiltration and albumin exchange have emerged as promising liver support therapies for liver failure and other toxic syndromes. They are designed to remove a broad range of blood-borne toxins and to restore normal functions of the circulating albumin by replacing defective forms of albumin and albumin molecules saturated with toxins with normal albumin. In view of the ongoing worldwide shortage and high cost of human albumin (native and recombinant), new usage criteria, protocols, and guidelines for appropriate utilization

  11. Freeze-drying of HI-6-loaded recombinant human serum albumin nanoparticles for improved storage stability.

    PubMed

    Dadparvar, Miriam; Wagner, Sylvia; Wien, Sascha; Worek, Franz; von Briesen, Hagen; Kreuter, Jörg

    2014-10-01

    Severe intoxications with organophosphates require the immediate administration of atropine in combination with acetyl cholinesterase (AChE) reactivators such as HI-6. Although this therapy regimen enables the treatment of peripheral symptoms, the blood-brain barrier (BBB) restricts the access of the hydrophilic antidotes to the central nervous system which could lead to a fatal respiratory arrest. Therefore, HI-6-loaded albumin nanoparticles were previously developed to enhance the transport across this barrier and were able to reactivate organophosphate-(OP)-inhibited AChE in an in vitro BBB model. Since HI-6 is known to be moisture-sensitive, the feasibility of freeze-drying of the HI-6-loaded nanoparticles was investigated in the present study using different cryo- and lyoprotectants at different concentrations. Trehalose and sucrose (3%, w/v)-containing formulations were superior to mannitol concerning the physicochemical parameters of the nanoparticles whereas trehalose-containing samples were subject of a prolonged storage stability study at temperatures between -20°C and +40°C for predetermined time intervals. Shelf-life computations of the freeze-dried HI-6 nanoparticle formulations revealed a shelf-life time of 18 months when stored at -20°C. The formulations' efficacy was proven in vitro by reactivation of OP-inhibited AChE after transport over a porcine brain capillary endothelial cell layer model.

  12. Recombinant human albumin supports development of somatic cell nuclear transfer embryos in mice: toward the establishment of a chemically defined cloning protocol.

    PubMed

    Cavaleri, F; Gentile, L; Schöler, H R; Boiani, M

    2006-01-01

    Culturing embryos in different media is a useful approach to characterize their nature in regard to "memory" of the donor nucleus and its "reprogramming" after somatic cell nuclear transfer (SCNT). However, efforts to elucidate the mechanisms of reprogramming are seriously undermined when embryo culture conditions are not completely defined. Using recombinant human albumin (rHA) is a step toward establishing defined culture conditions for mouse cloning. Recombinant HA supports blastocyst formation of cumulus cell-derived clones at a rate comparable with two types of bovine serum albumin (BSA); following transfer of blastocysts to the genital tract, rates of development to midgestation (10.5 dpc) were indistinguishable. rHA also supports the derivation of germline competent embryonic stem (ES) cells from SCNT blastocysts at a substantial rate compared with BSA counterparts and with zygotic blastocysts. Unlike the developmental parameters, the gene expression patterns of clones cultured in rHA or BSA were not superimposed; identical patterns were observed for zygotic blastocysts in the two albumins. In summary, the present study demonstrates that (1) rHA can replace BSA, proving a defined protein source for SCNT in mice; (2) although using rHA is similar to BSA, it is not equal (rHA leaves a mark on gene expression of clones but not zygotes). Future studies that investigate reprogramming after SCNT will need to consider not only the implications of culture media for cloning but also the supplement choice.

  13. Development of expression systems for the production of recombinant human serum albumin using the MOX promoter in Hansenula polymorpha DL-1.

    PubMed

    Kang, H A; Kang, W; Hong, W K; Kim, M W; Kim, J Y; Sohn, J H; Choi, E S; Choe, K B; Rhee, S K

    2001-09-01

    To optimize the secretory expression of recombinant human serum albumin (HSA) under the control of methanol oxidase (MOX) promoter in the methylotrophic yeast Hansenula polymorpha DL-1, we analyzed several parameters affecting the expression of HSA from the MOX promoter. Removal of the 5'-untranslated region derived from HSA cDNA in the expression cassette led to at least a fivefold improvement of HSA expression efficiency at the translational level. With the optimized expression cassette, the gene dosage effect on HSA expression was abolished and thus, a single copy of the expression vector integrated into the MOX locus became sufficient for the maximal expression of HSA. Northern blot analysis revealed that the levels of HSA transcript did not increase any further upon increasing copy number. The mox-disrupted (mox Delta) transformant was constructed, in which the genomic MOX gene was transplaced with the HSA expression cassette, to examine the effect of the methanol oxidase-deficient phenotype of the host on HSA expression. The mox Delta transformant showed higher levels of HSA production in shake-flask cultures than the MOX wild-type transformant, especially at low concentrations of methanol and a twofold higher specific HSA production rate in fed-batch fermentation with an abrupt induction mode. The native prepro signal sequence of HSA secreted in H. polymorpha was correctly processed and the mature recombinant protein had a pI value identical to that of the authentic HSA. Our results suggest that the H. polymorpha expression systems developed in this study are suitable for large-scale production of recombinant albumin.

  14. Generation of fatty acids from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/cardiolipin liposomes that stabilize recombinant human serum albumin.

    PubMed

    Frahm, Grant E; Cameron, Brooke E; Smith, Jeffrey C; Johnston, Michael J W

    2013-06-01

    At elevated temperatures, studies have shown that serum albumin undergoes irreversible changes to its secondary structure. Anionic fatty acids and/or anionic surfactants have been shown to stabilize human serum albumin (HSA) against thermal denaturation through bridging hydrophobic domains and cationic amino acids residues of the protein. As albumin can readily interact with a variety of liposomes, this study proposes that cardiolipin delivered via 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes can improve the thermal stability of recombinant HSA produced in Saccharomyces cerevisiae (ScrHSA) in a similar manner to anionic fatty acids. Thermal stability and structure of ScrHSA in the absence and presence of DPPC/cardiolipin liposomes was assessed with U/V circular dichroism spectropolarimetry and protein thermal stability was confirmed with differential scanning calorimetry. Although freshly prepared DPPC/cardiolipin liposomes did not improve the stability of ScrHSA, DPPC/cardiolipin liposomes incubated at room temperature for 7 d (7dRT) dramatically improved the thermal stability of the protein. Mass spectrometry analysis identified the presence of fatty acids in the 7dRT liposomes, not identified in freshly prepared liposomes, to which the improved stability was attributed. The generation of fatty acids is attributed to either the chemical hydrolysis or oxidative cleavage of the unsaturated acyl chains of cardiolipin. By modulating the lipid composition through the introduction of lipids with higher acyl chain unsaturation, it may be possible to generate the stabilizing fatty acids in a more rapid manner.

  15. Determination of supplier-to-supplier and lot-to-lot variability in glycation of recombinant human serum albumin expressed in Oryza sativa.

    PubMed

    Frahm, Grant E; Smith, Daryl G S; Kane, Anita; Lorbetskie, Barry; Cyr, Terry D; Girard, Michel; Johnston, Michael J W

    2014-01-01

    The use of different expression systems to produce the same recombinant human protein can result in expression-dependent chemical modifications (CMs) leading to variability of structure, stability and immunogenicity. Of particular interest are recombinant human proteins expressed in plant-based systems, which have shown particularly high CM variability. In studies presented here, recombinant human serum albumins (rHSA) produced in Oryza sativa (Asian rice) (OsrHSA) from a number of suppliers have been extensively characterized and compared to plasma-derived HSA (pHSA) and rHSA expressed in yeast (Pichia pastoris and Saccharomyces cerevisiae). The heterogeneity of each sample was evaluated using size exclusion chromatography (SEC), reversed-phase high-performance liquid chromatography (RP-HPLC) and capillary electrophoresis (CE). Modifications of the samples were identified by liquid chromatography-mass spectrometry (LC-MS). The secondary and tertiary structure of the albumin samples were assessed with far U/V circular dichroism spectropolarimetry (far U/V CD) and fluorescence spectroscopy, respectively. Far U/V CD and fluorescence analyses were also used to assess thermal stability and drug binding. High molecular weight aggregates in OsrHSA samples were detected with SEC and supplier-to-supplier variability and, more critically, lot-to-lot variability in one manufactures supplied products were identified. LC-MS analysis identified a greater number of hexose-glycated arginine and lysine residues on OsrHSA compared to pHSA or rHSA expressed in yeast. This analysis also showed supplier-to-supplier and lot-to-lot variability in the degree of glycation at specific lysine and arginine residues for OsrHSA. Both the number of glycated residues and the degree of glycation correlated positively with the quantity of non-monomeric species and the chromatographic profiles of the samples. Tertiary structural changes were observed for most OsrHSA samples which correlated well

  16. Determination of Supplier-to-Supplier and Lot-to-Lot Variability in Glycation of Recombinant Human Serum Albumin Expressed in Oryza sativa

    PubMed Central

    Frahm, Grant E.; Smith, Daryl G. S.; Kane, Anita; Lorbetskie, Barry; Cyr, Terry D.; Girard, Michel; Johnston, Michael J. W.

    2014-01-01

    The use of different expression systems to produce the same recombinant human protein can result in expression-dependent chemical modifications (CMs) leading to variability of structure, stability and immunogenicity. Of particular interest are recombinant human proteins expressed in plant-based systems, which have shown particularly high CM variability. In studies presented here, recombinant human serum albumins (rHSA) produced in Oryza sativa (Asian rice) (OsrHSA) from a number of suppliers have been extensively characterized and compared to plasma-derived HSA (pHSA) and rHSA expressed in yeast (Pichia pastoris and Saccharomyces cerevisiae). The heterogeneity of each sample was evaluated using size exclusion chromatography (SEC), reversed-phase high-performance liquid chromatography (RP-HPLC) and capillary electrophoresis (CE). Modifications of the samples were identified by liquid chromatography-mass spectrometry (LC-MS). The secondary and tertiary structure of the albumin samples were assessed with far U/V circular dichroism spectropolarimetry (far U/V CD) and fluorescence spectroscopy, respectively. Far U/V CD and fluorescence analyses were also used to assess thermal stability and drug binding. High molecular weight aggregates in OsrHSA samples were detected with SEC and supplier-to-supplier variability and, more critically, lot-to-lot variability in one manufactures supplied products were identified. LC-MS analysis identified a greater number of hexose-glycated arginine and lysine residues on OsrHSA compared to pHSA or rHSA expressed in yeast. This analysis also showed supplier-to-supplier and lot-to-lot variability in the degree of glycation at specific lysine and arginine residues for OsrHSA. Both the number of glycated residues and the degree of glycation correlated positively with the quantity of non-monomeric species and the chromatographic profiles of the samples. Tertiary structural changes were observed for most OsrHSA samples which correlated well

  17. Safety and PK/PD correlation of TV-1106, a recombinant fused human albumin-growth hormone, following repeat dose administration to monkeys.

    PubMed

    Ashkenazi, Nurit; Rosenstock, Moti; Hallak, Hussein; Bassan, Merav; Rasamoelisolo, Michele; Leuschner, Jost; Shinar, Doron

    TV-1106 is a recombinant human albumin genetically fused to growth hormone which is intended to reduce the frequency of injections for GH therapy users. We report the safety, tolerability, pharmacokinetics and pharmacodynamics of repeated subcutaneous injections of TV-1106 in Cynomolgus monkeys. Cynomolgus monkeys received four weekly subcutaneous injections of 0, 5, 10 or 20mg/kg TV-1106 and were monitored for safety signals throughout the study. Serum levels of TV-1106 and insulin-like growth factor 1 (IGF-1) were assayed. Treated animals showed no adverse effects or histopathological changes. TV-1106 serum concentrations showed sustained exposure to the drug. Exposure increased in a dose-dependent manner with peak concentrations at approximately 24h post-dosing and elimination half-lives in the range of 12 to 24h. IGF-1 serum concentrations were elevated throughout the entire study duration, indicative of the pharmacological response. There was a clear correlation between change in IGF-1 levels and dose or exposure to TV-1106. The safety, pharmacokinetic and pharmacodynamic findings support the further development of TV-1106 as a once-weekly administered treatment for patients with GHD. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Investigation of the differences in thermal stability of two recombinant human serum albumins with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine liposomes by UV circular dichroism spectropolarimetry.

    PubMed

    Frahm, Grant E; Cyr, Terry D; Smith, Daryl G S; Walrond, Lisa D; Johnston, Michael J W

    2012-04-19

    Previous studies have demonstrated that liposome-protein interactions can result in changes to the thermal stability of the protein. We utilized far-UV circular dichroism spectropolarimetry and fluorescence spectroscopy to investigate the interaction of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes with two recombinant human serum albumins (rHSA). We demonstrate that rHSA expressed in Oryza sativa (OsrHSA) has improved secondary structure thermal stability compared to rHSA expressed in Pichia pastoris (PprHSA). A similar stability profile was observed when comparing bovine serum albumin (BSA) and defatted bovine serum albumin (dfBSA), suggesting the presence of fatty acids may be responsible for the improved stability of OsrHSA. Addition of DPPC liposomes reduced the thermal stability of both OsrHSA and BSA, but not of PprHSA or dfBSA. DPPC liposomes may disrupt stabilizing native fatty acids on OsrHSA and BSA.

  19. Evaluation of an Albumin-Binding Domain Protein Fused to Recombinant Human IL-2 and Its Effects on the Bioactivity and Serum Half-Life of the Cytokine

    PubMed Central

    Adabi, Elham; Saebi, Fateme; Hasan-Abad, Amin Moradi; Teimoori-Toolabi, Ladan; Kardar, Gholam Ali

    2017-01-01

    Background: Cancer immunotherapy is a promising strategy for cancer treatment. In this strategy, the immune system is triggered to destroy cancer cells. IL-2 is an important factor in passive cancer immunotherapy that helps modulating some important immune functions. One of the IL-2 limitations is low serum half-life; therefore, repetitive high doses of the injections are required to maintain effective concentrations. High-dose IL-2 therapy results in severe side effects; thus, improvement of its serum half-life would provide therapeutic benefits. Methods: We have investigated a strategy that is able to utilize an albumin-binding domain (ABD) from streptococcal protein G. In this strategy, the fusion protein ABD-rIL-2 binds to serum albumin, which results in improvement of the IL-2 serum half-life. PET26b+ plasmid was used as an expression vector, which encoded rIL-2 and ABD-rIL-2, both fused to pelB secretion signal under the control of the strong bacteriophage T7 promoter. The constructs were expressed in E. coli Rosetta (DE3), and the recombinant proteins were purified from periplasmic fractions. Results: The analysis of in vitro bioactivity proved that the fusion of ABD to rIL-2 does not interfere with its bioactivity. ABD-rIL-2 fusion protein indicated higher serum half-life compared to rIL-2, when it was tested in the BALB/c mice. Conclusion: The current study provides an alternative strategy to extend the half-life and improve pharmacokinetic properties of rIL-2 without reducing its bioactivity in vitro. PMID:27805072

  20. Production of recombinant albumin by a herd of cloned transgenic cattle.

    PubMed

    Echelard, Yann; Williams, Jennifer L; Destrempes, Margaret M; Koster, Julie A; Overton, Susan A; Pollock, Daniel P; Rapiejko, Karen T; Behboodi, Esmail; Masiello, Nicholas C; Gavin, William G; Pommer, Jerry; Van Patten, Scott M; Faber, David C; Cibelli, Jose B; Meade, Harry M

    2009-06-01

    Purified plasma derived human albumin has been available as a therapeutic product since World War II. However, cost effective recombinant production of albumin has been challenging due to the amount needed and the complex folding pattern of the protein. In an effort to provide an abundant source of recombinant albumin, a herd of transgenic cows expressing high levels of rhA in their milk was generated. Expression cassettes efficiently targeting the secretion of human albumin to the lactating mammary gland were obtained and tested in transgenic mice. A high expressing transgene was transfected in primary bovine cell lines to produce karyoplasts for use in a somatic cell nuclear transfer program. Founder transgenic cows were produced from four independent cell lines. Expression levels varying from 1-2 g/l to more than 40 g/l of correctly folded albumin were observed. The animals expressing the highest levels of rhA exhibited shortened lactation whereas cows yielding 1-2 g/l had normal milk production. This herd of transgenic cattle is an easily scalable and well characterized source of rhA for biomedical uses.

  1. 21 CFR 640.80 - Albumin (Human).

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... a sterile solution of the albumin derived from human plasma. (b) Source material. The source material of Albumin (Human) shall be plasma recovered from Whole Blood prepared as prescribed in §§ 640.1 through 640.5, or Source Plasma prepared as prescribed in §§ 640.60 through 640.76. (c) Additives in...

  2. 21 CFR 640.80 - Albumin (Human).

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... a sterile solution of the albumin derived from human plasma. (b) Source material. The source material of Albumin (Human) shall be plasma recovered from Whole Blood prepared as prescribed in §§ 640.1 through 640.5, or Source Plasma prepared as prescribed in §§ 640.60 through 640.76. (c) Additives...

  3. 21 CFR 640.80 - Albumin (Human).

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... a sterile solution of the albumin derived from human plasma. (b) Source material. The source material of Albumin (Human) shall be plasma recovered from Whole Blood prepared as prescribed in §§ 640.1 through 640.5, or Source Plasma prepared as prescribed in §§ 640.60 through 640.76. (c) Additives in...

  4. 21 CFR 640.80 - Albumin (Human).

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... a sterile solution of the albumin derived from human plasma. (b) Source material. The source material of Albumin (Human) shall be plasma recovered from Whole Blood prepared as prescribed in §§ 640.1 through 640.5, or Source Plasma prepared as prescribed in §§ 640.60 through 640.76. (c) Additives in...

  5. 21 CFR 640.80 - Albumin (Human).

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... a sterile solution of the albumin derived from human plasma. (b) Source material. The source material of Albumin (Human) shall be plasma recovered from Whole Blood prepared as prescribed in §§ 640.1 through 640.5, or Source Plasma prepared as prescribed in §§ 640.60 through 640.76. (c) Additives in...

  6. 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.

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

    PubMed Central

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

    2015-01-01

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

  8. Polymerized soluble venom--human serum albumin

    SciTech Connect

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

    1985-03-01

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

  9. 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.

  10. 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

  11. Serial coupling of ion-exchange and size-exclusion chromatography to determine aggregation levels in mAbs in the presence of a proteinaceous excipient, recombinant human serum albumin.

    PubMed

    Weisbjerg, Paul Luigi Gargani; Caspersen, Mikael Bjerg; Cook, Ken; Van De Weert, Marco

    2015-02-01

    Aggregation levels of therapeutic proteins may be difficult to determine in mixtures containing other proteinaceous excipients. We performed a feasibility study of using serial coupling of an anion exchange and size exclusion column to determine the aggregation levels of four different model monoclonal antibodies (mAb) mixed with the model proteinaceous excipient recombinant human serum albumin (rHSA). For three of the four mAbs suitable elution conditions could be established. From the limitations imposed by the pI of the rHSA, the pI of the mAb and the nature of the columns used, it was possible to propose a set of general conditions that allows quantification of the aggregation level of a therapeutic protein in the presence of a proteinaceous excipient: The excipient protein and protein of interest should differ in pI by a minimum of 0.5 units, and the pI of the protein of interest should not be higher than ca. 8.5.

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

    NASA Astrophysics Data System (ADS)

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

    2017-05-01

    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 human FcRn (hFcRn) engagement using recombinant human albumin (rHA) of either a wild type (WT) or an engineered human FcRn high binding variant (HB). Albumin-aptamer 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 albumin-aptamer conjugation, however, substantially compromized binding to hFcRn, 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 hFcRn binding can be increased using a HB engineered albumin.

  13. Pseudo-esterase Activity of Human Albumin

    PubMed Central

    Lockridge, Oksana; Xue, Weihua; Gaydess, Andrea; Grigoryan, Hasmik; Ding, Shi-Jian; Schopfer, Lawrence M.; Hinrichs, Steven H.; Masson, Patrick

    2008-01-01

    Human albumin is thought to hydrolyze esters because multiple equivalents of product are formed for each equivalent of albumin. Esterase activity with p-nitrophenyl acetate has been attributed to turnover at tyrosine 411. However, p-nitrophenyl acetate creates multiple, stable, acetylated adducts, a property contrary to turnover. Our goal was to identify residues that become acetylated by p-nitrophenyl acetate and determine the relationship between stable adduct formation and turnover. Fatty acid-free human albumin was treated with 0.5 mm p-nitrophenyl acetate for 5 min to 2 weeks, or with 10 mm p-nitrophenyl acetate for 48 h to 2 weeks. Aliquots were digested with pepsin, trypsin, or GluC and analyzed by mass spectrometry to identify labeled residues. Only Tyr-411 was acetylated within the first 5 min of reaction with 0.5 mm p-nitrophenyl acetate. After 0.5–6 h there was partial acetylation of 16–17 residues including Asp-1, Lys-4, Lys-12, Tyr-411, Lys-413, and Lys-414. Treatment with 10 mm p-nitrophenyl acetate resulted in acetylation of 59 lysines, 10 serines, 8 threonines, 4 tyrosines, and Asp-1. When Tyr-411 was blocked with diisopropylfluorophosphate or chlorpyrifos oxon, albumin had normal esterase activity with β-naphthyl acetate as visualized on a nondenaturing gel. However, after 82 residues had been acetylated, esterase activity was almost completely inhibited. The half-life for deacetylation of Tyr-411 at pH 8.0, 22 °C was 61 ± 4 h. Acetylated lysines formed adducts that were even more stable. In conclusion, the pseudo-esterase activity of albumin is the result of irreversible acetylation of 82 residues and is not the result of turnover. PMID:18577514

  14. 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.

  15. (PCG) Protein Crystal Growth Human Serum Albumin

    NASA Technical Reports Server (NTRS)

    1989-01-01

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

  16. Recombinant human milk proteins.

    PubMed

    Lönnerdal, Bo

    2006-01-01

    Human milk provides proteins that benefit newborn infants. They not only provide amino acids, but also facilitate the absorption of nutrients, stimulate growth and development of the intestine, modulate immune function, and aid in the digestion of other nutrients. Breastfed infants have a lower prevalence of infections than formula-fed infants. Since many women in industrialized countries choose not to breastfeed, and an increasing proportion of women in developing countries are advised not to breastfeed because of the risk of HIV transmission, incorporation of recombinant human milk proteins into infant foods is likely to be beneficial. We are expressing human milk proteins known to have anti-infective activity in rice. Since rice is a normal constituent of the diet of infants and children, limited purification of the proteins is required. Lactoferrin has antimicrobial and iron-binding activities. Lysozyme is an enzyme that is bactericidal and also acts synergistically with lactoferrin. These recombinant proteins have biological activities identical to their native counterparts. They are equally resistant to heat processing, which is necessary for food applications, and to acid and proteolytic enzymes which are needed to maintain their biological activity in the gastrointestinal tract of infants. These recombinant human milk proteins may be incorporated into infant formulas, baby foods and complementary foods, and used with the goal to reduce infectious diseases.

  17. 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.

  18. Recombinant albumins containing additional peptide sequences smaller than barbourin retain the ability of barbourin-albumin to inhibit platelet aggregation.

    PubMed

    Sheffield, William P; Wilson, Brianna; Eltringham-Smith, Louise J; Gataiance, Sharon; Bhakta, Varsha

    2005-05-01

    The previously described fusion protein BLAH(6) (Marques JA et al.,Thromb Haemost 2001; 86: 902-8) is a recombinant protein that combines the small disintegrin barbourin with hexahistidine-tagged rabbit serumalbumin (RSA) produced in Pichia pastoris yeast. We sought to determine: (1) if BLAH(6) was immunogenic; and (2) if its barbourin domain could be productively replaced with smaller peptides. Purified BLAH(6) was injected into rabbits, and anti-barbourin antibodies were universally detected in plasma 28 days later; BLAH(6) was, however, equally effective in reducing platelet aggregation in both naive and pre-treated rabbits. Thrombocytopenia was not observed, and complexing BLAH(6) to alpha(IIb)beta(3) had no effect on antibody detection. The barbourin moiety of BLAH(6) was replaced with each of four sequences: Pep I (VCKGDWPC); PepII (VCRGDWPC); PepIII (bar-bourin 41-54); and PepIV (LPSPGDWR). The corresponding fusion proteins were tested for their ability to inhibit ADP-induced platelet aggregation. PepIII-LAH(6) inhibited neither rabbit nor human platelets. PepI-LAH(6) and PepIV-LAH(6) inhibited rabbit platelet aggregation as effectively as BLAH(6), but PepIV-LAH(6) did not inhibit human platelet aggregation. PepI-LAH(6) and PepIILAH(6) inhibited human platelet aggregation with IC(50)s 10- and 20-fold higher than BLAH(6). Cross-immunoprecipitation assays with human platelet lysates confirmed that all proteins and peptides interacted with the platelet integrin alpha(IIb)beta(3), but with greatly varying affinities. Our results suggest that the antiplatelet activity of BLAH(6) can be retained in albumin fusion proteins in which smaller peptides replace the barbourin domain; these proteins may be less immunogenic than BLAH(6).

  19. 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.

  20. 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.

  1. Extending the pharmacokinetic half-life of coagulation factors by fusion to recombinant albumin.

    PubMed

    Metzner, H J; Pipe, S W; Weimer, T; Schulte, S

    2013-11-01

    The prophylactic treatment of haemophilia B and the management of haemophilia A or B with inhibitors demand frequent administrations of coagulation factors due to the suboptimal half-lives of the products commercially available and currently in use, e.g. recombinant factor IX (rFIX) and recombinant factor VIIa (rFVIIa), respectively. The extension of the half-lives of rFIX and rFVIIa could allow for longer intervals between infusions and could thereby improve adherence and clinical outcomes and may improve quality of life. Albumin fusion is one of a number of different techniques currently being examined to prolong the half-life of rFIX and rFVIIa. Results from a phase I clinical trial demonstrated that the recombinant fusion protein linking FIX to albumin (rIX-FP) has a five-times longer half-life than rFIX, and preclinical studies with the recombinant fusion protein linking FVIIa to albumin (rVIIa-FP) suggest that rVIIa-FP possesses a significantly extended half-life versus rFVIIa. In this review, we describe albumin fusion technology and examine the recent progress in the development of rIX-FP and rVIIa-FP.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. Egg on their faces. The story of human albumin solution.

    PubMed

    Roberts, Ian; Bunn, Frances

    2002-03-01

    In 1998, the Cochrane Injuries Group published the results of a systematic review of human albumin administration in critically ill patients. The results showed that the risk of death in patients receiving albumin was 14%, and the risk of death in patients not receiving albumin was 8%, suggesting that for every 17 critically ill patients treated with albumin there is one extra death. The results were widely reported in the television and print media throughout the world and stimulated an immediate response from the drug regulatory agencies, the plasma products industry, and the medical profession. Despite vigorous attempts by the plasma products industry to limit the effect of the systematic review on albumin sales, the use of albumin declined steeply, showing that evidence from systematic reviews can have an important effect on clinical care.

  7. 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

  8. Production of Human Albumin in Pigs Through CRISPR/Cas9-Mediated Knockin of Human cDNA into Swine Albumin Locus in the Zygotes.

    PubMed

    Peng, Jin; Wang, Yong; Jiang, Junyi; Zhou, Xiaoyang; Song, Lei; Wang, Lulu; Ding, Chen; Qin, Jun; Liu, Liping; Wang, Weihua; Liu, Jianqiao; Huang, Xingxu; Wei, Hong; Zhang, Pumin

    2015-11-12

    Precise genome modification in large domesticated animals is desirable under many circumstances. In the past it is only possible through lengthy and burdensome cloning procedures. Here we attempted to achieve that goal through the use of the newest genome-modifying tool CRISPR/Cas9. We set out to knockin human albumin cDNA into pig Alb locus for the production of recombinant human serum albumin (rHSA). HSA is a widely used human blood product and is in high demand. We show that homologous recombination can occur highly efficiently in swine zygotes. All 16 piglets born from the manipulated zygotes carry the expected knockin allele and we demonstrated the presence of human albumin in the blood of these piglets. Furthermore, the knockin allele was successfully transmitted through germline. This success in precision genomic engineering is expected to spur exploration of pigs and other large domesticated animals to be used as bioreactors for the production of biomedical products or creation of livestock strains with more desirable traits.

  9. Nephroprotective Potential of Human Albumin Infusion: A Narrative Review

    PubMed Central

    Wiedermann, Christian J.; Joannidis, Michael

    2015-01-01

    Albumin infusion improves renal function in cirrhosis; however, mechanisms are incompletely understood. In clinical practice, human albumin is used in various intensive care unit indications to deal with a wide range of problems, from volume replacement in hypovolemic shock, or sepsis, to treatment of ascites in patients with liver cirrhosis. Against the background of the results of recent studies on the use of human albumin in septic patients, the importance of the natural colloid in these critically ill patients is being redefined. In addition to the hemodynamic effects of administration of human albumin impacting on sympathetic tone, attention is being paid to other effects in which its pharmacodynamics is associated with the physiological importance of endogenous albumin. The morbidity and mortality data discussed in this paper support the importance of both the hemodynamic and the pharmacological effects of the administration of human albumin in various indications. The contribution that human albumin could make towards the maintenance of renal function in the course and treatment of severe sepsis and cirrhosis of the liver is the subject of this narrative review. PMID:26136776

  10. 99M-technetium labeled macroaggregated human serum albumin pharmaceutical

    DOEpatents

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

    1977-05-17

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

  11. Atomic structure and chemistry of human serum albumin

    NASA Technical Reports Server (NTRS)

    He, Xiao M.; Carter, Daniel C.

    1992-01-01

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

  12. 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.

  13. 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

  14. 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.

  15. Selection of Recombinant Human Antibodies.

    PubMed

    Tomszak, Florian; Weber, Susanne; Zantow, Jonas; Schirrmann, Thomas; Hust, Michael; Frenzel, André

    2016-01-01

    Since the development of therapeutic antibodies the demand of recombinant human antibodies is steadily increasing. Traditionally, therapeutic antibodies were generated by immunization of rat or mice, the generation of hybridoma clones, cloning of the antibody genes and subsequent humanization and engineering of the lead candidates. In the last few years, techniques were developed that use transgenic animals with a human antibody gene repertoire. Here, modern recombinant DNA technologies can be combined with well established immunization and hybridoma technologies to generate already affinity maturated human antibodies. An alternative are in vitro technologies which enabled the generation of fully human antibodies from antibody gene libraries that even exceed the human antibody repertoire. Specific antibodies can be isolated from these libraries in a very short time and therefore reduce the development time of an antibody drug at a very early stage.In this review, we describe different technologies that are currently used for the in vitro and in vivo generation of human antibodies.

  16. Angiographic patency study of an albumin-free recombinant streptokinase formulation in acute myocardial infarction.

    PubMed

    Llerena, Lorenzo D; Quirós, Juan J; Sainz, Benito; Valdés, José A; Zorio, Biolkys; Villanueva, Luis H; Filgueiras, César E; Cabrera, Francisco; Echarte, Julio C; Pérez del Todo, Jesús M; Guerrero, Israel; López, Leonardo; García, Ernesto J; Nadal, Betty; Betancourt, Blas Y; Díaz-Rojo, Gisou; García, Ana I; López-Saura, Pedro

    2004-11-26

    Fibrinolytic therapy restores coronary patency and reduces mortality in patients with acute myocardial infarction. Albumin is present in most of the streptokinase formulation as a stabilizer but it is not known whether it plays a role in the product's efficacy and safety profiles. The aim of this study was to assess 90 minutes-coronary patency of a new albumin-free recombinant streptokinase (rSK) formulation. METHODS . Patients with ischemic chest pain and ST-segment elevation, less than 12 hours after symptoms onset, without contraindications for fibrinolytic therapy, were included to receive 1.5 x 10(6) IU of rSK in a one-hour intravenous infusion. Angiography was performed 90 minutes after and coronary patency was classified according to the TIMI flow scales. The study enrolled 25 patients, 59.4 +/- 9.2 years-old, 88% men and 92% white. The mean time interval between the symptoms onset and rSK infusion was 3.0 +/- 2.0 hours. Patency rate (TIMI 2-3) of the infarct-related vessel was 72% (18/25). Partial or complete ST-segment resolution was achieved in 17 patients (68%). Hypotension and nauseas were the most frequent adverse events. Haemorrhage or in-hospital deaths were not reported. This study suggests that intravenous albumin-free rSK is a safe and appropriate therapy to get early (90-minute) coronary patency in patients with acute myocardial infarction.

  17. VOLUME RESUSCITATION FROM HEMORRHAGIC SHOCK WITH ALBUMIN AND HEXA PEGYLATED HUMAN SERUM ALBUMIN

    PubMed Central

    Cabrales, Pedro; Tsai, Amy G.; Ananda, K.; Acharya, Seetharama A.; Intaglietta, Marcos

    2009-01-01

    The effect of restoring intravascular volume with polyethylene glycol (PEG) conjugated to human serum albumin (PEG-Alb) on systemic parameters and microvascular hemodynamics after hemorrhagic shock resuscitation was studied in the hamster window chamber model. Moderate hemorrhagic shock was induced by controlled arterial bleeding of 50% of blood volume, and hypovolemia was maintained for 1 hour. Fluid resuscitation was accomplished by infusion of 25% of blood volume and recovery was followed over 90 minutes. The PEG-Alb (six chains of maleimide phenyl PEG conjugated human serum albumin at 4%) resuscitation group was compared human serum albumin (HSA) at 5% (HSA5) and 10% (HSA10) protein concentrations. Systemic parameters, microvascular perfusion and capillary perfusion (functional capillary density, FCD) were measured by noninvasive methods. Hyperoncotic solutions provided rapid restoration of blood pressure, blood gas parameters and microvascular perfusion. Systemic and microvascular recovery was best and most rapid with PEG-Alb and followed by HSA10 and HSA5 rapid. Only recovery with PEG-Alb was sustained beyond 90 min. Hemodynamic functional benefits of PEG-Alb and the potential disadvantages associated with HSA, suggest PEG-Alb as better resuscitation solution. PMID:18621463

  18. TALEN-mediated modification of the bovine genome for large-scale production of human serum albumin.

    PubMed

    Moghaddassi, Shaida; Eyestone, Will; Bishop, Colin E

    2014-01-01

    As an initial step towards creating genetically modified cattle as a biopharming source of recombinant human serum albumin (rHSA), we report modification of the bovine albumin (bA) locus by transcription activator-like effector nuclease (TALEN)-stimulated homology-directed repair (HDR). Pedigreed bovine fibroblasts were co-transfected with TALENs and an 11.5-kb human serum albumin (HSA) minigene donor construct, designed to simultaneously disrupt and replace bovine serum albumin (BSA) expression with controlled rHSA expression in both the liver and the milk. Targeted integration of the HSA minigene was confirmed in transfected fibroblasts at a frequency of approximately 11% and transgenic bovine embryos were produced from targeted fibroblasts using somatic cell nuclear transfer (SCNT). The research delineated here lays the foundation for the future generation of transgenic rHSA cattle with the potential to provide a large-scale, reliable, and quality-controlled source of rHSA.

  19. The exclusion of human serum albumin by human dermal collagenous fibres and within human dermis.

    PubMed Central

    Bert, J L; Mathieson, J M; Pearce, R H

    1982-01-01

    Preparations of dermal collagenous fibres and slices of human dermis have been equilibrated with 125I-labelled monomeric human serum albumin. The space inaccessible to the albumin in the fibres and in the dermis was determined by subtraction of the accessible space, calculated from the radioactivity of the specimen, from its total fluid. For a fibre preparation examined in detail, the fluid exclusion was independent of the concentration of either albumin or collagen. Binding of albumin to the fibres was not demonstrable. Three fibre preparations excluded albumin from 3.75 +/- 0.96, 3.55 +/- 0.67, and 2.05 +/- 0.39 g of fluid/g of collagen (+/-S.D.). Slices from three specimens of dermis excluded albumin from 1.45 +/- 0.08 g of fluid/g of insoluble solids or 1.57 +/- 0.11 g of fluid/g of collagen (+/-S.D.). Thus the exclusion of albumin by dermis was much less than expected from its content of collagenous fibres. On the basis of these data and the published composition of dermis, the concentration of albumin in the accessible interstitial space was estimated to be close to that in the plasma. PMID:7082298

  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. Affinity of human serum albumin for bilirubin varies with albumin concentration and buffer composition: results of a novel ultrafiltration method.

    PubMed

    Weisiger, R A; Ostrow, J D; Koehler, R K; Webster, C C; Mukerjee, P; Pascolo, L; Tiribelli, C

    2001-08-10

    Albumin binding is a crucial determinant of bilirubin clearance in health and bilirubin toxicity in certain disease states. However, prior attempts to measure the affinity of albumin for bilirubin have yielded highly variable results, reflecting both differing conditions and the confounding influence of impurities. We therefore have devised a method based on serial ultrafiltration that successively removes impurities in [(14)C]bilirubin until a stable binding affinity is achieved, and then we used it to assess the effect of albumin concentration and buffer composition on binding. The apparent binding affinity of human serum albumin for [(14)C]bilirubin was strongly dependent on assay conditions, falling from (5.09 +/- 0.24) x 10(7) liters/mol at lower albumin concentrations (15 microm) to (0.54 +/- 0.05) x 10(7) liters/mol at higher albumin concentrations (300 microm). To determine whether radioactive impurities were responsible for this change, we estimated impurities in the stock bilirubin using a novel modeling approach and found them to be 0.11-0.13%. Formation of new impurities during the study and their affinity for albumin were also estimated. After correction for impurities, the binding affinity remained heavily dependent on the albumin concentration (range (5.37 +/- 0.26) x 10(7) liters/mol to (0.65 +/- 0.03) x 10(7) liters/mol). Affinities decreased by about half in the presence of chloride (50 mm). Thus, the affinity of human albumin for bilirubin is not constant, but varies with both albumin concentration and buffer composition. Binding may be considerably less avid at physiological albumin concentrations than previously believed.

  2. Development of Recombinant Lactococcus lactis Displaying Albumin-Binding Domain Variants against Shiga Toxin 1 B Subunit

    PubMed Central

    Zadravec, Petra; Marečková, Lucie; Petroková, Hana; Hodnik, Vesna; Perišić Nanut, Milica; Anderluh, Gregor; Štrukelj, Borut; Malý, Petr; Berlec, Aleš

    2016-01-01

    Infections with shiga toxin-producing bacteria, like enterohemorrhagic Escherichia coli and Shigella dysenteriae, represent a serious medical problem. No specific and effective treatment is available for patients with these infections, creating a need for the development of new therapies. Recombinant lactic acid bacterium Lactococcus lactis was engineered to bind Shiga toxin by displaying novel designed albumin binding domains (ABD) against Shiga toxin 1 B subunit (Stx1B) on their surface. Functional recombinant Stx1B was produced in Escherichia coli and used as a target for selection of 17 different ABD variants (named S1B) from the ABD scaffold-derived high-complex combinatorial library in combination with a five-round ribosome display. Two most promising S1Bs (S1B22 and S1B26) were characterized into more details by ELISA, surface plasmon resonance and microscale thermophoresis. Addition of S1Bs changed the subcellular distribution of Stx1B, completely eliminating it from Golgi apparatus most likely by interfering with its retrograde transport. All ABD variants were successfully displayed on the surface of L. lactis by fusing to the Usp45 secretion signal and to the peptidoglycan-binding C terminus of AcmA. Binding of Stx1B by engineered lactococcal cells was confirmed using flow cytometry and whole cell ELISA. Lactic acid bacteria prepared in this study are potentially useful for the removal of Shiga toxin from human intestine. PMID:27606705

  3. Conjugation of organoruthenium(II) 3-(1H-benzimidazol-2-yl)pyrazolo[3,4-b]pyridines and indolo[3,2-d]benzazepines to recombinant human serum albumin: a strategy to enhance cytotoxicity in cancer cells.

    PubMed

    Stepanenko, Iryna N; Casini, Angela; Edafe, Fabio; Novak, Maria S; Arion, Vladimir B; Dyson, Paul J; Jakupec, Michael A; Keppler, Bernhard K

    2011-12-19

    Following our strategy of coupling cyclin-dependent kinase (Cdk) inhibitors with organometallic moieties to improve their physicochemical properties and bioavailability, five organoruthenium complexes (1c-5c) of the general formula [RuCl(η(6)-arene)(L)]Cl have been synthesized in which the arene is 4-formylphenoxyacetyl-η(6)-benzylamide and L is a Cdk inhibitor [3-(1H-benzimidazol-2-yl)-1H-pyrazolo[3,4-b]pyridines (L1-L3) and indolo[3,2-d]benzazepines (L4 and L5)]. All of the compounds were characterized by spectroscopic and analytical methods. Upon prolonged standing (2-3 months) at room temperature, the dimethyl sulfoxide (DMSO) solutions of 1c and 2c(-HCl) afforded residues, which after recrystallization from EtOH and EtOH/H(2)O, respectively, were shown by X-ray diffraction to be cis,cis-[Ru(II)Cl(2)(DMSO)(2)(L1)]·H(2)O and mer-[Ru(II)Cl(DMSO)(3)(L2-H)]·H(2)O. Compound 5c, with a coordinated amidine unit, undergoes E/Z isomerization in solution. The antiproliferative activities and effects on the cell cycle of the new compounds were evaluated. Complexes 1c-5c are moderately cytotoxic to cancer cells (CH1, SW480, A549, A2780, and A2780cisR cell lines). Therefore, in order to improve their antiproliferative effects, as well as their drug targeting and delivery to cancer cells, 1c-5c were conjugated to recombinant human serum albumin, potentially exploiting the so-called "enhanced permeability and retention" effect that results in the accumulation of macromolecules in tumors. Notably, a marked increase in cytotoxicity of the albumin conjugates was observed in all cases.

  4. Interaction of glucocorticoids and progesterone derivatives with human serum albumin.

    PubMed

    Abboud, Rola; Akil, Mohammad; Charcosset, Catherine; Greige-Gerges, Hélène

    2017-10-01

    Glucocorticoids (GCs) and progesterone derivatives (PGDs) are steroid hormones with well-known biological activities. Their interaction with human serum albumin (HSA) may control their distribution. Their binding to albumin is poorly studied in literature. This paper deals with the interaction of a series of GCs (cortisol, cortisone, prednisolone, prednisone, 6-methylprednisolone and 9-fluorocortisol acetate) and PGDs (progesterone, hydroxylated PGDs, methylated PGDs and dydrogesterone) with HSA solution (pH 7.4) at molar ratios steroid to HSA varying from 0 to 10. Similar titrations were conducted using Trp aqueous solution. Fluorescence titration method and Fourier transform infrared spectroscopy (FTIR) are used. PGDs (except dydrogesterone), cortisone and 9-fluorocortisol acetate affected weakly the fluorescence of Trp in buffer solution while they decreased in a dose-dependent manner that of HSA. Their binding constants to HSA were then calculated. Moreover, displacement experiment was performed using bilirubin as a site marker. The binding constant of bilirubin to albumin was determined in the absence and presence of a steroid at a molar ratio steroid to HSA of 1. The results indicate that the steroids bind to HSA at site I in a pocket different from that of bilirubin. Furthermore, the peak positions of amide I and amide II bands of HSA were shifted in the presence of progesterone, dydrogesterone and GCs. Also a variation was observed in amide I region indicating the formation of hydrogen bonding between albumin and steroids. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. 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.

  6. Large-scale production of functional human serum albumin from transgenic rice seeds.

    PubMed

    He, Yang; Ning, Tingting; Xie, Tingting; Qiu, Qingchuan; Zhang, Liping; Sun, Yunfang; Jiang, Daiming; Fu, Kai; Yin, Fei; Zhang, Wenjing; Shen, Lang; Wang, Hui; Li, Jianjun; Lin, Qishan; Sun, Yunxia; Li, Hongzhen; Zhu, Yingguo; Yang, Daichang

    2011-11-22

    Human serum albumin (HSA) is widely used in clinical and cell culture applications. Conventional production of HSA from human blood is limited by the availability of blood donation and the high risk of viral transmission from donors. Here, we report the production of Oryza sativa recombinant HSA (OsrHSA) from transgenic rice seeds. The level of OsrHSA reached 10.58% of the total soluble protein of the rice grain. Large-scale production of OsrHSA generated protein with a purity >99% and a productivity rate of 2.75 g/kg brown rice. Physical and biochemical characterization of OsrHSA revealed it to be equivalent to plasma-derived HSA (pHSA). The efficiency of OsrHSA in promoting cell growth and treating liver cirrhosis in rats was similar to that of pHSA. Furthermore, OsrHSA displays similar in vitro and in vivo immunogenicity as pHSA. Our results suggest that a rice seed bioreactor produces cost-effective recombinant HSA that is safe and can help to satisfy an increasing worldwide demand for human serum albumin.

  7. Large-scale production of functional human serum albumin from transgenic rice seeds

    PubMed Central

    He, Yang; Ning, Tingting; Xie, Tingting; Qiu, Qingchuan; Zhang, Liping; Sun, Yunfang; Jiang, Daiming; Fu, Kai; Yin, Fei; Zhang, Wenjing; Shen, Lang; Wang, Hui; Li, Jianjun; Lin, Qishan; Sun, Yunxia; Li, Hongzhen; Zhu, Yingguo; Yang, Daichang

    2011-01-01

    Human serum albumin (HSA) is widely used in clinical and cell culture applications. Conventional production of HSA from human blood is limited by the availability of blood donation and the high risk of viral transmission from donors. Here, we report the production of Oryza sativa recombinant HSA (OsrHSA) from transgenic rice seeds. The level of OsrHSA reached 10.58% of the total soluble protein of the rice grain. Large-scale production of OsrHSA generated protein with a purity >99% and a productivity rate of 2.75 g/kg brown rice. Physical and biochemical characterization of OsrHSA revealed it to be equivalent to plasma-derived HSA (pHSA). The efficiency of OsrHSA in promoting cell growth and treating liver cirrhosis in rats was similar to that of pHSA. Furthermore, OsrHSA displays similar in vitro and in vivo immunogenicity as pHSA. Our results suggest that a rice seed bioreactor produces cost-effective recombinant HSA that is safe and can help to satisfy an increasing worldwide demand for human serum albumin. PMID:22042856

  8. Fluorescence lifetime measurements of native and glycated human serum albumin and bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Joshi, Narahari V.; Joshi, Virgina O. d.; Contreras, Silvia; Gil, Herminia; Medina, Honorio; Siemiarczuk, Aleksander

    1999-05-01

    Nonenzymatic glycation, also known as Maillard reaction, plays an important role in the secondary complications of the diabetic pathology and aging, therefore, human serum albumin (HSA) and bovine serum albumin (BSA) were glycated by a conventional method in our laboratory using glucose as the glycating agent. Fluorescence lifetime measurements were carried out with a laser strobe fluorometer equipped with a nitrogen/dye laser and a frequency doubler as a pulsed excitation source. The samples were excited at 295 nm and the emission spectra were recorded at 345 nm. The obtained decay curves were tried for double and triple exponential functions. It has been found that the shorter lifetime increases for glycated proteins as compared with that of the native ones. For example, in the case of glycated BSA the lifetime increased from 1.36 ns to 2.30 ns. Similarly, for HSA, the lifetime increases from 1.58 ns to 2.26 ns. Meanwhile, the longer lifetime changed very slightly for both proteins (from 6.52 ns to 6.72 ns). The increase in the lifetime can be associated with the environmental effect; originated from the attachment of glucose to some lysine residues. A good example is Trp 214 which is in the cage of Lys 225, Lys 212, Lys 233, Lys 205, Lys 500, Lys 199 and Lys 195. If fluorescence lifetime technique is calibrated and properly used it could be employed for assessing glycation of proteins.

  9. Predictive Value of Serum Albumin Level for the Prognosis of Severe Sepsis Without Exogenous Human Albumin Administration.

    PubMed

    Yin, Mei; Si, Lei; Qin, Weidong; Li, Chen; Zhang, Jianning; Yang, Hongna; Han, Hui; Zhang, Fan; Ding, Shifang; Zhou, Min; Wu, Dawei; Chen, Xiaomei; Wang, Hao

    2016-01-01

    The prognostic significance of serum albumin levels in patients with sepsis has previously been reported; however, these studies have not excluded the potential confounding effect of exogenous albumin administration. In this study, we investigate the predictive value of serum albumin for the prognosis of severe sepsis without the interference of exogenous albumin administration. A prospective cohort study was conducted from April to November 2014 in the internal and surgical intensive care units of a tertiary care hospital. During the study period, due to a supply shortage, patients were not treated with human albumin. Serum albumin levels were measured, and laboratory and clinical data were collected at the onset of severe sepsis. Prognostic factors were analyzed using receiver operating characteristic curve and multivariate Cox proportional hazard regression analysis. Survival was assessed by Kaplan-Meier method. One hundred sixteen patients were included in the study. The overall 28-day mortality was 26.7%. The most common infection sources were lower respiratory tract, abdomen/pelvis, and bloodstream. Compared to patients who survived, those who died had lower serum albumin levels and higher Acute Physiology and Chronic Health Evaluation II (APACHE II) and Sequential Organ Failure Assessment (SOFA) scores. Receiver operating characteristic curves demonstrate that albumin level is a strong predictor of 28-day mortality, and the optimal cutoff value maximizing sensitivity and specificity is 29.2 g/L. Through multivariate Cox regression analysis, low serum albumin levels (<29.2 g/L) and APACHE II scores are identified as independent risk factors for mortality. Patients with lower serum albumin levels more often had abdominal/pelvic sources of infection, acute kidney or liver injury, septic shock, and higher APACHE II and SOFA scores. The 28-day survival rate was lower for patients with serum albumin below 29.2 g/L than for patients with serum albumin at or above

  10. Transforming the treatment for hemophilia B patients: update on the clinical development of recombinant fusion protein linking recombinant coagulation factor IX with recombinant albumin (rIX-FP).

    PubMed

    Santagostino, Elena

    2016-05-01

    Recombinant fusion protein linking recombinant coagulation factor IX with recombinant albumin (rIX-FP; Idelvion®(†)) is an innovative new treatment designed to extend the half-life of factor IX (FIX) and ease the burden of care for hemophilia B patients. The rIX-FP clinical development program - PROLONG-9FP - is in its advanced phases, with pivotal studies in previously treated adults, adolescents, and pediatrics now completed. Across all age groups studied, rIX-FP has demonstrated a markedly improved pharmacokinetic profile compared with plasma-derived and recombinant FIX treatments, with a 30-40% higher incremental recovery, an approximately 5-fold longer half-life, a lower clearance, and a greater area under the curve. rIX-FP has been very well tolerated with an excellent safety profile. In the pivotal studies, there have been no reports of FIX inhibitors or antidrug antibodies, and few treatment-related adverse events have been observed. Prophylactic regimens of rIX-FP administered once weekly to once every 14 days have been highly effective. When used for surgical prophylaxis, a single infusion of rIX-FP has been sufficient to maintain hemostasis, even during major orthopedic surgery. An ongoing study is now enrolling previously untreated patients and evaluating the possibility of extending the dosing interval to every 21 days. There is little doubt that rIX-FP will transform the treatment of hemophilia B. © 2016 Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

  11. Homogeneous substrate-labeled fluorescent immunoassay for human serum albumin.

    PubMed

    Patinkin, J; Inbar, D; Ben-Gigi, C; Derfler, S; Klausner, Y; Fridlender, B

    1983-01-01

    A homogeneous substrate-labeled fluorescent immunoassay for human serum albumin (HSA) has been developed, similar to previously described immunoassays for Immunoglobulin G and Immunoglobulin M. HSA was covalently linked to 6-(7-beta-galactosylcoumarin-3-carboxamide) hexylamine. The resulting conjugate had minimal fluorescence at 450 nm (with excitation at 400 nm). However, when the acetal linkage of the galactosyl moiety was hydrolyzed by beta-galactosidase, a substantial increase in the fluorescence was obtained. This increase was specifically inhibited by antibody to HSA. A competitive binding immunoassay was established by letting the conjugate compete with HSA in the serum for the limited number of antibody-binding sites. The level of fluorescence resulting from the addition of enzyme was proportional to the amount of HSA in the serum. Precision, analytical recovery and serum dilution studies were carried out on the assay. The immunoassay was compared to an albumin assay using the dye-binding method.

  12. Sequences Of Amino Acids For Human Serum Albumin

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C.

    1992-01-01

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

  13. 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.

  14. Multiple fatty acid binding to albumin in human blood plasma.

    PubMed

    Brodersen, R; Andersen, S; Vorum, H; Nielsen, S U; Pedersen, A O

    1990-04-30

    Binding equilibria of long-chain fatty acids to human serum albumin, in serum or plasma, were studied by a dialysis exchange rate technique. Palmitate was added to citrated plasma in vitro and it was observed that between six and ten palmitate molecules were bound to albumin with nearly equal affinity. Observations in vivo gave similar results in the following series: (a) in two volunteers with increased fatty acid concentrations after fasting, exercise, and a cold shower: (b) in three male volunteers in whom high concentrations of non-esterified fatty acids, up to 4.6 mM, were induced by intravenous administration of a preparation of lecithin/glycocholate mixed micelles, and (c) in 81 patients with diabetes mellitus, type I. The binding pattern of palmitate in serum or plasma is essentially different from that observed with palmitate added to buffered solutions of pure albumin when two molecules are tightly bound and about four additional molecules with lower affinity. The differences may partly be explained by the presence of chloride ions in blood plasma, reducing the affinity for binding of the first two fatty acid molecules, and partly by facilitated binding of several molecules of mixed fatty acids, as found in plasma.

  15. Interaction of human serum albumin and its clinically relevant modification with oligoribonucleotides.

    PubMed

    Gerasimova, Yuliya V; Erchenko, Irina A; Shakirov, Makhmut M; Godovikova, Tatyana S

    2008-08-15

    Human serum albumin (HSA) was shown to mediate oligoribonucleotide cleavage. Nonenzymatic glycation of HSA decreased the ribonuclease-like activity of the protein. According to (31)P NMR data, both native and glycated albumins induced hydrolysis of RNA molecule through 2',3'-cyclophosphate intermediates. A feasible mechanism of RNA hydrolysis by native albumin and its clinically relevant modification was discussed.

  16. Novel Transgenic Mouse Model for Studying Human Serum Albumin as a Biomarker of Carcinogenic Exposure.

    PubMed

    Sheng, Jonathan; Wang, Yi; Turesky, Robert J; Kluetzman, Kerri; Zhang, Qing-Yu; Ding, Xinxin

    2016-05-16

    Albumin is a commonly used serum protein for studying human exposure to xenobiotic compounds, including therapeutics and environmental pollutants. Often, the reactivity of albumin with xenobiotic compounds is studied ex vivo with human albumin or plasma/serum samples. Some studies have characterized the reactivity of albumin with chemicals in rodent models; however, differences between the orthologous peptide sequences of human and rodent albumins can result in the formation of different types of chemical-protein adducts with different interaction sites or peptide sequences. Our goal is to generate a human albumin transgenic mouse model that can be used to establish human protein biomarkers of exposure to hazardous xenobiotics for human risk assessment via animal studies. We have developed a human albumin transgenic mouse model and characterized the genotype and phenotype of the transgenic mice. The presence of the human albumin gene in the genome of the model mouse was confirmed by genomic PCR analysis, whereas liver-specific expression of the transgenic human albumin mRNA was validated by RT-PCR analysis. Further immunoblot and mass spectrometry analyses indicated that the transgenic human albumin protein is a full-length, mature protein, which is less abundant than the endogenous mouse albumin that coexists in the serum of the transgenic mouse. The transgenic protein was able to form ex vivo adducts with a genotoxic metabolite of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, a procarcinogenic heterocyclic aromatic amine formed in cooked meat. This novel human albumin transgenic mouse model will facilitate the development and validation of albumin-carcinogen adducts as biomarkers of xenobiotic exposure and/or toxicity in humans.

  17. Photoexcited riboflavin induces oxidative damage to human serum albumin

    NASA Astrophysics Data System (ADS)

    Hirakawa, Kazutaka; Yoshioka, Takuto

    2015-08-01

    Photoexcited riboflavin induced damage of human serum albumin (HSA), a water soluble protein, resulting in the diminishment of fluorescence from the tryptophan residue. Because riboflavin hardly photosensitized singlet oxygen generation and sodium azide, a singlet oxygen quencher, did not inhibit protein damage, electron transfer-mediated oxidation of HSA was speculated. Fluorescence lifetime of riboflavin was not affected by HSA, suggesting that the excited triplet state of riboflavin is responsible for protein damage through electron transfer. In addition, the preventive effect of xanthone derivatives, triplet quenchers, on photosensitized protein damage could be evaluated using this photosensitized reaction system of riboflavin and HSA.

  18. Three-dimensional structure of human serum albumin

    NASA Technical Reports Server (NTRS)

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

    1989-01-01

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

  19. Biomolecular Interaction Study of Cyclolinopeptide A with Human Serum Albumin

    PubMed Central

    Rempel, Ben; Gui, Bo; Maley, Jason; Reaney, Martin; Sammynaiken, Ramaswami

    2010-01-01

    The kinetics, energetics, and structure of Cyclolinopeptide A binding with Human Serum Albumin were investigated with surface plasmon resonance and circular dichroism. The complex is formed through slow recognition kinetics that is temperature sensitive in the range of 20°C–37°C. The overall reaction was observed to be endothermic (ΔH = 204 kJ mol−1) and entropy driven (ΔS = 746 J mol−1K−1) with overall small changes to the tertiary structure. PMID:21436992

  20. THERMODYNAMIC CHARACTERIZATION OF METAL PHTHALOCYANINES-HUMAN SERUM ALBUMIN INTERACTIONS

    PubMed Central

    Jones, Cecil L.

    2012-01-01

    The temperature dependence of the binding constants for human serum albumin and sulfonated metal-phthalocyanines were estimated by fluorescence spectroscopy. Stern-Volmer’s analysis and Chipman’s fits provided binding data for van’t Hoff calculations of the thermodynamic parameters governing these interactions. Results show that the formations of the HSA- phthalocyanine complexes are favorable processes by both the ΔH° and ΔS°. However, the formation of HSA-AIPcS4 has a stronger dependence than HSA-ZnPcS 4on ΔS°. PMID:24058291

  1. Generation of a double transgenic humanized neonatal Fc receptor (FcRn)/albumin mouse to study the pharmacokinetics of albumin-linked drugs.

    PubMed

    Viuff, Dorthe; Antunes, Filipa; Evans, Leslie; Cameron, Jason; Dyrnesli, Hans; Thue Ravn, Birgitte; Stougaard, Magnus; Thiam, Kader; Andersen, Birgitte; Kjærulff, Søren; Howard, Kenneth A

    2016-02-10

    Human serum albumin (HSA) is a natural carrier protein possessing multiple ligand binding sites with a plasma half-life ~19days, facilitated by interaction with the human neonatal Fc receptor (FcRn), that promotes it as a highly attractive drug delivery technology. A lack of adequate rodent models, however, is a major challenge in the preclinical development of albumin-linked therapeutics. This work describes the first double transgenic mouse model bearing both human FcRn and HSA genes (hFcRn(+/+), hAlb(+/+)) under the control of an endogenous promoter. Human FcRn was shown by immunohistochemical and qPCR analysis to be ubiquitously expressed in the major organs. Physiological levels of HSA were detected in the blood that exhibited similar FcRn binding kinetics to recombinant or human serum-derived HSA. The circulatory half-life (t1/2) was shown to be dependent on FcRn binding affinity that increased from low affinity (t1/2 29h), to wild type (t1/2 50h), to high affinity (t1/2 80h) variants, that validates the application of the model for optimizing the pharmacokinetics of drug carriers who's circulatory half-life is dependent in some manner upon interaction with endogenous FcRn. This study presents a novel mouse model that better mimics the human physiological conditions and, thus, has potential wide applications in the development of albumin-linked drugs or conventional drugs whose action is influenced by reversible binding to endogenous HSA. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. A New Application for Albumin Dialysis in Extracorporeal Organ Support: Characterization of a Putative Interaction Between Human Albumin and Proinflammatory Cytokines IL-6 and TNFα.

    PubMed

    Pfensig, Claudia; Dominik, Adrian; Borufka, Luise; Hinz, Michael; Stange, Jan; Eggert, Martin

    2016-04-01

    Albumin dialysis in extracorporeal organ support is often performed in the treatment of liver failure as it facilitates the removal of toxic components from the blood. Here, we describe a possible effect of albumin dialysis on proinflammatory cytokine levels in vitro. Initially, albumin samples were incubated with different amounts of cytokines and analyzed by enzyme-linked immunosorbent assay (ELISA). Analysis of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNFα) levels indicated that increased concentrations of albumin reduce the measureable amount of the respective cytokines. This led to the hypothesis that the used proinflammatory cytokines may interact with albumin. Size exclusion chromatography of albumin spiked with cytokines was carried out using high-performance liquid chromatography analysis. The corresponding fractions were evaluated by immunoblotting. We detected albumin and cytokines in the same fractions indicating an interaction of the small-sized cytokines IL-6 and TNFα with the larger-sized albumin. Finally, a two-compartment albumin dialysis in vitro model was used to analyze the effect of albumin on proinflammatory cytokines in the recirculation circuit during 6-h treatment. These in vitro albumin dialysis experiments indicated a significant decrease of IL-6, but not of TNFα, when albumin was added to the dialysate solution. Taken together, we were able to show a putative in vitro interaction of human albumin with the proinflammatory cytokine IL-6, but with less evidence for TNFα, and demonstrated an additional application for albumin dialysis in liver support therapy where IL-6 removal might be indicated.

  3. Comparison of albumin receptors expressed on bovine and human group G streptococci.

    PubMed Central

    Raeder, R; Otten, R A; Boyle, M D

    1991-01-01

    The albumin receptor expressed by bovine group G streptococci was extracted and affinity purified. The protein was characterized for species reactivity, and monospecific antibodies were prepared to the purified receptor. The bovine group G albumin receptor was compared functionally, antigenically, and for DNA homology with the albumin-binding protein expressed by human group G streptococci. In agreement with previous reports, the albumin-binding activity of human strains was mediated by a unique domain of the type III immunoglobulin G-Fc-binding molecule, protein G. The albumin receptor expressed by bovine group G strains was found to lack any immunoglobulin G-binding potential but displayed a wider profile of species albumin reactivity than protein G. Both albumin receptors could inhibit the binding of the other to immobilized human serum albumin, and each displayed similar binding properties. Antigenic comparison of the two albumin receptors demonstrated a low level of cross-reactivity; however comparison at the DNA level, using an oligonucleotide probe specific for the albumin-binding region of protein G, demonstrated that the two albumin receptors expressed by human and bovine group G streptococcal strains do not display significant homology. Images PMID:1846128

  4. DNA recombination. Recombination initiation maps of individual human genomes.

    PubMed

    Pratto, Florencia; Brick, Kevin; Khil, Pavel; Smagulova, Fatima; Petukhova, Galina V; Camerini-Otero, R Daniel

    2014-11-14

    DNA double-strand breaks (DSBs) are introduced in meiosis to initiate recombination and generate crossovers, the reciprocal exchanges of genetic material between parental chromosomes. Here, we present high-resolution maps of meiotic DSBs in individual human genomes. Comparing DSB maps between individuals shows that along with DNA binding by PRDM9, additional factors may dictate the efficiency of DSB formation. We find evidence for both GC-biased gene conversion and mutagenesis around meiotic DSB hotspots, while frequent colocalization of DSB hotspots with chromosome rearrangement breakpoints implicates the aberrant repair of meiotic DSBs in genomic disorders. Furthermore, our data indicate that DSB frequency is a major determinant of crossover rate. These maps provide new insights into the regulation of meiotic recombination and the impact of meiotic recombination on genome function.

  5. Human Insulin from Recombinant DNA Technology

    NASA Astrophysics Data System (ADS)

    Johnson, Irving S.

    1983-02-01

    Human insulin produced by recombinant DNA technology is the first commercial health care product derived from this technology. Work on this product was initiated before there were federal guidelines for large-scale recombinant DNA work or commercial development of recombinant DNA products. The steps taken to facilitate acceptance of large-scale work and proof of the identity and safety of such a product are described. While basic studies in recombinant DNA technology will continue to have a profound impact on research in the life sciences, commercial applications may well be controlled by economic conditions and the availability of investment capital.

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

    NASA Technical Reports Server (NTRS)

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

    1994-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1994-01-01

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

  8. Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine

    Cancer.gov

    This page contains brief information about recombinant human papillomavirus (HPV) quadrivalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

  9. Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine

    Cancer.gov

    This page contains brief information about recombinant human papillomavirus (HPV) nonavalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

  10. Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine

    Cancer.gov

    This page contains brief information about recombinant human papillomavirus (HPV) quadrivalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

  11. Recombinant Human Papillomavirus (HPV) Bivalent Vaccine

    Cancer.gov

    This page contains brief information about recombinant human papillomavirus (HPV) bivalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

  12. Single particle conformations of human serum albumin by electron microscopy.

    PubMed

    Ueno, Yutaka; Mio, Muneyo; Sato, Chikara; Mio, Kazuhiro

    2007-06-01

    Using single particle images taken by electron microscopy, pH-dependent conformational changes of human serum albumin were investigated. Despite the noisy particle images of negatively stained serum albumin (67 kDa), our novel algorithm for automated particle picking and reference-free classification resulted in the appropriate grouping of the particle images. Iteratively aligned particle images in the same group provided recognizable image features for individual groups. In a pH 7.0 study, monomer images were consistent with an available crystal structure model; the dimer images were separated into different classes. At pH 3.5, the monomer images were similar to those at pH 7.0; slight differences included a small number of elongated conformations and increased population of larger multimers. Our images were also compared with projection images of an atomic model from crystallography, and demonstrated consistency of the molecular conformation both at pH 7.0 and pH 3.5. Our classification method was effective in discriminating monomers from a mixture of different conformations of the protein, enabling the study of the conformational dynamics of small proteins, using the atomic model as a reference.

  13. Human mature adipocytes express albumin and this expression is not regulated by inflammation.

    PubMed

    Sirico, Maria Luisa; Guida, Bruna; Procino, Alfredo; Pota, Andrea; Sodo, Maurizio; Grandaliano, Giuseppe; Simone, Simona; Pertosa, Giovanni; Riccio, Eleonora; Memoli, Bruno

    2012-01-01

    Our group investigated albumin gene expression in human adipocytes, its regulation by inflammation and the possible contribution of adipose tissue to albumin circulating levels. Both inflamed and healthy subjects provided adipose tissue samples. RT-PCR, Real-Time PCR, and Western Blot analysis on homogenates of adipocytes and pre-adipocytes were performed. In sixty-three healthy subjects and fifty-four micro-inflamed end stage renal disease (ESRD) patients circulating levels of albumin were measured by nephelometry; all subjects were also evaluated for body composition, calculated from bioelectrical measurements and an thropometric data. A clear gene expression of albumin was showed in pre-adipocytes and, for the first time, in mature adipocytes. Albumin gene expression resulted significantly higher in pre-adipocytes than in adipocytes. No significant difference in albumin gene expression was showed between healthy controls and inflamed patients. A significant negative correlation was observed between albumin levels and fat mass in both healthy subjects and inflamed ESRD patients. In the present study we found first time evidence that human adipocytes express albumin. Our results also showed that systemic inflammation does not modulate albumin gene expression. The negative correlation between albumin and fat mass seems to exclude a significant contributing role of adipocyte in plasma albumin.

  14. Human Mature Adipocytes Express Albumin and This Expression Is Not Regulated by Inflammation

    PubMed Central

    Sirico, Maria Luisa; Guida, Bruna; Procino, Alfredo; Pota, Andrea; Sodo, Maurizio; Grandaliano, Giuseppe; Simone, Simona; Pertosa, Giovanni; Riccio, Eleonora; Memoli, Bruno

    2012-01-01

    Aims. Our group investigated albumin gene expression in human adipocytes, its regulation by inflammation and the possible contribution of adipose tissue to albumin circulating levels. Methods. Both inflamed and healthy subjects provided adipose tissue samples. RT-PCR, Real-Time PCR, and Western Blot analysis on homogenates of adipocytes and pre-adipocytes were performed. In sixty-three healthy subjects and fifty-four micro-inflamed end stage renal disease (ESRD) patients circulating levels of albumin were measured by nephelometry; all subjects were also evaluated for body composition, calculated from bioelectrical measurements and an thropometric data. Results. A clear gene expression of albumin was showed in pre-adipocytes and, for the first time, in mature adipocytes. Albumin gene expression resulted significantly higher in pre-adipocytes than in adipocytes. No significant difference in albumin gene expression was showed between healthy controls and inflamed patients. A significant negative correlation was observed between albumin levels and fat mass in both healthy subjects and inflamed ESRD patients. Conclusions. In the present study we found first time evidence that human adipocytes express albumin. Our results also showed that systemic inflammation does not modulate albumin gene expression. The negative correlation between albumin and fat mass seems to exclude a significant contributing role of adipocyte in plasma albumin. PMID:22675238

  15. Interaction of triterpenoids with human serum albumin: A review.

    PubMed

    Abboud, Rola; Charcosset, Catherine; Greige-Gerges, Hélène

    2017-10-01

    Triterpenoids are a large group of natural and synthetic products. This review deals with the current state of knowledge on their interaction with serum albumin. The binding of drugs to albumin may control their distribution in tissues. In literature, different techniques were used to investigate the albumin-triterpenoid interaction and include fluorescence spectroscopy, Fourier transform infrared spectroscopy, circular dichroism, calorimetric techniques and molecular modeling. Changes in fluorescence intensity of albumin were observed upon triterpenoid-albumin complex formation. Thermodynamic analyses proved that hydrophobic interactions and hydrogen bonds were the mainly binding forces in triterpenoid-albumin systems. Molecular docking and site marker competitive experimental results revealed that triterpenoids bound to Sudlow's site I of albumin. Furthermore, Fourier transform infrared spectroscopy and circular dichroism spectra analysis indicated that the native conformation of the protein is affected upon binding to triterpenoids. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Human serum albumin incorporating synthetic heme: red blood cell substitute without hypertension by nitric oxide scavenging.

    PubMed

    Tsuchida, Eishun; Komatsu, Teruyuki; Matsukawa, Yasuko; Nakagawa, Akito; Sakai, Hiromi; Kobayashi, Koichi; Suematsu, Makoto

    2003-02-01

    The administration of extracellular, hemoglobin-based oxygen carriers often elicits an acute increase in blood pressure by vasoconstriction. This side effect is now recognized to be due to the depletion of nitric oxide (endothelial-derived relaxing factor) by the extravasuated hemoglobins. We have recently found that the administration of a recombinant human serum albumin (rHSA)-based oxygen carrier involving synthetic tetraphenyporphinatoiron(II) derivative (FeP) (rHSA-FeP) does not induce such hypertensive action, because of its low permeability through the vascular endothelium. The heart rate responses after the rHSA-FeP injection were also negligibly small. Visualization of the intestinal microcirculatory changes clearly revealed the widths of the venule and arteriole to be fairly constant. The entirely synthetic rHSA-FeP becomes a promising material as a new type of red blood cell substitute. Copyright 2002 Wiley Periodicals Inc. J Biomed Mater Res 64A: 257-261, 2003

  17. A monoclonal IgM protein with antibody-like activity for human albumin.

    PubMed

    Hauptman, S; Tomasi, T B

    1974-03-01

    The serum of a patient (L'ec) with an IgM lambda monoclonal protein was noted to bind albumin on immunoelectrophoresis. Analytical ultracentrifugation of the L'ec serum demonstrated 23S and 12S peaks, but no 4S (albumin) boundary. Immunologically identical 20S and 9S IgM proteins were isolated from the serum and the addition in vitro of either the patient's albumin or albumin isolated from normal serum was shown to reconstitute the 23S and 12S boundaries. The binding of high molecular weight IgM to albumin was demonstated by Sephadex G200 chromatography with (125)I-labeled albumin and isolated IgM. Immunoelectrophoresis of the L'ec IgM developed with aggregated albumin (reverse immunoelectrophoresis) also demonstrated the binding of albumin to IgM. That all of the patient's IgM complexed with albumin was shown by affinity chromatography employing an aggregated albumin-immunoadsorbent column. Binding was shown to be of the noncovalent type by polyacrylamide gel electrophoresis in 8 M urea. With hot trypsin proteolysis, Fabmu and Fcmu5 fragments were isolated, and monomer albumin was shown to complex only with the Fabmu fragment by both analytical ultracentrifugation and molecular sieve chromatogaphy employing (125)I-labeled Fab fragments. 1 mol of Fabmu fragment bound 1 mol of monomer albumin. Polymers of human albumin, produced by heat aggregation, precipitated with the isolated L'ec protein on gel diffusion analysis and, when coated on sheep red blood cells, gave a hemagglutination titer greater than 1 million with the whole L'ec serum. 50 additional monoclonal IgM, 33 IgA, and 80 IgG sera failed to show precipitation or hemagglutination with aggregated albumin. Native monomer albumin inhibited precipitation only at high concentrations (> 50 mg/ml); dimer albumin or fragments of albumin produced by trypsin digestion inhibited at low concentrations (0.4 mg/ml). No reactivity occurred with the albumin of five other mammalian species, including bovine. The L

  18. Hydrodynamic characterization of recombinant human fibrinogen species

    PubMed Central

    Raynal, Bertrand; Cardinali, Barbara; Grimbergen, Jos; Profumo, Aldo; Lord, Susan T.; England, Patrick; Rocco, Mattia

    2013-01-01

    Introduction Fibrinogen is a key component of the blood coagulation system and plays important, diverse roles in several relevant pathologies such as thrombosis, hemorrhage, and cancer. It is a large glycoprotein whose three-dimensional molecular structure is not fully known. Furthermore, circulating fibrinogen is highly heterogeneous, mainly due to proteolytic degradation and alternative mRNA processing. Recombinant production of human fibrinogen allows investigating the impact on the three-dimensional structure of specific changes in the primary structure. Methods We performed analytical ultracentrifugation analyses of a full-length recombinant human fibrinogen, its counterpart purified from human plasma, and a recombinant human fibrinogen with both Aα chains truncated at amino acid 251, thus missing their last 359 amino acid residues. Results We have accurately determined the translational diffusion and sedimentation coefficients (Dt(20,w)0, s(20,w)0) of all three species. This was confirmed by derived molecular weights within 1% for the full length species, and 5% for the truncated species, as assessed by comparison with SDS-PAGE/Western blot analyses and primary structure data. No significant differences in the values of Dt(20,w)0 and s(20,w)0 were found between the recombinant and purified full length human fibrinogens, while slightly lower and higher values, respectively, resulted for the recombinant truncated human fibrinogen compared to a previously characterized purified human fibrinogen fragment X obtained by plasmin digestion. Conclusions Full-length recombinant fibrinogen is less polydisperse but hydrodynamically indistinguishable from its counterpart purified from human plasma. Recombinant Aα251-truncated human fibrinogen instead behaves differently from fragment X, suggesting a role for the Bβ residues 1–52 in inter-molecular interactions. Overall, these new hydrodynamic data will constitute a reliable benchmark against which models of

  19. Investigation of the interaction between flavonoids and human serum albumin

    NASA Astrophysics Data System (ADS)

    Bi, Shuyun; Ding, Lan; Tian, Yuan; Song, Daqian; Zhou, Xin; Liu, Xia; Zhang, Hanqi

    2004-10-01

    The interactions of flavonoids, including quercetin, rutin, hyperin and baicalin, with human serum albumin (HSA) were studied. Fluorescence emission spectra of HSA in the presence of the investigated compounds, recorded at the excitation and emission wavelength of 280 and 290-500 nm, respectively, clearly showed that these compounds quenched the fluorescence of HSA. The binding constants and the number of binding sites of the flavonoids with HSA were obtained by three different calculation methods and the results obtained by these methods were compared. The effects of various metal ions on the binding constants of these flavonoids with HSA were also studied. Based on the mechanism of the Förster energy transference, the energy transfer efficiency between the acceptors and HSA were found. The relationship between the structure characteristics of these compounds and binding properties of the flavonoids and HSA was explored.

  20. 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.

  1. Hydrophobic conjugated microporous polymers for sorption of human serum albumin

    NASA Astrophysics Data System (ADS)

    Zheng, Chunli; Du, Miaomiao; Feng, Shanshan; Sun, Hanxue; Li, An; He, Chi; Zhang, TianCheng; Wang, Qiaorui; Wei, Wei

    2016-02-01

    This paper investigated the sorption of human serum albumin (HSA) from water by three kinds of conjugated microporous polymers (CMPs) with surface hydrophobicity and intrinsic porosity. It was found that the three CMPs captured HSA with fast sorption kinetics and good working capacity. Equilibrium was obtained at 80 min for all the tests, and the maximum sorption quantity (qm) ranged from 0.07 to 0.14 mg/mg. With the increase in the particle external surface area of the CMPs, a greater extent of HSA sorption was achieved. Moreover, promoting the dispersion of CMPs in HSA aqueous solution was also beneficial to the extraction. Attenuated Total Reflection Fourier Transform Infrared spectroscopy verified the interactions between the CMPs and the Nsbnd H, Cdbnd O, and Csbnd N groups of HSA. This paper might provide fundamental guidance for the practical application of CMPs to proteins separation and recovery.

  2. Targeted Quantification of the Glycated Peptides of Human Serum Albumin.

    PubMed

    Vannuruswamy, Garikapati; Korwar, Arvind M; Jagadeeshaprasad, Mashanipalya G; Kulkarni, Mahesh J

    2017-01-01

    Glycated human serum albumin (HSA) serves as an important marker for monitoring the glycemic status. Developing methods for unambiguous identification and quantification of glycated peptides of HSA using high-throughput technologies such as mass spectrometry has a great clinical significance. The following protocol describes the construction of reference spectral libraries for Amadori-modified lysine (AML), N(ε)-(carboxymethyl) lysine (CML)-, and N(ε)-(carboxyethyl)lysine (CEL)-modified peptides of synthetically modified HSA using high-resolution mass spectrometers. The protocol also describes work flows, for unambiguous identification and quantification of glycated modified peptides of HSA in clinical plasma using standard spectral libraries by various mass spectrometry approaches such as parallel reaction monitoring (PRM), sequential window acquisition of all theoretical fragment ion spectra (SWATH), and MS(E).

  3. Human serum albumin crystals and method of preparation

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C. (Inventor)

    1989-01-01

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

  4. A Micrograting Sensor for DNA Hybridization and Antibody Human Serum Albumin-Antigen Human Serum Albumin Interaction Experiments

    NASA Astrophysics Data System (ADS)

    Chathirat, Naphat; Atthi, Nithi; Hruanun, Charndet; Poyai, Amporn; Leasen, Suthisa; Osotchan, Tanakorn; Hodak, Jose H.

    2011-01-01

    A biosensor structure comprising silicon nitride (Si3N4) micrograting arrays coated with a spin-on-glass (SOG) material was investigated. This grating structure was located on a silicon groove, which was etched by a deep reactive ion etching (DRIE) process. The biosensor was used as a specific detector of DNA molecules and antibody-antigen interactions. In our DNA sensing experiments, the first step was the activation of the grating surface with amine functional groups, followed by attachment of a 23-base oligonucleotide probe layer for hybridization with a complementary target DNA. The sensing device was tested for detecting specific antigen/antibody interactions for human serum albumin (HSA) and antigen bovine serum albumin (BSA). The readout system consisted of a white light lamp that illuminated a small spot on the grating surface at normal incidence through a fiber optic probe with a spectrometer used to collect the reflected light through a second fiber. We show that these sensing devices have the capability to detect DNA as well as antigen-antibody binding for HSA. The detection sensitivity for HSA was better than that for DNA mainly owing to the larger size and concomitant refractive index changes upon binding to the sensor. We show that it is possible to quantify the amount of biomolecules bound to the grating surface by measuring the wavelength shift of the reflectance spectra upon exposure to the samples.

  5. [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

  6. Pyrogen reactions to human serum albumin during plasma exchange.

    PubMed

    Pool, M; McLeod, B C

    1995-01-01

    Reactions to human serum albumin (HSA) in therapeutic plasma exchange (TPE) are rare. Nevertheless, older literature describes possible adverse effects, including specific immune responses to albumin or other proteins, and reactions due to contaminating organisms or pyrogen. During an eight day period three patients in our unit had unusual reactions after infusion of 1.5-2 L of HSA. Patient 1 had trembling that persisted for 20 min. Patient 2 had shaking for 40 min despite calcium gluconate infusion, and fever to 100.8 degrees F. Patient 3 had severe rigors that subsided after 90 min when meperidine was finally given, and fever to 103.5 degrees F. Record reviews revealed that all three patients had received HSA from the same lot, and that only one other TPE patient had received HSA from that lot. Neither our pharmacy nor the manufacturer was aware of other reactions associated with that lot. Material from a bottle only partially infused to patient 3 was negative in culture and was negative for pyrogen when retested by the manufacturer. Nevertheless, because patients 1 and 2 had each had multiple previous uneventful TPEs and because all three patients tolerated subsequent TPEs without incident when another brand of HSA was used, we conclude that these patients had pyrogen reactions to the implicated HSA lot. This experience illustrates the value of cluster recognition in arousing suspicion of unusual reactions to HSA and the value of recorded lot numbers in pursuing such suspicions. Apheresis personnel should be aware of the potential for pyrogen reactions with HSA and should record lot numbers of all fluids infused during TPE.

  7. Investigation of Human Albumin-Induced Circular Dichroism in Dansylglycine

    PubMed Central

    Graciani, Fernanda S.; Ximenes, Valdecir F.

    2013-01-01

    Induced circular dichroism (ICD), or induced chirality, is a phenomenon caused by the fixation of an achiral substance inside a chiral microenvironment, such as the hydrophobic cavities in proteins. Dansylglycine belongs to a class of dansylated amino acids, which are largely used as fluorescent probes for the characterization of the binding sites in albumin. Here, we investigated the ICD in dansylglycine provoked by its binding to human serum albumin (HSA). We found that the complexation of HSA with dansylglycine resulted in the appearance of an ICD band centred at 346 nm. Using this ICD signal and site-specific ligands of HSA, we confirmed that dansylglycine is a site II ligand. The intensity of the ICD signal was dependent on the temperature and revealed that the complexation between the protein and the ligand was reversible. The induced chirality of dansylglycine was susceptive to the alteration caused by the oxidation of the protein. A comparison was made between hypochlorous acid (HOCl) and hypobromous acid (HOBr), and revealed that site II in the protein is more susceptible to alteration provoked by the latter oxidant. These findings suggest the relevance of the aromatic amino acids in the site II, since HOBr is a more efficient oxidant of these residues in proteins than HOCl. The three-dimensional structure of HSA is pH-dependent, and different conformations have been characterised. We found that HSA in its basic form at pH 9.0, which causes the protein to be less rigid, lost the capacity to bind dansylglycine. At pH 3.5, HSA retained almost all of its capacity for binding to dansylglycine. Since the structure of HSA at pH 3.5 is expanded, separating the domain IIIA from the rest of the molecule, we concluded that this separation did not alter its binding capacity to dansylglycine. PMID:24146932

  8. Surface-bound bovine serum albumin carrier protein as present in recombinant cytokine preparations amplifies T helper 17 cell polarization

    PubMed Central

    Dong, Lei; Helmke, Alexandra; Waisman, Ari; Haller, Hermann; Pich, Andreas; von Vietinghoff, Sibylle

    2016-01-01

    Understanding of T helper 17 lineage (TH17) polarization has been significantly promoted by cell culture experiments that reduce the complexity of the in vivo environment. We here investigated TH17 amplification by coating of cytokine preparations. Cytokine preparations coated to the surface compared to the same amount given in solution significantly enhanced TH17 polarization assessed by flow cytometry and interleukin (IL)-17A, IL-17F and RORγt mRNA expression. T cell proliferation and TH1 polarization were similarly enhanced while TREG polarization was impeded. TH17 amplification was replicated by coating the plate with low amounts of FCS or albumin as used as carrier protein for cytokines (0.5 μl 0.1%). It was unaltered by filtration, protein digestion and arylhydrocarbon receptor blockade, not replicated by LPS and independent of integrin stimulation. TH17 amplification required anti-CD3 stimulation and was T cell intrinsic. Supernatants of CD4+ cells polarized on coated cytokine preparations with carrier albumin conferred amplification to fresh splenocytes. Coating markedly elevated CD4+ IL-22 mRNA expression and IL-22 blockade significantly reduced TH17 amplification. Our data show TH17 amplification by coated albumin in the low amounts present in recombinant cytokine preparations. This unexpected adjuvant like effect underscores the need for controls also for temporal and spatial factors in cell culture. PMID:27808281

  9. 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.

  10. Non-enzymatic glucosylation induced neo-epitopes on human serum albumin: A concentration based study

    PubMed Central

    Neelofar, Km; Arif, Zarina; Ahmad, Jamal; Alam, Khursheed

    2017-01-01

    Hyperglycaemia induced non enzymatic glycation is accelerated in diabetic patients and aggressively involved in diabetes progression. Human serum albumin (HSA) is the most abundant protein in blood circulation. In hyperglycaemia, it undergoes fast glycation and results in the impairment of structure. Our previous study has demonstrated structural alterations in Amadori-albumin modified with different glucose concentrations from physiological to pathophysiological range. Here, we focused on immunological characterization of Amadori-albumin. Immunogenicity of Amadori-albumin was analysed by direct binding and competitive ELISA. Amadori-albumin was found to be highly immunogenic (expect albumin modified with 5mM) and induced high titre antibodies depending upon the extent of modification. Very high titre antibodies were obtained with albumin modified with 75mM glucose as compared to native albumin. Anti-Amadori-albumin-IgG from rabbit sera exhibited increased recognition of Amadori-albumin than native albumin in competitive immunoassay. Alteration induced in albumin after glucosylation has made it highly immunogenic. Induced antibodies were quite specific for respective immunogens but showed cross-reaction with other Amadori/native proteins. It suggests that glucosylation has generated highly immunogenic epitopes on albumin. Formation of high molecular weight immune complex with retarded mobility further supports specificity of anti-Amadori-albumin-IgG towards Amadori-albumin. It may be concluded that due to early glycation, an array of modification occurred in HSA structure. Such gross structural changes might favour polymerization of most of the native epitopes into potent immunogenic neo-epitopes, but some original epitopes were still active and has contributed in the immunogenicity. It could be concluded that induction of anti-Amadori-albumin antibodies may be due to protection of glucose modified albumin from protiolytic breakdown. We assumed that this type of

  11. Gold nanoparticle shape effects on human serum albumin corona interface: a molecular dynamic study

    NASA Astrophysics Data System (ADS)

    Ramezani, Fatemeh; Amanlou, Massoud; Rafii-Tabar, Hashem

    2014-07-01

    In recent years, there has been considerable progress in the design and study of gold nanoparticles that geared toward biomedical applications. In most imaging and therapeutic applications, gold nanoparticles enter the bloodstream directly by intravenous administration forming molecular complexes with encountered proteins termed as the protein corona. Since albumin is the most abundant protein in human blood plasma, in this study, gold nanoparticle interactions and its shape effects on human serum albumin were studied by molecular dynamic simulation. These results revealed that in the interaction of albumin with any shapes of gold nanoparticle, human serum albumin unfolds and helix amount decreases. Cubic gold nanoparticles showed stronger unfolding effects on the albumin than the spherical gold nanoparticles.

  12. Antihemophilic factor (recombinant) plasma/albumin-free method for the management and prevention of bleeding episodes in patients with hemophilia A

    PubMed Central

    Pipe, Steven

    2009-01-01

    Hemophilia is a rare genetic bleeding disorder that, if not adequately controlled, is associated with life-threatening bleeding events and serious and costly complications, primarily from joint damage. The advent of effective clotting factor replacement therapy for patients with hemophilia is considered one of the foremost medical advances of the 20th century. The last 3 decades of experience in hemophilia care have witnessed the effectiveness of the care of patients with hemophilia within specialized comprehensive care centers, advances in factor replacement therapies, the benefits of prophylaxis over on-demand replacement therapy, and the role of aggressive management of joint disease to prevent dysfunction. Ongoing challenges, including the management of inhibitors to factor therapies and the consequences of thousands of patients with hemophilia becoming infected with human immunodeficiency virus and hepatitis C virus in the 1980s from contaminated plasma-derived factor concentrates, have highlighted the need for vigilance with respect to clotting factor product safety, access to care, and a full complement of choice of factor replacement therapies. Advate® (antihemophilic factor [recombinant] plasma/albumin-free method [rAHF-PFM]) is the first recombinant factor VIII therapy manufactured without human or animal protein additives to eliminate the risk of pathogen transmission that could be carried by these additives. Preclinical studies established bioequivalence with recombinant antihemophilic factor (Recombinate®), a product with 16 years of clinical experience. Currently licensed in 44 countries worldwide, rAHF-PFM has over 7 years of clinical research within 5 global studies supporting its safety and efficacy in the treatment of patients with hemophilia A. PMID:19707401

  13. Effect of human serum albumin on drug metabolism: structural evidence of esterase activity of human serum albumin.

    PubMed

    Yang, Feng; Bian, Chuanbing; Zhu, Lili; Zhao, Gengxiang; Huang, Zixiang; Huang, Mingdong

    2007-02-01

    Human serum albumin (HSA) is the most abundant plasma protein in the human body with a plasma concentration of 0.6mM. HSA plays an important role in drug transport and metabolism. Enzymatic activity of HSA on different substrates or drugs has been studied and documented. The structural mechanism of this activity, however, is unknown. In this study, we have determined the crystal structures of HSA-myristate in a complex of aspirin and of salicylic acid, respectively. The crystal structure of HSA-myristate-aspirin illustrates that aspirin transfers acetyl group to Lys199 and is hydrolyzed into salicylic acid by HSA. The hydrolysis product, salicylic acid, remains bound to HSA at a similar location, but it shows a very different orientation when compared with the salicylic acid in the HSA-myristate-salicylic acid ternary complex. These results not only provide the structural evidence of esterase activity of HSA, and demonstrate the conformational plasticity of HSA on drug binding, but also may provide structural information for the modulation of HSA-drug interaction by computational approach based on HSA-drug structure.

  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. 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.

  16. Interaction of perfluorooctanoic acid with human serum albumin

    PubMed Central

    Wu, Ling-Ling; Gao, Hong-Wen; Gao, Nai-Yun; Chen, Fang-Fang; Chen, Ling

    2009-01-01

    Background Recently, perfluorooctanoic acid (PFOA) has become a significant issue in many aspects of environmental ecology, toxicology, pathology and life sciences because it may have serious effects on the endocrine, immune and nervous systems and can lead to embryonic deformities and other diseases. Human serum albumin (HSA) is the major protein component of blood plasma and is called a multifunctional plasma carrier protein because of its ability to bind an unusually broad spectrum of ligands. Results The interaction of PFOA with HSA was investigated in the normal physiological condition by equilibrium dialysis, fluorospectrometry, isothermal titration calorimetry (ITC) and circular dichroism (CD). The non-covalent interaction is resulted from hydrogen bond, van der Waals force and hydrophobic stack. PFOA binding to HSA accorded with two-step binding model with the saturation binding numbers of PFOA, only 1 in the hydrophobic intracavity of HSA and 12 on the exposed outer surface. The interaction of PFOA with HSA is spontaneous and results in change of HSA conformation. The possible binding sites were speculated. Conclusion The present work suggested a characterization method for the intermolecular weak interaction. It is potentially useful for elucidating the toxigenicity of perfluorochemicals when combined with biomolecular function effect, transmembrane transport, toxicological testing and the other experiments. PMID:19442292

  17. 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.

  18. 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.

  19. 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.

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

    PubMed

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

    2008-04-01

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

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

    SciTech Connect

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

    2009-07-31

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

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

    PubMed

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

    2016-01-01

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

  3. Cooperative binding of drugs on human serum albumin

    NASA Astrophysics Data System (ADS)

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

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

  4. 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.

  5. 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.

  6. Interaction of Human Serum Albumin with Metal Protoporphyrins

    NASA Astrophysics Data System (ADS)

    Hu, Jie; Brancaleon, Lorenzo

    2015-03-01

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

  7. Superhydrophobic Effect on the Adsorption of Human Serum Albumin

    PubMed Central

    Leibner, Evan S.; Barnthip, Naris; Chen, Weinan; Baumrucker, Craig R.; Badding, John V.; Pishko, Michael; Vogler, Erwin A.

    2009-01-01

    Analytical protocol greatly influences measurement of human-serum albumin (HSA) adsorption to commercial expanded polytetrafluororethylene (ePTFE) exhibiting superhydrophobic wetting properties. Degassing of buffer solutions and evacuation of ePTFE adsorbent to remove trapped air immediately prior to contact with protein solutions are shown to be essential. Results obtained with ePTFE as a prototypical superhydrophobic test material suggest that vacuum degassing should be applied in the measurement of protein adsorption to any surface exhibiting superhydrophobicity. Solution depletion quantified using radiometry (I-125 labeled HSA) or electrophoresis yield different measures of adsorption, with nearly four-fold higher surface concentrations of unlabeled HSA measured by the electrophoresis method. This outcome is attributed to the influence of the radiolabel on HSA hydrophilicity which decreases radiolabeled-HSA affinity for a hydrophobic adsorbent in comparison to unlabeled HSA. These results indicate that radiometry underestimates the actual amount of protein adsorbed to a particular material. Removal of radiolabeled HSA adsorbed to ePTFE by 3X serial buffer rinses also shows that the remaining “bound fraction” was about 35% lower than the amount measured by radiometric depletion. This observation implies that measurement of protein bound after surface rinsing significantly underestimates the actual amount of protein concentrated by adsorption into the surface region of a protein-contacting material. PMID:19135420

  8. Multiple geminate ligand recombinations in human hemoglobin.

    PubMed Central

    Esquerra, R M; Goldbeck, R A; Reaney, S H; Batchelder, A M; Wen, Y; Lewis, J W; Kliger, D S

    2000-01-01

    The geminate ligand recombination reactions of photolyzed carbonmonoxyhemoglobin were studied in a nanosecond double-excitation-pulse time-resolved absorption experiment. The second laser pulse, delayed by intervals as long as 400 ns after the first, provided a measure of the geminate kinetics by rephotolyzing ligands that have recombined during the delay time. The peak-to-trough magnitude of the Soret band photolysis difference spectrum measured as a function of the delay between excitation pulses showed that the room temperature kinetics of geminate recombination in adult human hemoglobin are best described by two exponential processes, with lifetimes of 36 and 162 ns. The relative amounts of bimolecular recombination to T- and R-state hemoglobins and the temperature dependence of the submicrosecond kinetics between 283 and 323 K are also consistent with biexponential kinetics for geminate recombination. These results are discussed in terms of two models: geminate recombination kinetics modulated by concurrent protein relaxation and heterogeneous kinetics arising from alpha and beta chain differences. PMID:10827999

  9. 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

  10. Protein Crystal Recombinant Human Insulin

    NASA Technical Reports Server (NTRS)

    1994-01-01

    The comparison of protein crystal, Recombiant Human Insulin; space-grown (left) and earth-grown (right). On STS-60, Spacehab II indicated that space-grown crystals are larger and of greater optical clarity than their earth-grown counterparts. Recombiant Human Insulin facilitates the incorporation of glucose into cells. In diabetics, there is either a decrease in or complete lack of insulin, thereby leading to several harmful complications. Principal Investigator is Larry DeLucas.

  11. Protein Crystal Recombinant Human Insulin

    NASA Technical Reports Server (NTRS)

    1994-01-01

    The comparison of protein crystal, Recombiant Human Insulin; space-grown (left) and earth-grown (right). On STS-60, Spacehab II indicated that space-grown crystals are larger and of greater optical clarity than their earth-grown counterparts. Recombiant Human Insulin facilitates the incorporation of glucose into cells. In diabetics, there is either a decrease in or complete lack of insulin, thereby leading to several harmful complications. Principal Investigator is Larry DeLucas.

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

    PubMed Central

    Levitt, David G; Levitt, Michael D

    2016-01-01

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

  13. The Role of Recombinant Genetics in Humanism.

    ERIC Educational Resources Information Center

    Jacobs, Troy A.

    1983-01-01

    To eliminate the public's fear of recombinant genetics the important link between science and the humanities should be part of the educational system. Universal applied genetics guidelines are needed that encompass philosophical and technical issues. Biological advances can revitalize humankind in the future. (AM)

  14. Human serum albumin complexes with chlorophyll and chlorophyllin.

    PubMed

    Ouameur, A Ahmed; Marty, R; Tajmir-Riahi, H A

    2005-02-15

    Porphyrins and their metal derivatives are strong protein binders. Some of these compounds have been used for radiation sensitization therapy of cancer and are targeted to interact with cellular DNA and protein. The presence of several high-affinity binding sites on human serum albumin (HSA) makes it possible target for many organic and inorganic molecules. Chlorophyll a and chlorophyllin (a food-grade derivative of chlorophyll), the ubiquitous green plant pigment widely consumed by humans, are potent inhibitors of experimental carcinogenesis and interact with protein and DNA in many ways. This study was designed to examine the interaction of HSA with chlorophyll (Chl) and chlorophyllin (Chln) in aqueous solution at physiological conditions. Fourier transform infrared, UV-visible, and CD spectroscopic methods were used to determine the pigment binding mode, the binding constant, and the effects of porphyrin complexation on protein secondary structure. Spectroscopic results showed that chlorophyll and chlorophyllin are located along the polypeptide chains with no specific interaction. Stronger protein association was observed for Chl than for Chln, with overall binding constants of K(Chl) = 2.9 x 10(4)M(-1) and K(Chln) = 7.0 x 10(3)M(-1). The protein conformation was altered (infrared data) with reduction of alpha-helix from 55% (free HSA) to 41-40% and increase of beta-structure from 22% (free HSA) to 29-35% in the pigment-protein complexes. Using the CDSSTR program (CD data) also showed major reduction of alpha-helix from 66% (free HSA) to 58 and 55% upon complexation with Chl and Chln, respectively.

  15. Anti-hemophilic factor (recombinant), plasma/albumin-free method (octocog-alpha; ADVATE) in the management of hemophilia A.

    PubMed

    Shapiro, Amy D

    2007-01-01

    Removal of blood-based additives from recombinant clotting factor concentrates continues to be advocated by the hemophilia community due to the history of infectious disease transmission with previous blood-derived clotting factor concentrates. In 2003, octocog-alpha, antihemophilic factor (recombinant), plasma/albumin-free method (ADVATE) was introduced, providing the first third-generation recombinant factor VIII (rFVIII) concentrate. Completed clinical trials have demonstrated ADVATE to be safe and effective in adult and pediatric subjects utilizing both prophylactic and on-demand treatment regimens, and for perioperative hemostatic coverage. In the five completed studies involving more than 200 previously treated patients (PTPs), a single incidence of low-titer, non-persistent inhibitor was reported. Active post authorization safety surveillance (PASS) is being performed to expand the efficacy and safety profile of ADVATE in routine clinical practice. Laboratory studies have documented the storage and post-reconstitution stability of ADVATE, conferring the desired versatility for home treatment. The evolving real-world experience and ongoing studies will provide further insight into ADVATE pharmacokinetics, alternative prophylactic dosing regimens, methods for perioperative hemostatic management, and utility in immune tolerance induction. Experience with ADVATE, and its place in today's treatment paradigm, is the focus of this article.

  16. Influence of myristic acid on furosemide binding to bovine serum albumin. Comparison with furosemide-human serum albumin complex

    NASA Astrophysics Data System (ADS)

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

    2010-06-01

    Fluorescence studies on furosemide (FUR) binding to bovine serum albumin (BSA) showed the existence of three or four binding sites in the tertiary structure of the protein. Two of them are located in subdomain IIA, while the others in subdomains IB and/or IIIA. Furosemide binding in subdomain IB is postulated on the basis of run of Stern-Volmer plot indicating the existence of two populations of tryptophans involved in the interaction with FUR. In turn, the significant participation of tyrosil residues in complex formation leads to the consideration of the subdomain IIIA as furosemide low-affinity binding site. The effect of increasing concentration of fatty acid on FUR binding in all studied binding sites was also investigated and compared with the previous results obtained for human serum albumin (HSA). For BSA the lesser impact of fatty acid on affinity between drug and albumin was observed. This is probably a result of more significant role of tyrosines in the complex formation and different polarity of microenvironment of the fluorophores when compared HSA and BSA. The most distinct differences between FUR-BSA and FUR-HSA binding parameters are observed when third fatty acid molecule is bound with the protein and rotation of domains I and II occurs. However these structural changes mostly affect FUR low affinity binding sites.

  17. Influence of myristic acid on furosemide binding to bovine serum albumin. Comparison with furosemide-human serum albumin complex.

    PubMed

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

    2010-06-01

    Fluorescence studies on furosemide (FUR) binding to bovine serum albumin (BSA) showed the existence of three or four binding sites in the tertiary structure of the protein. Two of them are located in subdomain IIA, while the others in subdomains IB and/or IIIA. Furosemide binding in subdomain IB is postulated on the basis of run of Stern-Volmer plot indicating the existence of two populations of tryptophans involved in the interaction with FUR. In turn, the significant participation of tyrosil residues in complex formation leads to the consideration of the subdomain IIIA as furosemide low-affinity binding site. The effect of increasing concentration of fatty acid on FUR binding in all studied binding sites was also investigated and compared with the previous results obtained for human serum albumin (HSA). For BSA the lesser impact of fatty acid on affinity between drug and albumin was observed. This is probably a result of more significant role of tyrosines in the complex formation and different polarity of microenvironment of the fluorophores when compared HSA and BSA. The most distinct differences between FUR-BSA and FUR-HSA binding parameters are observed when third fatty acid molecule is bound with the protein and rotation of domains I and II occurs. However these structural changes mostly affect FUR low affinity binding sites. Copyright 2010 Elsevier B.V. All rights reserved.

  18. 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.

  19. Low molecular weight silicones particularly facilitate human serum albumin denaturation.

    PubMed

    Nayef, Lamees M; Khan, Madiha F; Brook, Michael A

    2015-04-01

    There is a market trend towards the administration of therapeutic proteins using sterilized, pre-filled glass syringes lubricated with silicone oil. It has been widely reported that initially clear solutions of proteins can become turbid during transport and storage, with unclear outcomes with respect to bioefficacy. While the basic processes of interactions of proteins with hydrophobic entities, leading to denaturation and aggregation, are increasingly well understood, the apparently random occurrence of such processes in syringes is not. To better understand the parameters that may be responsible for this change, we report the systematic examination of a series of factors that can affect the behavior of the protein human serum albumin (HSA) when in contact with silicone oil in water. Fluorescence spectroscopy showed that greater mixing times and greater concentrations of silicones (polydimethylsiloxane (PDMS)), especially lower molecular weight hydrophobic silicones like octamethyltetracyclosiloxane (D4), were associated with increased protein denaturation. The turbidity of HSA solutions, due to the formation both of silicone oil-in-water (O/W) emulsions and protein aggregates, was also facilitated by the presence of D4. A series of mixtures of silicone oils, all of which exhibited a viscosity of 1000 cSt but which were comprised of different silicone constituents, clearly showed a correlation between the presence of lower molecular silicones and enhanced solution turbidity. While the addition of a non-ionic silicone-polyether surfactant led to greater turbidity by increasing the number of stabilized oil droplets, it was not accompanied by protein denaturation. These results are consistent with HSA denaturation and subsequent aggregation as a consequence of contact particularly with low molecular weight, hydrophobic silicones that are more mobile, leading to more efficient protein/silicone contact.

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

    SciTech Connect

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

    1988-01-01

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

  1. Behavior of human serum albumin on strong cation exchange resins: I. experimental analysis.

    PubMed

    Voitl, Agnes; Butté, Alessandro; Morbidelli, Massimo

    2010-08-20

    Experiments with human serum albumin on the strong cation exchange resin Fractogel EMD SE Hicap (M) were carried out. Even though human serum albumin was used at high purity, two peaks in gradient elution experiments occurred. The obtained data can be explained by considering that human serum albumin binds to Fractogel EMD SE Hicap (M) in two different binding conformations: the protein adsorbs instantaneously in the first conformation and then changes into the second one with a kinetic limitation. The two-peak behavior of human serum albumin was analyzed in detail, especially at various gradient lengths, concentrations and temperatures. Breakthrough curves were performed at four modifier concentrations and three velocities. The characteristic adsorption behavior, found for gradient experiments, was confirmed by the breakthrough curves. The two-peak elution pattern of human serum albumin was also found for other strong cation exchange resins, but not for weak cation exchange resins. It is concluded that the described behavior is peculiar for the interaction of human serum albumin with the strong cation exchange ligand of the resin.

  2. Actions of serum and plasma albumin on intracellular Ca2+ in human endothelial cells.

    PubMed Central

    Fuentes, E; Nadal, A; Jacob, R; McNaughton, P

    1997-01-01

    1. The effects of serum and plasma albumin on [Ca2+]i in human endothelial cells were examined using single-cell Ca2+ imaging. Two types of endothelial cell were used: human umbilical vein endothelial cells (HUVEC) in primary culture, and the endothelial-derived cell line ECV304. 2. Serum albumin caused a large and transient rise in [Ca2+]i, due to Ca2+ release from an IP3-sensitive internal store, followed by a maintained elevation in [Ca2+]i attributable to Ca2+ influx from the external medium. A half-maximal rise in [Ca2+]i was produced by a concentration of serum albumin of about 1 microgram ml-1. 3. The Ca(2+)-releasing action of serum albumin is abolished by methanol extraction and is therefore attributable to an attached polar lipid. A possible candidate is lysophosphatidic acid, known to be released from platelets during blood coagulation, which produced similar effects to those of serum albumin. 4. In HUVEC, plasma albumin caused a sustained decrease in [Ca2+]i from the mean resting level of 114 nM to 58 nM. No effect of plasma albumin was observed in ECV304 cells. 5. The decrease in [Ca2+]i caused by plasma albumin is due to an uptake into intracellular stores. The store loading substantially potentiates the action of Ca(2+)-releasing agonists such as histamine. 6. The results show that normal plasma albumin, which carries few lipids, lowers [Ca2+]i and potentiates the actions of Ca(2+)-releasing agonists by promoting Ca2+ uptake into intracellular stores. When converted to the serum form, by binding lysophosphatidic acid released during blood coagulation, albumin has a potent effect in elevating [Ca2+]i. Blood coagulation may therefore play a role in regulating vascular tone and capillary permeability. PMID:9365906

  3. 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.

  4. Safety and pharmacokinetics of a novel recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in hemophilia B patients

    PubMed Central

    Negrier, Claude; Klamroth, Robert; Tiede, Andreas; Pabinger-Fasching, Ingrid; Voigt, Christine; Jacobs, Iris; Morfini, Massimo

    2012-01-01

    A recombinant fusion protein linking coagulation factor IX (FIX) with human albumin (rIX-FP) has been developed to facilitate hemophilia B treatment by less frequent FIX dosing. This first-in-human dose-escalation trial in 25 previously treated subjects with hemophilia B (FIX ≤ 2 IU/dL) examined the safety and pharmacokinetics of 25, 50, and 75 IU/kg rIX-FP. Patients in the 50-IU/kg cohort underwent a comparative pharmacokinetics assessment with their previous FIX product (plasma-derived or recombinant). No allergic reactions or inhibitors were observed. Four mild, possibly treatment-related adverse events were reported. In the 50-IU/kg cohort (13 subjects), the mean half-life of rIX-FP was 92 hours, more than 5 times longer than the subjects' previous FIX product. After 25 or 50 IU/kg rIX-FP administration, the baseline-corrected mean FIX activity remained elevated at day 7 (7.4 IU/dL and 13.4 IU/dL, respectively) and day 14 (2.5 IU/dL and 5.5 IU/dL, respectively). The incremental recovery of rIX-FP was higher than both recombinant and plasma-derived FIX (1.4 vs 0.95 and 1.1 IU/dL per IU/kg, respectively). These results demonstrated both the safety and improved pharmacokinetics of rIX-FP, thus indicating this new product with extended half-life as possibly able to control and prevent bleeding with less frequent injection. The trial was registered at www.clinicaltrials.gov as no. NCT01233440. PMID:22859609

  5. 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).

  6. Binding of aluminum to human serum transferrin, human serum albumin and rat serum proteins

    SciTech Connect

    El-Sebae, A.K.H.; Zeid, M.M.A.; Abdel-Rahman, F.H.; Saleh, M.A. . Environmental Chemistry and Toxicology Lab.)

    1994-01-01

    Human serum transferrin (HSTF), human serum albumin (HSA) and rat serum were compared for their interaction with AlCl[sub 3], in a Tris-HCl buffer solutions. The AlCl[sub 3] was tested in series of concentrations in the range of 50 [mu]M up to 500 [mu]M. HSTF, HSA and their 1:1 mixture and rat serum were incubated at 37 C with series of AlCl[sub 3] concentrations. The protein profile of the incubated solutions were compared to control using SDS-PAGE and FPLC tests. The results indicated that HSTF was more specifically responsive to AlCl[sub 3] showing a characteristic increase in it UV absorption, peak and area dimensions. Simultaneously, HSA was less affected, but it showed a significant shift with an increase in molecular weight accompanied with a change in its profile. The respective bands of transferrin and albumin in rat serum behaved similarly.

  7. Separation of Albumin, Ceruloplasmin, and Transferrin from Human Plasma.

    ERIC Educational Resources Information Center

    Barnes, Grady; Frieden, Earl

    1982-01-01

    Procedures are provided for separating the principal metalloproteins (albumin, ceruloplasmin, and transferrin) from plasma using column chromatographic techniques. The experiment can be completed in two separate three-hour laboratory periods during which column chromatography is illustrated and the effect of pH on charge and affinity of a protein…

  8. Separation of Albumin, Ceruloplasmin, and Transferrin from Human Plasma.

    ERIC Educational Resources Information Center

    Barnes, Grady; Frieden, Earl

    1982-01-01

    Procedures are provided for separating the principal metalloproteins (albumin, ceruloplasmin, and transferrin) from plasma using column chromatographic techniques. The experiment can be completed in two separate three-hour laboratory periods during which column chromatography is illustrated and the effect of pH on charge and affinity of a protein…

  9. [Effects of union of albumin and egg yolk on human semen cryopreservation].

    PubMed

    Huang, Dong-Hui; Zhao, Hu; Xiong, Cheng-Liang; Sheng, Ji-Yun; Zhou, Jie-Ling

    2006-02-01

    To provide a more effective cryoprotective medium (CPM), effect of union of albumin and egg yolk on human sperm cryopreservation was studied. Egg yolk-glycerol-sodium citrate was regarded as CPM of the control group and egg yolk-glycerol-sodium citrate with different concentrations of albumin (1, 2, 3, 4, 5 g/L) were regarded as CPMs of experiment groups. Before and after cryopreservation, sperm movement parameters were assessed by using computer-aided sperm analyzer (CASA) system, and then egg yolk-glycerol-sodium citrate group added 1 g/L albumin was selected, whose movement parameters were the best among the experimental groups, and egg yolk-glycerol-sodium citrateto group as the control to compare sperm survival rate, membrane integrity, function of mitochondrion and ultrastruction. Sperm in egg yolk-glycerol-sodium citrate added I g/L albumin showed significantly higher motility, viability than those in the control group and other experimental groups (P < 0.05). Sperm in egg yolk-glycerol-sodium citrate group added 1 g/L albumin had significantly higher survival rate, head unpigmenting rate than those in control group (P < 0.05). Sperm in egg yolk-glycerol-sodium citrate group added I g/L albumin manifested significantly higher succinate dehydrogenase (SDH) activity than that in control group (P < 0.05) and had better ultrastructure than that in control group. Union of two kinds of albumin and egg yolk has better effects on human sperm cryopreservation than those of solitary use of egg yolk. The action of albumin is related to its concentration, and albumin combining with egg yolk may have plus and complementary effects on human sperm cryopreservation.

  10. Bayesian inference of shared recombination hotspots between humans and chimpanzees.

    PubMed

    Wang, Ying; Rannala, Bruce

    2014-12-01

    Recombination generates variation and facilitates evolution. Recombination (or lack thereof) also contributes to human genetic disease. Methods for mapping genes influencing complex genetic diseases via association rely on linkage disequilibrium (LD) in human populations, which is influenced by rates of recombination across the genome. Comparative population genomic analyses of recombination using related primate species can identify factors influencing rates of recombination in humans. Such studies can indicate how variable hotspots for recombination may be both among individuals (or populations) and over evolutionary timescales. Previous studies have suggested that locations of recombination hotspots are not conserved between humans and chimpanzees. We made use of the data sets from recent resequencing projects and applied a Bayesian method for identifying hotspots and estimating recombination rates. We also reanalyzed SNP data sets for regions with known hotspots in humans using samples from the human and chimpanzee. The Bayes factors (BF) of shared recombination hotspots between human and chimpanzee across regions were obtained. Based on the analysis of the aligned regions of human chromosome 21, locations where the two species show evidence of shared recombination hotspots (with high BFs) were identified. Interestingly, previous comparative studies of human and chimpanzee that focused on the known human recombination hotspots within the β-globin and HLA regions did not find overlapping of hotspots. Our results show high BFs of shared hotspots at locations within both regions, and the estimated locations of shared hotspots overlap with the locations of human recombination hotspots obtained from sperm-typing studies.

  11. Recombinant human intelectin binds bovine lactoferrin and its peptides.

    PubMed

    Shin, Kouichirou; Wakabayashi, Hiroyuki; Yamauchi, Koji; Yaeshima, Tomoko; Iwatsuki, Keiji

    2008-08-01

    Intelectin (IntL), a lectin that exists on the brush border membrane of the small intestine, plays a role in the innate immune response and also acts as a receptor for lactoferrin (LF), an iron-binding glycoprotein found in milk and other secretions. Similar to human LF (hLF), bovine LF (bLF) has been shown to induce proliferation and differentiation of human enterocytes and to modulate their cytokine productions. To evaluate the interaction between human IntL (hIntL) and bLF, recombinant hIntL (rhIntL) conjugated with a tag sequence was examined for its ligand-binding capacity by using microtiter plates coated with LF or other proteins. Interestingly, rhIntL showed higher binding for bLF than hLF. It also bound pepsin hydrolysate of bLF, but to a lower degree than native bLF. A very low binding of rhIntL was observed for bovine serum albumin or transferrin. These findings suggest that hIntL acts as a receptor for bLF and its digested fragments.

  12. 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

  13. Serum albumins - unusual allergens

    PubMed Central

    Chruszcz, Maksymilian; Mikolajczak, Katarzyna; Mank, Nicholas; Majorek, Karolina A.; Porebski, Przemyslaw J.; Minor, Wladek

    2015-01-01

    Background Albumins are multifunctional proteins present in the blood serum of animals. They can bind and transport a wide variety of ligands which they accommodate due to their conformational flexibility. Serum albumins are highly conserved both in amino acid sequence and three-dimensional structure. Several mammalian and avian serum albumins (SAs) are also allergens. Sensitization to one of the SAs coupled with the high degree of conservation between SAs may result in cross-reactive antibodies in allergic individuals. Sensitivity to SA generally begins with exposure to an aeroallergen, which can then lead to cross-sensitization to serum albumins present in food. Scope of Review This review focuses on the allergenicity of SAs presented in a structural context. Major Conclusions SA allergenicity is unusual taking into account the high sequence identity and similarity between SA from different species and human serum albumin. Cross-reactivity of human antibodies towards different SAs is one of the most important characteristics of these allergens. General Significance Establishing a relationship between sequence and structure of different SAs and their interactions with antibodies is crucial for understanding the mechanisms of cross-sensitization of atopic individuals. Structural information can also lead to better design and production of recombinant SAs to replace natural proteins in allergy testing and desensitization. Therefore, structural analyses are important for diagnostic and treatment purposes. PMID:23811341

  14. Vapor conjugation of toluene diisocyanate to specific lysines of human albumin

    PubMed Central

    Hettick, Justin M.; Siegel, Paul D.; Green, Brett J.; Liu, Jian; Wisnewski, Adam V.

    2012-01-01

    Exposure to toluene diisocyanate (TDI), an industrially important crosslinking agent used in the production of polyurethane products, can cause asthma in sensitive workers. Albumin has been identified as a major reaction target for TDI in vivo, and TDI–albumin reaction products have been proposed to serve as exposure biomarkers and to act as asthmagens, yet they remain incompletely characterized. In the current study, we used a multiplexed tandem mass spectrometry (MS/MS) approach to identify the sites of albumin conjugation by TDI vapors, modeling the air/liquid interface of the lung. Vapor phase TDI was found to react with human albumin in a dose-dependent manner, with up to 18 potential sites of conjugation, the most susceptible being Lys351 and the dilysine site Lys413–414. Sites of vapor TDI conjugation to albumin were quantitatively limited compared with those recently described for liquid phase TDI, especially in domains IIA and IIIB of albumin. We hypothesize that the orientation of albumin at the air/liquid interface plays an important role in vapor TDI conjugation and, thus, could influence biological responses to exposure and the development of in vitro assays for exposure and immune sensitivity. PMID:22206939

  15. Albumin inhibits human polymorphonuclear leucocyte luminol-dependent chemiluminescence: evidence for oxygen radical scavenging.

    PubMed Central

    Holt, M. E.; Ryall, M. E.; Campbell, A. K.

    1984-01-01

    Luminol-dependent chemiluminescence of normal human polymorphonuclear leucocytes (PMN) which were resting, or stimulated by unopsonized latex beads, opsonized zymosan or the chemotactic peptide N-formyl-met-leu-phe was decreased more than 80% in the presence of physiological concentrations of albumin (4%, w/v). This inhibition did not result from impairment of light transmission, cellular toxicity, luminol excited-state quenching or a dialysable contaminant in the albumin preparation, but was reduced by 30% when the fall induced by albumin in extracellular free Ca2+ concentration was corrected. The inhibition was most apparent in the larger second phase of the PMN chemiluminescent response to chemotactic peptide or opsonized zymosan stimulation. The smaller first phase of these responses was in fact enhanced by low concentrations of albumin (0.05-0.5%, w/v) and only inhibited up to 50% by 4% (w/v) albumin. Albumin in the range 0.1-4% (w/v) exerted a similar effect on chemiluminescence resulting from superoxide anion (O-2) and hydrogen peroxide (H2O2) production by xanthine oxidase catalysed oxidation of xanthine in the presence of luminol. We suggest that the effect of albumin on PMN luminol-dependent chemiluminescence is mediated by modification of the oxygen radical generating pathway, or oxygen radical scavenging. This previously undocumented property of the major extracellular protein requires further examination if oxygen radicals are to be established as important mediators of inflammation. PMID:6712882

  16. Caveolae may enable albumin to enter human renal glomerular endothelial cells.

    PubMed

    Moriyama, Takahito; Takei, Takashi; Itabashi, Mitsuyo; Uchida, Keiko; Tsuchiya, Ken; Nitta, Kosaku

    2015-06-01

    Caveolae on human renal glomerular endothelial cells (HRGECs) are increased in glomerular disease and correlate with the degree of albuminuria. To assess the mechanism by which caveolae contribute to albuminuria, we investigated whether albumin enters into HRGECs through caveolae. HRGECs were incubated with Alexa Fluor 488 labeled BSA or transferrin, followed by immunofluorescence localization with antibody to caveolin-1 (Cav-1), the main structural protein of caveolae, or clathrin, the major structural protein of clathrin coated pits, to assess whether BSA colocalized with Cav-1. HRGECs were also incubated with albumin and caveolae disrupting agents, including methyl beta cyclodextrin (MBCD) and nystatin, to determine whether disrupting caveolae interfered with albumin endocytosis into HRGECs. HRGECs were also incubated with albumin after transfection with Cav-1 small interfering RNAs (siRNAs). Labeled BSA colocalized with Cav-1, but not with clathrin. In contrast, labeled transferrin colocalized with clathrin, but not with Cav-1. Incubation of HRGECs with MBCD or nystatin, or transfection with Cav-1 siRNA, significantly reduced the intracellular amounts of albumin and Cav-1, relative to normal HRGECs, as shown by western blotting and immunofluorescence. These findings indicate that albumin enters HRGECs through the caveolae, suggesting that caveolae play an important role in the pathogenesis of albuminuria by providing a pathway through which albumin can enter glomerular endothelial cells.

  17. Revisiting Escherichia coli as microbial factory for enhanced production of human serum albumin.

    PubMed

    Sharma, Ashima; Chaudhuri, Tapan K

    2017-10-05

    Human serum albumin (HSA)-one of the most demanded therapeutic proteins with immense biotechnological applications-is a large multidomain protein containing 17 disulfide bonds. The current source of HSA is human blood plasma which is a limited and unsafe source. Thus, there exists an indispensable need to promote non-animal derived recombinant HSA (rHSA) production. Escherichia coli is one of the most convenient hosts which had contributed to the production of more than 30% of the FDA approved recombinant pharmaceuticals. It grows rapidly and reaches high cell density using inexpensive and simple subst-rates. E. coli derived recombinant products have more economic potential as fermentation processes are cheaper compared to the other expression hosts. The major bottleneck in exploiting E. coli as a host for a disulfide-rich multidomain protein is the formation of aggregates of overexpressed protein. The majority of the expressed HSA forms inclusion bodies (more than 90% of the total expressed rHSA) in the E. coli cytosol. Recovery of functional rHSA from inclusion bodies is not preferred because it is difficult to obtain a large multidomain disulfide bond rich protein like rHSA in its functional native form. Purification is tedious, time-consuming, laborious and expensive. Because of such limitations, the E. coli host system was neglected for rHSA production for the past few decades despite its numerous advantages. In the present work, we have exploited the capabilities of E. coli as a host for the enhanced functional production of rHSA (~ 60% of the total expressed rHSA in the soluble fraction). Parameters like intracellular environment, temperature, induction type, duration of induction, cell lysis conditions etc. which play an important role in enhancing the level of production of the desired protein in its native form in vivo have been optimized. We have studied the effect of assistance of different types of exogenously employed chaperone systems on the

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

    NASA Astrophysics Data System (ADS)

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

    2014-07-01

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

  19. An albumin leader sequence coupled with a cleavage site modification enhances the yield of recombinant C-terminal Mullerian Inhibiting Substance

    PubMed Central

    Pépin, D.; Hoang, M.; Nicolaou, F.; Hendren, K.; Benedict, L.A.; Al-Moujahed, A.; Sosulski, A.; Marmalidou, A.; Vavvas, D.; Donahoe, P.K.

    2014-01-01

    Mullerian Inhibiting Substance (MIS) has been shown to inhibit ovarian cancer cells both in-vitro and in-vivo. Furthermore, recent evidence suggests that MIS may effectively target a putative ovarian cancer progenitor cell population enriched by a panel of CD44+, CD24+, Ep-CAM+, and E-cadherin-cell surface markers. In order to accommodate clinical testing of MIS in ovarian cancer patients, the production of recombinant human MIS must be optimized to increase yield and purity. Here we show that, compared to wild type, the substitution of the MIS leader sequence to that of human serum albumin, combined with a modification of the endogenous cleavage site from RAQR/S to a furin/kex2 RARR/S consensus site results in high expression, increased C-terminus cleavage and a reduction in unwanted cryptic internal cleavage products when produced in CHO cells. Purified MIS containing these alterations retains its capacity to induce regression of the Mullerian duct in fetal rat embryonic urogenital ridge assays. PMID:24729676

  20. Human Albumin Prevents 6-Hydroxydopamine-Induced Loss of Tyrosine Hydroxylase in In Vitro and In Vivo

    PubMed Central

    Zhang, Li-Juan; Xue, Yue-Qiang; Yang, Chun; Yang, Wei-Hua; Chen, Long; Zhang, Qian-Jin; Qu, Ting-Yu; Huang, Shile; Zhao, Li-Ru; Wang, Xiao-Min; Duan, Wei-Ming

    2012-01-01

    Human albumin has recently been demonstrated to protect brain neurons from injury in rat ischemic brain. However, there is no information available about whether human albumin can prevent loss of tyrosine hydroxylase (TH) expression of dopaminergic (DA) neurons induced by 6-hydroxydopamine (6-OHDA) toxicity that is most commonly used to create a rat model of Parkinson's disease (PD). In the present study, two microliters of 1.25% human albumin were stereotaxically injected into the right striatum of rats one day before or 7 days after the 6-OHDA lesion in the same side. D-Amphetamine-induced rotational asymmetry was measured 7 days, 3 and 10 weeks after 6-OHDA lesion. We observed that intrastriatal administration of human albumin significantly reduced the degree of rotational asymmetry. The number of TH-immunoreactive neurons present in the substantia nigra was greater in 6-OHDA lesioned rats following human albumin-treatment than non-human albumin treatment. TH-immunoreactivity in the 6-OHDA-lesioned striatum was also significantly increased in the human albumin-treated rats. To examine the mechanisms underlying the effects of human albumin, we challenged PC12 cells with 6-OHDA as an in vitro model of PD. Incubation with human albumin prevented 6-OHDA-induced reduction of cell viability in PC12 cell cultures, as measured by MTT assay. Furthermore, human albumin reduced 6-OHDA-induced formation of reactive oxygen species (ROS) and apoptosis in cultured PC12 cells, as assessed by flow cytometry. Western blot analysis showed that human albumin inhibited 6-OHDA-induced activation of JNK, c-Jun, ERK, and p38 mitogen-activated protein kinases (MAPK) signaling in PC12 cultures challenged with 6-OHDA. Human albumin may protect against 6-OHDA toxicity by influencing MAPK pathway followed by anti-ROS formation and anti-apoptosis. PMID:22815976

  1. Human Innate Immune Responses to Hexamethylene Diisocyanate (HDI) and HDI-Albumin Conjugates

    PubMed Central

    Wisnewski, Adam V.; Liu, Qing; Liu, Jian; Redlich, Carrie A.

    2011-01-01

    Background Isocyanates, a leading cause of occupational asthma, are known to induce adaptive immune responses; however, innate immune responses, which generally precede and regulate adaptive immunity, remain largely uncharacterized. Objective Identify and characterize cellular, molecular and systemic innate immune responses induced by hexamethylene diisocyanate (HDI). Methods Human peripheral blood mononuclear cells (PBMCs) were stimulated in vitro with HDI-albumin conjugates or control antigen, and changes in phenotype, gene, and protein expression were characterized by flow cytometry, microarray, Western blot and ELISA. Cell uptake of isocyanate was visualized microscopically using HDI-albumin conjugates prepared with fluorescently-labeled albumin. In vivo, human HDI exposure was performed via specific inhalation challenge, and subsequent changes in PBMCs and serum proteins were measured by flow cytometry and ELISA. Genotypes were determined by PCR. Results Human monocytes take-up HDI-albumin conjugates and undergo marked changes in morphology and gene/protein expression in vitro. The most significant (p 0.007 – 0.05) changes in mircoarray gene expression were noted in lysosomal genes, especially peptidases and proton pumps involved in antigen processing. Chemokines that regulate monocyte/macrophage trafficking (MIF, MCP-1), and pattern recognition receptors that bind chitin (chitinases) and oxidized low-density lipoprotein (CD68) were also increased following isocyanate-albumin exposure. In vivo, HDI exposed subjects exhibited an acute increase in the percentage of PBMCs with the same HDI-albumin responsive phenotype characterized in vitro (HLA-DR+/CD11c+ with altered light scatter properties). An exposure-dependent decrease (46±11%; p<0.015) in serum concentrations of chitinase-3-like-1 was also observed, in individuals that lack the major (type 1) human chitinase (due to genetic polymorphism), but not in individuals possessing at least one functional

  2. Human innate immune responses to hexamethylene diisocyanate (HDI) and HDI-albumin conjugates.

    PubMed

    Wisnewski, A V; Liu, Q; Liu, J; Redlich, C A

    2008-06-01

    Isocyanates, a leading cause of occupational asthma, are known to induce adaptive immune responses; however, innate immune responses, which generally precede and regulate adaptive immunity, remain largely uncharacterized. The aim of the study was to identify and characterize the cellular, molecular and systemic innate immune responses induced by hexamethylene diisocyanate (HDI). Human peripheral blood mononuclear cells (PBMCs) were stimulated in vitro with HDI-albumin conjugates or control antigen, and changes in phenotype, gene and protein expression were characterized by flow cytometry, microarray, Western blot and ELISA. Cell uptake of isocyanate was visualized microscopically using HDI-albumin conjugates prepared with fluorescently labelled albumin. In vivo, human HDI exposure was performed via a specific inhalation challenge, and subsequent changes in PBMCs and serum proteins were measured by flow cytometry and ELISA. Genotypes were determined by PCR. Human monocytes take up HDI-albumin conjugates and undergo marked changes in morphology and gene/protein expression in vitro. The most significant (P-values 0.007-0.05) changes in microarray gene expression were noted in lysosomal genes, especially peptidases and proton pumps involved in antigen processing. Chemokines that regulate monocyte/macrophage trafficking (MIF, MCP-1) and pattern-recognition receptors that bind chitin (chitinases) and oxidized low-density lipoprotein (CD68) were also increased following isocyanate-albumin exposure. In vivo, HDI-exposed subjects exhibited a drastic increase in the percentage of PBMCs with the same HDI-albumin responsive phenotype characterized in vitro (HLA-DR(+)/CD11c(+) with altered light scatter properties). An exposure-dependent decrease (46+/-11%; P<0.015) in serum concentrations of chitinase 3-like-1 was also observed in individuals who lack the major (type 1) human chitinase (due to genetic polymorphism), but not in individuals possessing at least one functional

  3. Mycotoxin Adducts on Human Serum Albumin: Biomarkers of Exposure to Stachybotrys chartarum

    PubMed Central

    Yike, Iwona; Distler, Anne M.; Ziady, Assem G.; Dearborn, Dorr G.

    2006-01-01

    Objective Despite the growing body of evidence showing adverse health effects from inhalation exposure to the trichothecene-producing mold Stachybotrys chartarum, controversy remains. Currently, there are no reliable assays suitable for clinical diagnosis of exposure. We hypothesized that satratoxin G (SG)–albumin adducts may serve as biomarkers of exposure to this fungus. Design We studied the formation of adducts of SG with serum albumin in vitro using Western blots and mass spectrometry (MS) and searched for similar adducts formed in vivo using human and animal serum. Results Samples of purified human serum albumin that had been incubated with increasing concentrations of SG showed concentration-dependent albumin bands in Western blots developed with anti-SG antibodies. MS analysis found that as many as 10 toxin molecules can be bound in vitro to one albumin molecule. The sequencing of albumin-adduct tryptic peptides and the analysis of pronase/aminopeptidase digests demonstrated that lysyl, cysteinyl, and histidyl residues are involved in the formation of these adducts. Serum samples from three patients with documented exposure to S. chartarum similarly revealed lysine–, cysteine–, and histidine–SG adducts after exhaustive digestion, affinity column enrichment, and MS analysis. These adducts were also found in the sera from rats exposed to the spores of S. chartarum in contrast to control human subjects and control animals. Conclusions These data document the occurrence of SG–albumin adducts in both in vitro experiments and in vivo human and animal exposures to S. chartarum. Relevance to clinical practice SG–amino acid adducts may serve as reliable dosimeter biomarkers for detection of exposure to S. chartarum. PMID:16882529

  4. Mycotoxin adducts on human serum albumin: biomarkers of exposure to Stachybotrys chartarum.

    PubMed

    Yike, Iwona; Distler, Anne M; Ziady, Assem G; Dearborn, Dorr G

    2006-08-01

    Despite the growing body of evidence showing adverse health effects from inhalation exposure to the trichothecene-producing mold Stachybotrys chartarum, controversy remains. Currently, there are no reliable assays suitable for clinical diagnosis of exposure. We hypothesized that satratoxin G (SG) -albumin adducts may serve as biomarkers of exposure to this fungus. We studied the formation of adducts of SG with serum albumin in vitro using Western blots and mass spectrometry (MS) and searched for similar adducts formed in vivo using human and animal serum. Samples of purified human serum albumin that had been incubated with increasing concentrations of SG showed concentration-dependent albumin bands in Western blots developed with anti-SG antibodies. MS analysis found that as many as 10 toxin molecules can be bound in vitro to one albumin molecule. The sequencing of albumin-adduct tryptic peptides and the analysis of pronase/aminopeptidase digests demonstrated that lysyl, cysteinyl, and histidyl residues are involved in the formation of these adducts. Serum samples from three patients with documented exposure to S. chartarum similarly revealed lysine-, cysteine-, and histidine-SG adducts after exhaustive digestion, affinity column enrichment, and MS analysis. These adducts were also found in the sera from rats exposed to the spores of S. chartarum in contrast to control human subjects and control animals. These data document the occurrence of SG-albumin adducts in both in vitro experiments and in vivo human and animal exposures to S. chartarum. SG-amino acid adducts may serve as reliable dosimeter biomarkers for detection of exposure to S. chartarum.

  5. The secretion of human serum albumin from the yeast Saccharomyces cerevisiae using five different leader sequences.

    PubMed

    Sleep, D; Belfield, G P; Goodey, A R

    1990-01-01

    We demonstrate the secretion of human serum albumin into the culture supernatant from the yeast Saccharomyces cerevisiae. Studies with five KEX2 processed leader sequences, namely the S. cerevisiae alpha factor, the natural human serum albumin, the Kluyveromyces lactis killer, a natural human serum albumin/alpha factor fusion, and a Kluyveromyces lactis killer/alpha factor fusion leader, are described. We show that the leader sequence used to direct secretion influences the quantity and quality of the secreted product. In designing secretion systems for heterologous proteins, one aims to maximise both the yield and fidelity of the product. Our results indicate that the choice of leader sequence and its relationship to the structural protein under study are crucial to the success of this process.

  6. Antigenic changes in human albumin caused by reactivity with the occupational allergen diphenylmethane diisocyanate

    PubMed Central

    Wisnewski, Adam V.; Liu, Jian; Redlich, Carrie A.

    2010-01-01

    Diphenylmethane diisocyanate (MDI), the chemical commonly used as a cross-linking agent in commercial polyurethane production, is a well-recognized cause of asthma. Reaction products between MDI and “self” proteins are hypothesized to act as antigens capable of inducing airway inflammation and asthma; however, such MDI antigens remain incompletely understood. We used a variety of analytical methods to characterize the range of MDI–albumin reaction products that form under physiological conditions. Sites of MDI conjugation on antigenic MDI–albumin products, as defined by serum immunoglobulin G (IgG) from MDI-exposed workers, were determined by high-performance liquid chromatography (HPLC) followed by tandem mass spectrometry (MS/MS). The data identified 14 MDI conjugation sites (12 lysines and 2 asparagines) on human albumin and highlight reaction specificity for the second lysine in dilysine (KK) motifs, and this may be a common characteristic of “immune-sensitizing” chemicals. Several of the MDI conjugation sites are not conserved in albumin from other species, and this may suggest species differences in epitope specificity for self protein (albumin)–isocyanate conjugates. The study also describes new applications of contemporary proteomic methodology for characterizing and standardizing MDI–albumin conjugates destined for use in clinical research. PMID:20123080

  7. 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.

  8. The pharmacokinetics of albumin conjugates of D-penicillamine in humans.

    PubMed

    Joyce, D A; Day, R O; Murphy, B R

    1991-01-01

    D-penicillamine (D-PEN) is incompletely recovered during short-term balance studies, despite rapid elimination of D-PEN and its low molecular weight metabolites. Urinary excretion of metabolites of D-PEN also persists long after cessation of chronic therapy. A study was performed to determine whether the formation and later breakdown of a stable disulfide between D-PEN and plasma albumin could explain these aspects of D-PEN pharmacokinetics. Five human volunteers received D-penicillamine, 250 mg orally, daily for 21 days. Plasma concentration-time profiles for D-PEN and D-PEN-albumin disulfide (D-PEN-albumin) were determined during the first day and pre-dose concentrations were measured on five further occasions. The pharmacokinetics of D-PEN on the first day were similar to those reported previously. No D-PEN was found in any of the pre-dose specimens. The concentration of D-PEN-albumin rose rapidly during the first day, with an estimated 8.6% of the bioavailable D-PEN being transformed to D-PEN-albumin. Pseudo-steady-state concentrations of D-PEN-albumin were achieved in three subjects at between 14 and 21 days. The mean trough concentration of D-PEN-albumin at 21 days (19.5 microM) exceeded the peak concentration of D-PEN (during the first day) by 5.7-fold. The terminal elimination half-life of D-PEN-albumin was 1.65 +/- 0.29 days, which compared with an elimination half-life of 59 +/- 8.4 min for D-PEN.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. 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.

  10. 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.

  11. Monoclonal antibodies against methionyl recombinant human prolactin.

    PubMed

    Paris, N; Robert, R; Mercier, L

    1993-02-01

    Hybridoma cell lines producing monoclonal antibody (Mab) against recombinant human prolactin (rhPrl) were established from fusion between X63-Ag8 myeloma cells and Balb/c mice splenocytes. Four Mabs numbered I to IV were selected by ELISA, purified and characterized. All these Mabs were of the Ig1 kappa isotype and able to recognize oxidized as well as reduced rhPrl. As shown by a competitive inhibition assay, Mab IV did not compete with any of the three others. Moreover, both rhPrl and hPrl extracted from human pituitaries, were recognized equally by this Mab. Properties displayed by Mab IV make it very attractive for the evaluation of prolactin levels by sandwich immunoassays.

  12. Expression of Jug r 1, the 2S albumin allergen from walnut (Juglans regia), as a correctly folded and functional recombinant protein.

    PubMed

    Sordet, Camille; Culerrier, Raphaël; Granier, Claude; Rancé, Fabienne; Didier, Alain; Barre, Annick; Rougé, Pierre

    2009-07-01

    Jug r 1, the 2S albumin allergen from walnut, was isolated from ripe nuts as a native allergen and expressed in Escherichia coli using the Gateway technology as a recombinant allergen. The recombinant Jug r 1 (15 kDa) differs from the native allergen by the absence of cleavage of the polypeptide chain in two covalently associated light (3.5 kDa) and heavy (8 kDa) chains. Recombinant rJug r 1 adopts the canonical alpha-helical fold of plant 2S albumins as checked on CD spectra. Four IgE-binding epitopic stretches were identified along the amino acid sequence of Jug r 1 and localized on the molecular surface of the modeled allergen. Both native and recombinant allergens exhibit similar IgE-binding activity and similarly trigger the degranulation of a FcepsilonRI-expressing rat basophilic leukaemia cell line previously treated by IgE-containing sera. Native Jug r 1 resists to heat denaturation and to the proteolytic attack of trypsin and chymotrypsin but is readily hydrolyzed in the presence of pepsin at acidic pH after 1 h of incubation at 37 degrees C in vitro. Recombinant Jug r 1 could be used for a component-resolved diagnosis of food-allergy.

  13. Patterns of recombination on human chromosome 22

    SciTech Connect

    Schlumpf, K.S.; Kim, D.; Haines, J.L.

    1994-09-01

    Virtually all genetic linkage maps generated to date are gross averages across individuals, ages, and (often) sexes. In addition, although some level of positive interference has been assumed, until recently little evidence to support this in humans has been available. The major stumbling block has been the quality of the data available, since even a few genotypic errors can have drastic effects on both the map length and the number of apparent recombinants. In addition, variation in recombination by factors other than sex have pretty much been ignored. To explore recombination in more detail, we have generated a microsatellite marker map of human chromosome 22. This map includes 32 markers genotyped through 46 sibships of the Venezuelan Reference Pedigree (VRP). Extensive error checking and regenotyping was performed to remove as many genotypic errors as possible, but no genotypes were removed simply because they created unlikely events. The following 1000:1 odds map has been obtained: cen--F8VWFP1--11--S264--3-S311--4--S257--2--TOP1P2--3--S156--1--CRYB2--1--S258--2--S310--6--S193--1--S275--3--S268--1--S280--4--S304--3--S283--2--LiR1--3--IL2RB--3--S299--1--S302--1--S537--2--S270--4--PDGF--8--S274--qter. The female map (91 cM) is twice as long as the male map (46 cM) and the log-likelihood difference in the maps (22.3) is highly significant (P=0.001, df=22) and appears constant across the chromosome. Analysis of recombination with age showed no particular trends for either males or females when chromosomes were grouped into three categories (20, 20-30, 30+) by parental age at birth of child. Positive interference was found in maternally derived chromosomes ({chi}{sup 2}=30.5 (4), p<0.005), but not in paternally derived chromosomes ({chi}{sup 2}=6.24 (3), P=0.10). This contrasts to data from chromosomes 9 and 21 where positive interference was found for both sexes. More detailed analyses are in progress.

  14. Evaluation of human and bovine serum albumin on oxidation characteristics by a photosensitization reaction under complete binding of talaporfin sodium.

    PubMed

    Kurotsu, Mariko; Yajima, Masahiro; Takahashi, Mei; Ogawa, Emiyu; Arai, Tsunenori

    2015-09-01

    In order to investigate the therapeutic interaction of an extra-cellular photosensitization reaction, we evaluated the oxidation characteristics of human and bovine serum albumin by this reaction with talaporfin sodium under complete binding with albumin by spectroscopic analysis in a cell-free solution. The solution was composed of 20μg/ml talaporfin sodium and 2.1mg/ml human or bovine serum albumin. A 662nm laser light was used to irradiate the solution. Visible absorbance spectra of solutions were measured to obtain the oxidized and non-oxidized relative densities of albumin and talaporfin sodium before and after the photosensitization reaction. The defined oxidation path ratio of talaporfin sodium to albumin reflected the oxidation of the solution. Absorbance wavelengths at approximately 240 and 660nm were used to calculate normalized molecular densities of oxidized albumin and talaporfin sodium, respectively. The oxidation path ratio of talaporfin sodium to albumin when binding human serum albumin was approximately 1.8 times larger than that of bovine serum albumin during the photosensitization reaction from 1 to 50J/cm(2). We hypothesized that the oxidation path ratio results might have been caused by talaporfin sodium binding affinity or binding location difference between the two albumins, because the fluorescence lifetimes of talaporfin sodium bound to human and bovine serum albumin were 7.0 and 4.9ns, respectively. Therefore, the photodynamic therapeutic interaction might be stronger with human serum albumin than with bovine serum albumin in the case of extracellular photosensitization reaction. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. 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

  16. Effect of oxidatively modified and non-modified human serum albumin on luminol-dependent chemiluminescence of human peripheral blood leukocytes stimulated with opsonized zymosan.

    PubMed

    Piryazev, A P; Azizova, A P; Aseichev, A V; Sergienko, V I

    2014-07-01

    We studied the effects of native and oxidized human serum albumin on luminol-dependent chemiluminescence of human peripheral blood leukocytes stimulated with opsonized zymosan. Human serum albumin was added simultaneously with opsonized zymosan at the beginning of the chemiluminescent reaction. Otherwise, leukocytes were incubated with human serum albumin at 37°C for various periods before addition of opsonized zymosan. Oxidized human serum albumin was obtained by the method of metal-catalyzed oxidation. In control to non-modified albumin, oxidized albumin produced an inhibitory effect on luminol-dependent chemiluminescence of leukocytes. These changes were observed in experiments with addition of oxidized albumin at the beginning of a chemiluminescent reaction and after incubation of study agent with cells.

  17. An in-depth view of human serum albumin corona on gold nanoparticles.

    PubMed

    Ramezani, Fatemeh; Rafii-Tabar, Hashem

    2015-02-01

    Upon entering biological systems, such as the blood stream, nanoparticles form molecular complexes with the proteins encountered called protein coronas, which shield the surface of the exogenous nanoparticle. The most abundant blood proteins, such as albumin, initially occupy the surface of the nanoparticle. Owing to the widespread applications of gold nanoparticles in medicine, in this study, the docking of human serum albumin to gold nanoparticles was examined and the changes in protein structure were investigated by a molecular dynamic simulation and GOLP force field. The results showed that after the adsorption of albumin on the gold nanoparticle, human serum albumin was denatured and the amount of alpha-helix significantly decreased. Domain III, which has a large cavity of fatty acids binding sites, plays an important role in the adsorption on the gold nanoparticles. Lys464, Thr504, Phe505, and Leu581 are critical amino acids in HSA adsorption on the GNPs. After the adsorption of albumin on the surface of gold nanoparticles, the fluctuations in some of the domains of the protein increased. Variations in the helix properties, such as helix length, dipole, radius, average phi and psi angles, and the length of hydrogen bonds, were calculated in detail.

  18. Metal-catalyzed oxidation of human serum albumin: conformational and functional changes. Implications in protein aging.

    PubMed

    Meucci, E; Mordente, A; Martorana, G E

    1991-03-15

    Mild oxidative stress, as elicited by ascorbate, oxygen, and trace metals, affects the binding properties of human serum albumin via purely conformational changes. In fact, no gross alteration can be observed in the electrophoretic and chromatographic patterns of albumin, whereas localized modifications are indicated by the changes in absorption and fluorescence spectra and in polarization degree. The oxidized protein presents a small increase of bityrosine production and a time-dependent increase in the content of carbonyl groups, whereas proteolytic susceptibility is unchanged. A higher affinity for cis-parinaric acid and a slight loss of solubility in high salt indicate a greater surface hydrophobicity. Pinpoint denaturation of the albumin molecule is also suggested by a decreased "esterase" activity in the presence of p-nitrophenyl acetate. Conformational stability evaluated through thermal shock and addition of moderate amounts of guanidine indicate that the oxidized protein is more heat-resistant, less flexible, and more rigid than the native one. Although limited, structural damages afforded by the oxidative stress cause alterations of albumin binding properties as documented by experiments with probes and physiological ligands. The loss of biological activity of human serum albumin induced by ascorbate system appears of medical relevance, because it can affect drug metabolism and particularly drug tolerance in the elderly.

  19. Molecular Structure-Affinity Relationship of Flavonoids in Lotus Leaf (Nelumbo nucifera Gaertn.) on Binding to Human Serum Albumin and Bovine Serum Albumin by Spectroscopic Method.

    PubMed

    Tang, Xiaosheng; Tang, Ping; Liu, Liangliang

    2017-06-23

    Lotus leaf has gained growing popularity as an ingredient in herbal formulations due to its various activities. As main functional components of lotus leaf, the difference in structure of flavonoids affected their binding properties and activities. In this paper, the existence of 11 flavonoids in lotus leaf extract was confirmed by High Performance Liquid Chromatography (HPLC) analysis and 11 flavonoids showed various contents in lotus leaf. The interactions between lotus leaf extract and two kinds of serum albumins (human serum albumin (HSA) and bovine serum albumin (BSA)) were investigated by spectroscopic methods. Based on the fluorescence quenching, the interactions between these flavonoids and serum albumins were further checked in detail. The relationship between the molecular properties of flavonoids and their affinities for serum albumins were analyzed and compared. The hydroxylation on 3 and 3' position increased the affinities for serum albumins. Moreover, both of the methylation on 3' position of quercetin and the C₂=C₃ double bond of apigenin and quercetin decreased the affinities for HSA and BSA. The glycosylation lowered the affinities for HSA and BSA depending on the type of sugar moiety. It revealed that the hydrogen bond force played an important role in binding flavonoids to HSA and BSA.

  20. 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.

  1. Long-Acting Recombinant Fusion Protein Linking Coagulation Factor IX with Albumin (rIX-FP) in Children

    PubMed Central

    Chambost, Hervé; Male, Christoph; Lambert, Thierry; Halimeh, Susan; Chernova, Tatiana; Mancuso, Maria Elisa; Curtin, Julie; Voigt, Christine; Li, Yanyan; Jacobs, Iris; Santagostino, Elena

    2016-01-01

    Summary A global phase 3 study evaluated the pharmacokinetics, efficacy and safety of a recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in 27 previously treated male children (1–11 years) with severe and moderately severe haemophilia B (factor IX [FIX] activity ≤2 IU/dl). All patients received routine prophylaxis once every seven days for up to 77 weeks, and treated any bleeding episodes on-demand. The mean terminal half-life of rIX-FP was 91.4 hours (h), 4.3-fold longer than previous FIX treatment and clearance was 1.11 ml/h/kg, 6.4-fold slower than previous FIX treatment. The median (Q1, Q3) annualised spontaneous bleeding rate was 0.00 (0.00, 0.91) and was similar between the <6 years and ≥6 years age groups, with a weekly median prophylactic dose of 46 IU/kg. In addition, patients maintained a median trough level of 13.4 IU/dl FIX activity on weekly prophylaxis. Overall, 97.2% of bleeding episodes were successfully treated with one or two injections of rIX-FP (95% CI: 92% to 99%), 88.7% with one injection, and 96% of the treatments were rated effective (excellent or good) by the Investigator. No patient developed FIX inhibitors and no safety concerns were identified. These results indicate that rIX-FP is safe and effective for preventing and treating bleeding episodes in children with haemophilia B with weekly prophylaxis. Routine prophylaxis with rIX-FP at treatment intervals of up to 14 days are currently being investigated in children with severe and moderately severe haemophilia B. Clinicaltrials.gov (NCT01662531) PMID:27583313

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

    PubMed Central

    Altinok, Mahmut; Urfels, Stephan; Bauer, Johann

    2014-01-01

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

  3. Impact of bovine and human serum albumin on Curcumin in vitro activity against Staphylococcus aureus.

    PubMed

    Teow, Sin Yeang; Ali, Syed Atif

    2017-05-01

    This study evaluated the impact of pH (7.4 and 6.5), bovine serum albumin (BSA), and human serum albumin (HSA) on Curcumin activity against 2 reference, 1 clinical, and 10 environmental strains of Staphylococcus aureus (S. aureus). Minimal inhibitory concentrations (MICs) of Curcumin against S. aureus were statistically indifferent (p>0.05) at pH7.4 and pH6.5. Activity of Curcumin against S. aureus was reduced by two folds in the presence of 1.25-5% BSA/HSA.

  4. International Validation of Two Human Recombinant Estrogen ...

    EPA Pesticide Factsheets

    An international validation study has been successfully completed for 2 competitive binding assays using human recombinant ERa. Assays evaluated included the Freyberger-Wilson (FW) assay using a full length human ER, and the Chemical Evaluation and Research Institute (CERI) assay using a ligand-binding domain of the human ER. Twenty three compounds were tested in 6 laboratories for the FW assay and 5 for the CERJ assay, which included three controls (used with every run), 9 uncoded, and 14 coded chemicals across 3 subtasks. The overall goal of this validation study was to demonstrate the ability of each of the two assays to reliably classify the test chemicals as binders or non-binders. Laboratories had little trouble with the ER binders that produced a full binding curve when using either the CERI or FW assays. As is typical with all ER competitive binding assays, the weak binders proved to be more challenging. However, overall results from both the FW and CERI assays were consistent and in agreement with expected classifications regardless of the form of the hrER (i.e., full length ER versus an ER ligand binding domain) or the subtle differences in the protocols for conducting each assay. The reproducibility and accuracy for classification of chemicals as potential ER binders and non- binders using the FW and CERI hrER binding assays were comparable to that of the U.S.EPA’s existing ER binding test guideline OPPTS 890.1250, while providing an improved, highe

  5. Kinetics of refolding of completely reduced human-serum albumin. Regain of immunochemical reactivity.

    PubMed

    Wichman, A; Svenson, A; Andersson, L O

    1977-10-03

    The kinetics of refolding of completely reduced human serum albumin has been studied by various methods including immunological techniques. The decrease in thiol content is very rapid in the beginning of the reoxidation process and rather slow in the later stages. Polyacrylamide gel electrophoresis studies show that, in the earlier stages of refolding, the main part of the albumin is present as various oligomers and that a slow conversion to monomer occurs as reoxidation proceeds. Rocket immunoelectrophoresis shows that the completely reduced protein is devoid of native albumin antigenic determinants but that a rapid regain of immunoprecipitability is obtained upon reoxidation. A new 'consumption' rocket immunoelectrophoretic method has been used to estimate the total regain of antigenicity. The data obtained indicate that there is a preferential rapid folding to native structure in certain parts of the molecule but that areas with wrong or incomplete foldings exist a considerable time after the inital refolding period.

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

    NASA Astrophysics Data System (ADS)

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

    1997-01-01

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

  7. [Preparation of recombinant human insulin--study of downstream process].

    PubMed

    Yu, Rong; Li, Xiaohong; Yang, Jiyu; Wu, Wutong

    2004-10-01

    This study was intended to establish a method of preparation of recombinant human insulin, with (His)6-Arg-Arg-human proinsulin (RRhPI) expressed by Escherichia coli. After DEAE-Sepharose Fast Flow ion-exchange chromatography, Sephadex G-25 chromatography and refolding, enzyme cleavage and Superdex 75 size exclusion chromatography,the RRhPI expressed by Escherichia coli in inclusion body form was converted to human insulin. The obtained recombinant human insulin was analyzed by SDS-PAGE, HPLC, amino acid composition analysis and bioidentity test (mouse convulsion test). The results indicate that our obtained preparation is highly purified, active recombinant human insulin.

  8. Human albumin in extracorporeal prime: effect on platelet function and bleeding.

    PubMed

    Kamra, C; Beney, A

    2013-11-01

    Synthetic starches have been positioned as an equivalent substitute for human albumin in extracorporeal prime, with both providing osmotic and oncotic pressure. Another effect of albumin is its ability to coat the synthetic surfaces of an extracorporeal circuit with a biopassivating protein monolayer. Whether this protein biopassivation has any benefit to the patient, assessed by platelet count, platelet function and 24-hour bleeding rate, is considered. Patients presenting for coronary artery bypass at a Canadian tertiary care hospital were randomized into two groups until a final study size of 20 patients was obtained. The Study Group received 2.5 g of human albumin in the extracorporeal prime and the Control Group remained protein free. Both groups included Voluven 6% as a synthetic starch. Blood samples were obtained at three intervals; Pre-bypass, During bypass (30 minutes after initiation of bypass), and Post-bypass. These samples were assayed for platelet function, platelet count and hemoglobin. Chest tube drainage over a 24-hour period was monitored. Platelet count was significantly higher in the During sample in the Study Group (196 ± 56.5 x 10(9)/ml versus 160 ± 18.5 x 10(9)/ml, p<0.05), however, this difference was no longer significant with the Post-bypass sample (135 ± 36.0 x 10(9)/mL versus 127 ± 1 9 x 10(9)/mL). Platelet function assays (PFA) showed no significant differences. Chest tube drainage after 24 hours was significantly lower in the Study Group (586 ± 131.8 ml/24 h versus 741 ± 272.5 ml/24 h, p<0.05). Human albumin can passivate the synthetic surfaces of the extracorporeal circuit, which is supported by observations of preserved platelet count and reduced chest tube drainage. Although some statistically significant benefits were observed, the practical benefits of passivating an extracorporeal circuit with human albumin may be minimal.

  9. Luminescent probe in the study of surfactant-induced structural changes in serum albumin in human blood plasma

    NASA Astrophysics Data System (ADS)

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

    2005-06-01

    The luminescence-kinetic technique of the monitoring of structural changes in albumins of human blood plasma that uses a luminescent probe-eosin is proposed. Phosphorescence of eosin bound to the globular proteins of blood plasma-albumins was recorded at room temperature. It is found that under the action of sodium dodecylsulfate on the albumins the rate constant of eosin phosphorescence decay grows and the intensity of eosin phosphorescence decreases. It is assumed that these changes are connected with the denaturing of blood plasma albumins by sodium dodecylsulfate.

  10. Albumin Redhill (-1 Arg, 320 Ala yields Thr): A glycoprotein variant of human serum albumin whose precursor has an aberrant signal peptidase cleavage site

    SciTech Connect

    Brennan, S.O.; Myles, T.; Peach, R.J.; George, P.M. ); Donaldson, D. )

    1990-01-01

    Albumin Redhill is an electrophoretically slow genetic variant of human serum albumin that does not bind {sup 63}Ni{sup 2+} and has a molecular mass 2.5 kDa higher than normal albumin. Its inability to bind Ni{sup 2+} was explained by the finding of an additional residue of Arg at position -1. This did not explain the molecular basis of the genetic variation or the increase in apparent molecular mass. Fractionation of tryptic digests on concanavalin A-Sepharose followed by peptide mapping of the bound and unbound fractions and sequence analysis of the glycopeptides identified a mutation of 320 Ala {yields} Thr. This introduces as Asn-Tyr-Thr oligosaccharide attachment sequence centered on Asn-318 and explains the increase in molecular mass. This, however, did not satisfactorily explain the presence of the additional Arg residue at position -1. DNA sequencing of polymerase chain reaction-amplified genomic DNA encoding the prepro sequence of albumin indicated an additional mutation of -2 Arg {yields} Cys. The authors propose that the new Phe-Cys-Arg sequence in the propeptide is an aberrant signal peptidase cleavage site and that the signal peptidase cleaves the propeptide of albumin Redhill in the lumen of the endoplasmic reticulum before it reaches the Golgi vesicles, the site of the diarginyl-specific proalbumin convertase.

  11. Structural specificity requirements in the binding of beta lactam antibiotics to human serum albumin.

    PubMed

    Nerli, B; Romanini, D; Picó, G

    1997-05-02

    The binding of some cephalosporins of pharmacological interest, to human serum albumin was studied using ultrafiltration method. The identification of the binding sites in albumin was also performed using probes for the so-called sites I, II, bilirubin and fatty acids binding sites. Cephalosporins were classified into three groups according to their affinity for albumin: low affinity (K = 10-10(2) M-1), medium affinity (K = 10(3) M-1) and high affinity (K = 10(4) M-1). Cephalosporin binding to albumin produced a perturbation of several basic amino acids of the protein such as histidine and lysine. It was found that only cefuroxime, ceftazidime and cefoperazone interact slightly with site I on serum albumin, while site II possesses capacity to bind: cephradine, cephalexin, ceftazidime, ceftriaxone, cefoperazone, cefaclor and cefsulodin. The bilirubin binding site showed capacity to interact with a great number of cephalosporins: ceftriaxone, cefazolin, cephaloglycin, cefamandole, cefotaxime, cefoxitin, cefuroxime, cefoperazone and cefadroxil. Ceftriaxone showed capacity to bind to the fatty acid binding site on HSA. No relation was found between the displacement of the marker and the chemical nature of the substituents at R1 and R2. Cephalosporins interact with HSA at the binding region that involves: tyrosyl 411, histidyl 146 and lysyls 195, 199, 225, 240 and 525 residues. The chemical modification of specific amino acids showed that the interaction of these amino acids with beta lactam antibiotics is not carried out to the same extent for all the cephalosporins tested. The results obtained revealed that the binding sites for cephalosporins on albumin are structurally heterogeneous, having different amino acids in the vicinity of the ligand molecule.

  12. Albumin infusion in humans does not model exercise induced hypervolaemia after 24 hours

    NASA Technical Reports Server (NTRS)

    Haskell, A.; Gillen, C. M.; Mack, G. W.; Nadel, E. R.

    1998-01-01

    We rapidly infused 234 +/- 3 mL of 5% human serum albumin in eight men while measuring haematocrit, haemoglobin concentration, plasma volume (PV), albumin concentration, total protein concentration, osmolality, sodium concentration, renin activity, aldosterone concentration, and atrial natriuretic peptide concentration to test the hypotheses that plasma volume expansion and plasma albumin content expansion will not persist for 24 h. Plasma volume and albumin content were expanded for the first 6 h after infusion (44.3 +/- 1.9-47.2 +/- 2.0 mL kg-1 and 1.9 +/- 0.1-2.1 +/- 0.1 g kg-1 at pre-infusion and 1 h, respectively, P < 0.05), but by 24 h plasma volume and albumin content decreased significantly from 1 h post-infusion and were not different from pre-infusion (44.8 +/- 1.9 mL kg-1 and 1.9 +/- 0.1 g kg-1, respectively). Plasma aldosterone concentration showed a significant effect of time over the 24 h after infusion (P < 0.05), and showed a trend to decrease at 2 h after infusion (167.6 +/- 32.5(-1) 06.2 +/- 13.4 pg mL-1, P = 0.07). These data demonstrate that a 6.8% expansion of plasma volume and 10.5% expansion of plasma albumin content by infusion does not remain in the vascular space for 24 h and suggest a redistribution occurs between the intravascular space and interstitial fluid space.

  13. Albumin infusion in humans does not model exercise induced hypervolaemia after 24 hours

    NASA Technical Reports Server (NTRS)

    Haskell, A.; Gillen, C. M.; Mack, G. W.; Nadel, E. R.

    1998-01-01

    We rapidly infused 234 +/- 3 mL of 5% human serum albumin in eight men while measuring haematocrit, haemoglobin concentration, plasma volume (PV), albumin concentration, total protein concentration, osmolality, sodium concentration, renin activity, aldosterone concentration, and atrial natriuretic peptide concentration to test the hypotheses that plasma volume expansion and plasma albumin content expansion will not persist for 24 h. Plasma volume and albumin content were expanded for the first 6 h after infusion (44.3 +/- 1.9-47.2 +/- 2.0 mL kg-1 and 1.9 +/- 0.1-2.1 +/- 0.1 g kg-1 at pre-infusion and 1 h, respectively, P < 0.05), but by 24 h plasma volume and albumin content decreased significantly from 1 h post-infusion and were not different from pre-infusion (44.8 +/- 1.9 mL kg-1 and 1.9 +/- 0.1 g kg-1, respectively). Plasma aldosterone concentration showed a significant effect of time over the 24 h after infusion (P < 0.05), and showed a trend to decrease at 2 h after infusion (167.6 +/- 32.5(-1) 06.2 +/- 13.4 pg mL-1, P = 0.07). These data demonstrate that a 6.8% expansion of plasma volume and 10.5% expansion of plasma albumin content by infusion does not remain in the vascular space for 24 h and suggest a redistribution occurs between the intravascular space and interstitial fluid space.

  14. The thiol pool in human plasma: The central contribution of albumin to redox processes

    PubMed Central

    Turell, Lucía; Radi, Rafael; Alvarez, Beatriz

    2013-01-01

    The plasma compartment has particular features regarding the nature and concentration of low and high molecular weight thiols and oxidized derivatives. Plasma is relatively poor in thiol-based antioxidants; thiols are in lower concentrations than in cells and mostly oxidized. The different thiol-disulfide pairs are not in equilibrium and the steady-state concentrations of total thiols as well as reduced versus oxidized ratios are maintained by kinetic barriers, including the rates of reactions and transport processes. The single thiol of human serum albumin (HSA-SH) is the most abundant plasma thiol. It is an important target for oxidants and electrophiles due to its reactivity with a wide variety of species and its relatively high concentration. A relatively stable sulfenic (HSA-SO3H) acid can be formed in albumin exposed to oxidants. Plasma increases in mixed disulfides (HSA-SSR) or in sulfinic (HSA-SO2H) and sulfonic (HSA-SO3H) acids are associated with different pathologies and may constitute biomarkers of the antioxidant role of the albumin thiol. In this work we provide a critical review of the plasma thiol pool with a focus on human serum albumin. PMID:23747983

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

    SciTech Connect

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

    1987-07-01

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

  16. Where does the Albumin go? Human Albumin Solution usage following the implementation of a demand management programme.

    PubMed

    Yazdani, M S; Retter, A; Maggs, T; Li, P; Robson, M G; Reid, C; Holmes, P; Garood, T; Robinson, S E

    2017-06-01

    To outline the Guy's and St Thomas' NHS Foundation Trust (GSTFT) and Evelina London Children's Hospital (ELCH) demand management plan for human albumin solution (HAS) and usage. There is no UK-wide guidance governing the use of HAS. A severe shortage in 2015 prompted a Trust demand management programme. Indications were categorised according to locally agreed colour code and ASFA categories. Following the implementation of the demand management programme, a 6-month audit of HAS usage was completed. A total of 1303.1 L of HAS was used in 1139 infusions; 737 infusions were 20% HAS, accounting for 175.7 L (13.5%) in 181 patients. Indications for 20% HAS were red in 53.9% (94.7 L), blue in 26.5% (46.5 L) and grey in 19.6% (34.5 L). The remaining 1127.4 L (86.5%) infused were of 4.5 and 5 % HAS. A total of 1102.3 L (97.8%) was used for plasma exchange, 941.4 L (85.4%) ASFA category I, 93.7 L (8.5%) category II, 25.5 L (2.3%) category IV and 41.7 L (3.8%) for indications not specified according to ASFA; 25.1 L (2.2%) were used for a grey indication (volume resuscitation for hypovolaemia). The demand management programme provides surveillance of indications and retrospective verification of appropriate use. The majority of HAS indications were appropriate. Plasma exchange accounted for 84.6% of HAS usage and will be the focus of further demand management strategies. The demand management programme whilst aiming to promote best transfusion practice also ensures a tool to manage future shortages according to indication and available supply. © 2017 British Blood Transfusion Society.

  17. Mast cell degranulation via MRGPRX2 by isolated human albumin fragments.

    PubMed

    Karhu, T; Akiyama, K; Vuolteenaho, O; Bergmann, U; Naito, T; Tatemoto, K; Herzig, K-H

    2017-08-24

    Mast cells are important modulators of the human immune system via their release of several inflammatory mediators and proteases. The release can be activated by different pathways: the classical immunoglobulin E-dependent pathway and by the non-immunological immunoglobulin E-independent pathway. MAS-related G protein-coupled receptor X2 (MRGPRX2) is expressed in mast cells and it is one of the endogenous receptor responsible for the IgE-independent activation of human mast cell. The MRGPRX2 is classified as orphan receptor and unlike most GPCRs, the MRGPRX2 recognizes a wide range of basic molecules. Thus, there still might be several unknown ligands for the receptor. MRGPRX2 activating peptides were isolated from human plasma using consecutive HPLC purification steps. The isolation process was monitored with MRGPRX2 transfected HEK 293 cells. The isolated peptides were sequenced by MS and synthetized. The synthetic peptides were used to determine degranulation of the human LAD 2 mast cell line by measuring β-hexosaminidase release. Three endogenous MRGPRX2 activating peptides were isolated from human plasma. These peptides are identified as fragments of albumin. The isolated fragments activate MRGPRX2 and degranulate MRGPRX2 expressing LAD 2 cells in dose-dependent manner. The isolated basic peptides generated from human albumin are able to degranulate mast cells via the MRGPRX2. These endogenous albumin fragments, cleaved from albumin by mast cell secreted proteases, provide a possible pathway for self-perpetuating mast cell dependent inflammation. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Differential effects of insulin deficiency on albumin and fibrinogen synthesis in humans.

    PubMed Central

    De Feo, P; Gaisano, M G; Haymond, M W

    1991-01-01

    Insulin deficiency decreases tissue protein synthesis, albumin mRNA concentration, and albumin synthesis in rats. In contrast, insulin deficiency does not change, or, paradoxically, increases estimates of whole body protein synthesis in humans. To determine if such estimates of whole body protein synthesis could obscure potential differential effects of insulin on the synthetic rates of individual proteins, we determined whole body protein synthesis and albumin and fibrinogen fractional synthetic rates using 5-h simultaneous infusions of [14C]leucine and [13C]bicarbonate, in six type 1 diabetics during a continuous i.v. insulin infusion (to maintain euglycemia) and after short-term insulin withdrawal (12 +/- 2 h). Insulin withdrawal increased (P less than 0.03) whole body proteolysis by approximately 35% and leucine oxidation by approximately 100%, but did not change 13CO2 recovery from NaH13CO3 or estimates of whole body protein synthesis (P = 0.21). Insulin deficiency was associated with a 29% decrease (P less than 0.03) in the albumin fractional synthetic rate but a 50% increase (P less than 0.03) in that of fibrinogen. These data provide strong evidence that albumin synthesis in humans is an insulin-sensitive process, a conclusion consistent with observations in rats. The increase in fibrinogen synthesis during insulin deficiency most likely reflects an acute phase protein response due to metabolic stress. These data suggest that the absence of changes in whole body protein synthesis after insulin withdrawal is the result of the summation of differential effects of insulin deficiency on the synthesis of specific body proteins. PMID:1909352

  19. Photosensitizer-Conjugated Human Serum Albumin Nanoparticles for Effective Photodynamic Therapy

    PubMed Central

    Jeong, Hayoung; Huh, MyungSook; Lee, So Jin; Koo, Heebeom; Kwon, Ick Chan; Jeong, Seo Young; Kim, Kwangmeyung

    2011-01-01

    Photodynamic therapy (PDT) is an emerging theranostic modality for various cancers and diseases. The focus of this study was the development of tumor-targeting albumin nanoparticles containing photosensitizers for efficient PDT. To produce tumor-targeting albumin nanoparticles, the hydrophobic photosensitizer, chlorin e6 (Ce6), was chemically conjugated to human serum albumin (HSA). The conjugates formed self-assembled nanoparticle structures with an average diameter of 88 nm under aqueous conditions. As expected, the Ce6-conjugated HSA nanoparticles (Ce6-HSA-NPs) were nontoxic in their native state, but upon illumination with the appropriate wavelength of light, they produced singlet oxygen and damaged target tumor cells in a cell culture system. Importantly, when the nanoparticles were injected through the tail vein into tumor-bearing HT-29 mice, Ce6-HSA-NPs compared with free Ce6 revealed enhanced tumor-specific biodistribution and successful therapeutic results following laser irradiation. These results suggest that highly tumor-specific albumin nanoparticles have the potential to serve not only as efficient therapeutic agents, but also as photodynamic imaging (PDI) reagents in cancer treatment. PMID:21562630

  20. Purification of soluble recombinant human FcgammaRII (CD32).

    PubMed

    Gruel, N; Chapiro, J; Fridman, W H; Teillaud, J L

    2001-11-01

    The present study describes the methodology used to purify human recombinant low-affinity FcgammaRIIa2 produced in E. coli and to evaluate its binding to surface IgG. The recombinant molecule was purified by a two-step chromatographic procedure, including affinity chromatography using IV.3 anti-FcgammaRIIa1/2 immunosorbent, followed by gel filtration chromatography. Using this method, the purified recombinant FcgammaRIIa2 was 99% pure. It exhibited an isoeletric point of 5.2. Binding studies demonstrated a specific binding of the purified recombinant molecule to surface IgG expressed by human B cells. Thus, we have set up a method which allows to purify functional human recombinant FcgammaRIIa2 for further characterization of its biological activities.

  1. 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.

  2. Recombinant expression of hydroxylated human collagen in Escherichia coli.

    PubMed

    Rutschmann, Christoph; Baumann, Stephan; Cabalzar, Jürg; Luther, Kelvin B; Hennet, Thierry

    2014-05-01

    Collagen is the most abundant protein in the human body and thereby a structural protein of considerable biotechnological interest. The complex maturation process of collagen, including essential post-translational modifications such as prolyl and lysyl hydroxylation, has precluded large-scale production of recombinant collagen featuring the biophysical properties of endogenous collagen. The characterization of new prolyl and lysyl hydroxylase genes encoded by the giant virus mimivirus reveals a method for production of hydroxylated collagen. The coexpression of a human collagen type III construct together with mimivirus prolyl and lysyl hydroxylases in Escherichia coli yielded up to 90 mg of hydroxylated collagen per liter culture. The respective levels of prolyl and lysyl hydroxylation reaching 25 % and 26 % were similar to the hydroxylation levels of native human collagen type III. The distribution of hydroxyproline and hydroxylysine along recombinant collagen was also similar to that of native collagen as determined by mass spectrometric analysis of tryptic peptides. The triple helix signature of recombinant hydroxylated collagen was confirmed by circular dichroism, which also showed that hydroxylation increased the thermal stability of the recombinant collagen construct. Recombinant hydroxylated collagen produced in E. coli supported the growth of human umbilical endothelial cells, underlining the biocompatibility of the recombinant protein as extracellular matrix. The high yield of recombinant protein expression and the extensive level of prolyl and lysyl hydroxylation achieved indicate that recombinant hydroxylated collagen can be produced at large scale for biomaterials engineering in the context of biomedical applications.

  3. Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511.

    PubMed

    Rodin, Sergey; Domogatskaya, Anna; Ström, Susanne; Hansson, Emil M; Chien, Kenneth R; Inzunza, José; Hovatta, Outi; Tryggvason, Karl

    2010-06-01

    We describe a system for culturing human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells on a recombinant form of human laminin-511, a component of the natural hES cell niche. The system is devoid of animal products and feeder cells and contains only one undefined component, human albumin. The hES cells self-renewed with normal karyotype for at least 4 months (20 passages), after which the cells could produce teratomas containing cell lineages of all three germ layers. When plated on laminin-511 in small clumps, hES cells spread out in a monolayer, maintaining cellular homogeneity with approximately 97% OCT4-positive cells. Adhesion of hES cells was dependent on alpha6beta1 integrin. The use of homogeneous monolayer hES or iPS cell cultures provides more controllable conditions for the design of differentiation methods. This xeno-free and feeder-free system may be useful for the development of cell lineages for therapeutic purposes.

  4. 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).

  5. Radiopharmaceutical development based on human blood albumin microspheres and 90Y

    NASA Astrophysics Data System (ADS)

    Petriev, V. M.; Vlasova, O. P.; Postnov, A. A.; Epstein, N. B.

    2017-01-01

    New radiopharmaceutial (RP) based on human serum albumin microspheres (MSA) and 90Y was developed for treatment of liver cancer. The optimized synthesis using chelation resulted in approximately 80% yield with high specific activity. The RP developed was tested in mice with inoculated sarcoma-37. In two weeks the tumor size reduced by 43% after the treatment with the dose of 500 μCi injected into the tumor site.

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

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

  7. Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins.

    PubMed

    Watanabe, Masaaki; Zemack, Helen; Johansson, Helene; Hagbard, Louise; Jorns, Carl; Li, Meng; Ellis, Ewa

    2016-01-01

    Refined methods for maintaining specific functions of isolated hepatocytes under xeno-free and chemical defined conditions is of great importance for the development of hepatocyte research and regenerative therapy. Laminins, a large family of heterotrimeric basement membrane adhesion proteins, are highly cell and tissue type specific components of the extracellular matrix and strongly influence the behavior and function of associated cells and/or tissues. However, detailed biological functions of many laminin isoforms are still to be evaluated. In this study, we determined the distribution of laminin isoforms in human liver tissue and isolated primary human hepatocytes by western blot analysis, and investigated the efficacy of different human recombinant laminin isoforms on hepatic functions during culture. Protein expressions of laminin-chain α2, α3, α4, β1, β3, γ1, and γ2 were detected in both isolated human hepatocytes and liver tissue. No α1 and α5 expression could be detected in liver tissue or hepatocytes. Hepatocytes were isolated from five different individual livers, and cultured on human recombinant laminin isoforms -111, -211, -221, -332, -411, -421, -511, and -521 (Biolamina AB), matrigel (extracted from Engelbreth-Holm-Swarm sarcoma), or collagen type IV (Collagen). Hepatocytes cultured on laminin showed characteristic hexagonal shape in a flat cell monolayer. Viability, double stranded DNA concentration, and Ki67 expression for hepatocytes cultured for six days on laminin were comparable to those cultured on EHS and Collagen. Hepatocytes cultured on laminin also displayed production of human albumin, alpha-1-antitrypsin, bile acids, and gene expression of liver-enriched factors, such as hepatocyte nuclear factor 4 alpha, glucose-6-phosphate, cytochrome P450 3A4, and multidrug resistance-associated protein 2. We conclude that all forms of human recombinant laminin tested maintain cell viability and liver-specific functions of primary human

  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. 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.

  10. Porphyrin mediated photo-modification of the structure and function of human serum albumin

    NASA Astrophysics Data System (ADS)

    Rozinek, Sarah C.

    Photosensitization reactions involve irradiating (with visible light) molecules with a high efficiency for either electron transfer or entering an excited triplet state (photosensitizer). Such reactions are applied to photodynamic cancer therapy, many medical laser-treatments, and a potential array of disinfection and pest elimination techniques. To understand the biophysical mechanisms of how these applications are effective at the protein level, the group of Dr. Brancaleon (UTSA) has investigated the irradiation of several dye-protein combinations, and discovered effects on protein structure and function. To further that work, we have investigated irradiation of the protein, human serum albumin (HSA), photosensitized by either protoporphyrin IX (PPIX) or meso-tetrakis(4-sulfonatophenyl)porphyrin (TSPP). HSA is the most abundant plasma protein, making it a likely substrate in PDT, and it possesses a specific binding pocket for iron-PPIX (heme) and possibly other porphyrin derivatives. The results of our research are summarized as follows. First, a thorough characterization of the binding of each photosensitizer to albumin was completed, elucidating a probable binding location for TSPP. Next, fluorescence lifetime emission of the single tryptophan residue, alongside circular dichroism, found tertiary structural changes around tryptophan and an overall 20% decrease in protein secondary structure after irradiation with TSPP bound. Finally, to determine if protein function was lost after photosensitization, size exclusion chromatography found modified albumin still recognizable by its receptor-protein, and comparative ex vivo up-take studies revealed that modified albumin is not processed the same way as native albumin in live tapeworm larva (Mesocestoides corti). Thus we found that visible light can induce partial unfolding of a protein by using a photo-activated ligand. These small structural modifications were sufficient to affect the protein's biological function.

  11. A highly efficient modified human serum albumin signal peptide to secrete proteins in cells derived from different mammalian species.

    PubMed

    Attallah, Carolina; Etcheverrigaray, Marina; Kratje, Ricardo; Oggero, Marcos

    2017-01-10

    Signal peptides (SPs) are key elements in the production of recombinant proteins; however, little information is available concerning different SP in mammalian cells other than CHO. In order to study the efficiency of different SPs to direct the traffic along the secretory pathway of the green fluorescence protein (GFP) and a scFv-Fc fusion protein; CHO-K1, HEK293 and NS0 cell lines were transfected in a transient and stable way. SP of human azurocidin (AZ), modified human albumin (mSA), modified Cricetulus griseus Ig kappa chain V III region MOPC 63 like (mIgκ C) and modified human Ig kappa chain V III region VG (mIgκ H) were evaluated. The efficiency of SPs to translocate a propeptide across the ER membrane was evaluated by fluorescence microscopy and flow cytometry for the GFP inside the secretory pathway, and by antigen-specific indirect ELISA for the scFv-Fc outside the cell. The mSA SP was successful in directing the secretion of the active proteins in these different types of mammalian cells, regardless of the transgene copy number. The goal of this work was to demonstrate that a modified version of SA SP might be used in different mammalian cells employing the same expression vector.

  12. α-Galactosidase delivery using 30Kc19-human serum albumin nanoparticles for effective treatment of Fabry disease.

    PubMed

    Lee, Hong Jai; Park, Hee Ho; Sohn, Youngsoo; Ryu, Jina; Park, Ju Hyun; Rhee, Won Jong; Park, Tai Hyun

    2016-12-01

    Fabry disease is a genetic lysosomal storage disease caused by deficiency of α-galactosidase, the enzyme-degrading neutral glycosphingolipid that is transported to lysosome. Glycosphingolipid accumulation by this disease causes multi-organ dysfunction and premature death of the patient. Currently, enzyme replacement therapy (ERT) using recombinant α-galactosidase is the only treatment available for Fabry disease. To maximize the efficacy of treatment, enhancement of cellular delivery and enzyme stability is a challenge in ERT using α-galactosidase. In this study, protein nanoparticles using human serum albumin (HSA) and 30Kc19 protein, originating from silkworm, were used to enhance the delivery and intracellular α-galactosidase stability. 30Kc19-HSA nanoparticles loaded with the α-galactosidase were formed by desolvation method. 30Kc19-HSA nanoparticles had a uniform spherical shape and were well dispersed in cell culture media. 30Kc19-HSA nanoparticles had negligible toxicity to human cells. The nanoparticles exhibited enhanced cellular uptake and intracellular stability of delivered α-galactosidase in human foreskin fibroblast. Additionally, they showed enhanced globotriaosylceramide degradation in Fabry patients' fibroblasts. It is expected that 30Kc19-HSA protein nanoparticles could be used as an effective tool for efficient delivery and enhanced stability of drugs.

  13. [Effect of Human Serum Albumin on Endotoxin Scattering Photometry].

    PubMed

    Takahashi, Gaku; Shibata, Shigehiro; Kan, Shigenori; Inada, Katsuya; Endo, Shigeatsu

    2015-07-01

    Laser scattering photometry (ESP) is a newly developed plasma endotoxin assay method using horseshoe crab amebocyte lysate (AL) that recognizes small particles produced by polymerization of coagulin under the stirring conditions at 1000rpm. We elucidated the effect of human serum album (HSA) in the ESP method. AL was dissolved with 630μL of the specimen and a 200-μL aliquot was used for ESP; this conventional protocol was regarded as the ESP630 method. The ESP210 method was also used, i. e. AL was dissolved with 210μL of the specimen and a 200-μL aliquot was used for ESP. Water induced the agglutination, and HSA prolonged the agglutination time depending on its concentration especially in the ESP630 method. The water-induced agglutination was not inhibited by the addition of anti-factor C monoclonal antibody, and amidinophenyl benzoate hydrochloride, used as a clotting enzyme inhibitor, intensively inhibited the water-induced agglutination. Therefore, the water-induced agglutination was suggested to be a false-positive reaction to non-specific activation of the clotting enzyme. The HSA-induced prolongation of the reaction in the national health insurance-covered turbidimetric kinetic assay was not observed. HSA or plasma protein seemed to affect the result, especially in the ESP630 method, and a non-specific reaction was found to occur in the ESP methods.

  14. Two distinct receptors account for recognition of maleyl-albumin in human monocytes during differentiation in vitro.

    PubMed Central

    Haberland, M E; Rasmussen, R R; Olch, C L; Fogelman, A M

    1986-01-01

    A comparison of the receptor-mediated interaction of malondialdehyde-low density lipoprotein and maleyl-albumin has been examined in human monocytes during differentiation in vitro. The recognition of both ligands by the scavenger receptor of these cells has been confirmed. We now report that human monocytes express a second cellular surface receptor for maleyl-albumin that is distinct from the scavenger receptor. The activity of the maleyl-albumin receptor, determined by both binding and lysosomal hydrolytic assays, substantially exceeds that of the scavenger receptor in freshly isolated monocytes. A dramatic and rapid decline in the activity of the maleyl-albumin receptor occurs within 72 to 96 h during differentiation in vitro. At day 7, while only 5-10% of the original activity of the maleyl-albumin receptor remains, it is similar to that of the maximally expressed scavenger receptor. Both the binding and hydrolysis of ligand mediated by the maleyl-albumin receptor are specifically inhibited by alpha-casein and alkaline-treated albumin; neither of these proteins is recognized by the scavenger receptor. The occurrence of the exceptionally active maleyl-albumin receptor on freshly isolated human monocytes suggests that it participates in processes necessary to the function of the cells that diminish in importance after differentiation of the monocytes into macrophages in vitro. Furthermore, while maleyl-albumin is a useful adjunct to studies of cellular events mediated by the scavenger receptor, the presence of a second receptor for maleyl-albumin must be taken into account as a potential contributing and complicating event. Images PMID:3949974

  15. Purification and partial characterisation of recombinant human hepcidin.

    PubMed

    Wallace, Daniel F; Jones, Marc D; Pedersen, Palle; Rivas, Lucy; Sly, Lindsay I; Subramaniam, V Nathan

    2006-01-01

    Hepcidin is a liver-expressed antimicrobial and iron regulatory peptide. A number of studies have indicated that hepcidin is important for the correct regulation of body iron homeostasis. The aims of this study were to analyse the expression, trafficking and regulation of human hepcidin in an in vitro cell culture system. Human hepcidin was transfected into human embryonic kidney cells. Immunofluorescence and confocal microscopy analysis revealed that recombinant hepcidin localised to the Golgi complex. Recombinant hepcidin is secreted from the cell within 1 h of its synthesis. Recombinant hepcidin was purified from the cell culture medium using ion-exchange and metal-affinity chromatography and was active in antimicrobial assays. Amino-terminal sequence analysis of the secreted peptide revealed that it was the mature 25 amino acid form of hepcidin. Our results show that recombinant myc-His tagged human hepcidin was expressed, processed and secreted correctly and biologically active in antimicrobial assays.

  16. 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

  17. In vitro inhibition of human neutrophil elastase by oleic acid albumin formulations from derivatized cotton wound dressings.

    PubMed

    Edwards, J Vincent; Howley, Phyllis; Cohen, I Kelman

    2004-10-13

    Human neutrophil elastase (HNE) is elevated in chronic wounds. Oleic acid albumin formulations that inhibit HNE may be applicable to treatment modalities for chronic wounds. Oleic acid/albumin formulations with mole ratios of 100:1, 50:1, and 25:1 (oleic acid to albumin) were prepared and found to have dose response inhibition properties against HNE. The IC50 values for inhibition of HNE with oleic acid/albumin formulations were 0.029-0.049 microM. Oleic acid/albumin (BSA) formulations were bound to positively and negatively charged cotton wound dressings and assessed for elastase inhibition using a fiber bound formulation in an assay designed to mimic HNE inhibition in the wound. Cotton derivatized with both carboxylate and amine functional groups were combined with oleic acid/albumin formulations at a maximum loading of 0.030 mg oleic acid + 0.14 mg BSA/mg fiber. The IC50 values for inhibition of HNE with oleic acid/albumin formulations bound to derivatized cotton were 0.26-0.42 microM. Release of the oleic acid/albumin formulation from the fiber was measured by measuring oleic acid levels with quantitative GC analysis. Approximately, 35-50% of the fiber bound formulation was released into solution within the first 15 min of incubation. Albumin was found to enhance the rate of elastase hydrolysis of the substrate within a concentration range of 0.3-50 g/L. The acceleration of HNE substrate hydrolysis by albumin required increased concentration of inhibitor in the formulation to obtain complete inhibition of HNE. Oleic acid formulations prepared with albumin enable transport, solubility and promote dose response inhibition of HNE from derivatized cotton fibers under aqueous conditions mimicking the chronic wound.

  18. Dependence of the solubility of natural flavonoids in water on the concentration of miramistin, polyvinylpyrrolidone, and human serum albumin

    NASA Astrophysics Data System (ADS)

    Lipkovska, N. A.; Barvinchenko, V. N.; Fedyanina, T. V.

    2014-05-01

    In organized media of the cationic surfactant miramistin and the polymers polyvinylpyrrolidone and human serum albumin, the solubility of natural flavonoids quercetin and rutin increased by one or two orders of magnitude. The increase was more significant for hydrophobic quercetin than for hydrophilic rutin. The solubility also depended on the structure and self-organization of molecules in organized media and the site of flavonoids in them. The calculated binding constants increased in the series polyvinylpyrrolidone < miramistin < human serum albumin.

  19. Formation of albumin dimers induced by exposure to peroxides in human plasma: a possible biomarker for oxidative stress.

    PubMed

    Ogasawara, Yuki; Namai, Tomoyuki; Togawa, Tadayasu; Ishii, Kazuyuki

    2006-02-10

    Human serum albumin (HSA) has one free thiol residue at Cys-34 that is likely oxidized by various reactive oxygen species (ROS). We attempted to identify the oxidation product of Cys-34 of HSA following exposure of plasma to ROS. Oxidation induced by tert-butyl hydroperoxide (t-BuOOH) of this free cysteine residue in HSA was observed in detail. Analysis of oxidized albumin in a partially purified fraction obtained by affinity column chromatography clearly revealed the formation of albumin disulfide dimers following t-BuOOH exposure. Albumin disulfide dimer formation was observed in normal plasma following treatment with various peroxides, as well as in untreated plasma from patients on hemodialysis using SDS-PAGE and Western blot analysis. The present results indicate that albumin dimers are oxidative products derived from peroxides, and that their presence in plasma might be a marker of oxidative stress as secondary metabolites of peroxidation.

  20. Interactions of hybrid gold-tannic acid nanoparticles with human serum albumin.

    PubMed

    Sekowski, Szymon; Tomaszewska, Emilia; Soliwoda, Katarzyna; Celichowski, Grzegorz; Grobelny, Jaroslaw

    2017-01-01

    Nanoparticles present a wide spectrum of chemical, biological, and physical properties which result in their usage in many branches of science. We present an investigation of the interaction between human serum albumin and hybrid gold-tannic acid nanoparticles synthesized via a chemical reduction method. The results obtained demonstrate that tannic acid can be a very effective reducing and stabilizing agent and allows monodisperse hybrid gold nanomaterial to be obtained. The synthesized hybrid gold-tannic acid nanoparticles strongly interact with human serum albumin by formation of protein-corona complexes. The strength of the interaction with albumin depends on the number of tannic acid molecules on the surface of the nanoparticles and the presence of citric acid. Nanoparticles of large size and rich in tannic acid react more strongly with the protein [K SV = (8.00 ± 0.2) × 10(5) M(-1)] compared with smaller ones [K SV = (6.83 ± 0.5) × 10(4) M(-1)] containing citric acid and low concentration of tannic acid.

  1. 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.

  2. 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.

  3. Interaction of Cis- and Trans-RuCl 2(DMSO)4 With Human Serum Albumin

    PubMed Central

    Kozlowski, Henryk; Katsaros, Nikolas

    2000-01-01

    The interaction between cis- and trans- RuCl2(DMSO)4 and human serum albumin have been investigated through UV-Vis, circular dichroism, fluorescence spectroscopy and inductively couplet plasma atomic emission spectroscopy (ICP(AES)) method Albumin can specifically bind 1 mole of cis-isomer and 2 moles of the trans-isomer RuCl2(DMSO)4 complex. The interaction of RuCl2(DMSO)4 with HSA causes: a conformational change with the loss of helical stability of protein; the strong quenching of the Trp 214 fluorescence indicating that the conformational change of the hydrophobic binding pocked in subdomain IIA takes place; a local perturbation of the warfarin binding site and induce some conformational changes at neighbour domains, a changing of the binding abilities towards heme. PMID:18475961

  4. 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.

  5. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2016-10-01

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

  7. Effectiveness of Recombinant Human Growth Hormone for Pharyngocutaneous Fistula Closure

    PubMed Central

    Sari, Murat; Midi, Ahmet; Yumusakhuylu, Ali Cemal; Findik, Ozan; Binnetoglu, Adem

    2015-01-01

    Objectives In laryngeal cancer, which comprises 25% of head and neck cancer, chemotherapy has come into prominence with the increase in organ-protective treatments. With such treatment, salvage surgery has increased following recurrence; the incidence of pharyngocutaneous fistula has also increased in both respiratory and digestive system surgery. We investigated the effects of recombinant human growth hormone on pharyngocutaneous fistula closure in Sprague-Dawley rats, based on an increase in amino acid uptake and protein synthesis for wound healing, an increase in mitogenesis, and enhancement of collagen formation by recombinant human growth hormone. Methods This study was experimental animal study. Forty Sprague-Dawley rats were separated into two groups, and pharyngoesophagotomy was performed. The pharyngoesophagotomy was sutured with vicryl in both groups. Rats in group 1 (control group) received no treatment, while those in group 2 were administered a subcutaneous injection of recombinant human growth hormone daily. On day 14, the pharynx, larynx, and upper oesophagus were excised and examined microscopically. Results Pharyngocutaneous fistula exhibited better closure macroscopically in the recombinant human growth hormone group. There was a significant difference in collagen formation and epithelisation in the recombinant human growth hormone group compared to the control group. Conclusion This study is believed to be the first in which the effect of recombinant human growth hormone on pharyngocutaneous fistula closure was evaluated, and the findings suggest the potential of use of growth hormone for treatment of pharyngocutaneous fistula. PMID:26622960

  8. 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.

  9. 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).

  10. 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.

  11. Characterization of isomeric VX nerve agent adducts on albumin in human plasma using liquid chromatography-tandem mass spectrometry.

    PubMed

    Saeidian, Hamid; Mirkhani, Valioallah; Mousavi Faraz, Sajjad; Taghi Naseri, Mohammad; Babri, Mehran

    2015-01-01

    This study includes the characterization of isomeric VX organophosphorus adducts on albumin in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). VX or its structural isomers were spiked into a vial containing plasma in order to obtain phosphorylated albumin. After pronase and trypsin digestion, the resulting solutions were analyzed to confirm adduct formation with the amino acid tyrosine on the albumin in human plasma. The LC-MS/MS experiments show that VX and its isomers can be attached to tyrosine on the albumin in human blood. Mass spectrometric studies revealed some interesting fragmentation pathways during the ionization process, such as ethylene, formic acid and ammonia elimination and an intermolecular electrophilic aromatic substitution reaction. The proposed mechanisms for the formation of the fragments were confirmed through the analysis of fragments of deuterated adducts.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. Soluble copolymer of wasp venom with human albumin for venom immunotherapy.

    PubMed

    Gewurz, A; Grammer, L C; Shaughnessy, M A; Patterson, R

    1986-03-01

    Polymerization of allergens decreases allergenicity while retaining immunogenicity, as we have demonstrated for ragweed, grass, and tree pollens. We have also polymerized bee venom with human albumin to form soluble, high-molecular-weight copolymers that are immunogenic in rabbits. We now have prepared a soluble wasp venom-albumin polymer (WVAP), molecular weight greater than or equal to 240,000 daltons, by glutaraldehyde treatment and Sephacryl S-300 column fractionation. Rabbits immunized with WVAP produced IgG to both WVAP and wasp venom (WV), as measured by ELISA. IgG against WVAP was totally inhibitable by a mixture of WV and albumin, demonstrating both retention of native antigens and absence of new antigenic determinants in WVAP. IgG against WV in serum from patients receiving maintenance doses of WV immunotherapy was inhibited by WVAP. In summary, we have synthesized a soluble, high-molecular-weight copolymer of WV that retains the immunogenicity of native WV, contains no new antigenic determinants, and has potential value in the treatment of patients with WV anaphylaxis.

  18. Radioactive excretion in human milk following administration of /sup 99m/Tc macroaggregated albumin

    SciTech Connect

    Pittard, W.B.; Merkatz, R.; Fletcher, B.D.

    1982-08-01

    Albumin-tagged sodium pertechnetate (technetium) is routinely used in nuclear medicine for scanning procedures of the lung. The rate of excretion of this radionuclide into breast milk and the resultant potential radiation hazard to the nursing infant have received little attention. Therefore the milk from a nursing mother who required a lung scan because of suspected pulmonary emboli using an intravenous injection of 4 mCi of /sup 99m/Tc macroaggregated human serum albumin was monitored. Albumin tagging severely limited the entrance of technetium into her milk and the radioactivity of the milk returned to base line by 24 hours. A total of 2.02 muCi of technetium was measured in the 24-hour milk collection after technetium injection and 94% of this amount was excreted by 15.5 hours. This amount of technetium administered orally to a newborn would deliver a total body radiation dose of .3 mrad. Therefore, an infant would receive trivial doses of radiation if breast-feeding were resumed 15.5 hours after administration of the radionuclide to the mother and nursing can clearly be resumed safely 24 hours after injection.

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

    PubMed

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

    2015-02-01

    Chlorogenic acids and their derivatives are abundant in coffee and their composition changes between coffee species. Human serum albumin (HSA) interacts with this family of compounds with high affinity. We have studied by fluorescence spectroscopy the specific binding of HSA with eight compounds that belong to the coffee polyphenols family, four acids (caffeic acid, ferulic acid, 5-O-caffeoyl quinic acid, and 3,4-dimethoxycinnamic acid) and four lactones (3,4-O-dicaffeoyl-1,5-γ-quinide, 3-O-[3,4-(dimethoxy)cinnamoyl]-1,5-γ-quinide, 3,4-O-bis[3,4-(dimethoxy)cinnamoyl]-1,5-γ-quinide, and 1,3,4-O-tris[3,4-(dimethoxy)cinnamoyl]-1,5-γ-quinide), finding dissociation constants of the albumin-chlorogenic acids and albumin-quinides complexes in the micromolar range, between 2 and 30μM. Such values are comparable with those of the most powerful binders of albumin, and more favourable than the values obtained for the majority of drugs. Interestingly in the case of 3,4-O-dicaffeoyl-1,5-γ-quinide, we have observed the entrance of two ligand molecules in the same binding site, leading up to a first dissociation constant even in the hundred nanomolar range, which is to our knowledge the highest affinity ever observed for HSA and its ligands. The displacement of warfarin, a reference drug binding to HSA, by the quinide has also been demonstrated. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Highly sensitive quantitation of human serum albumin in clinical samples for hypoproteinemia using metal-enhanced fluorescence.

    PubMed

    Zhang, Yongxia; Mali, Buddha L; Aitken, Caroleann; Geddes, Chris D

    2013-01-01

    In this paper, we have explored metal-enhanced fluorescence (MEF) of the Human serum albumin indicators: Albumin Blue 580, Merocyanine 540 and Bromophenol Blue in close proximity to silver nano-particles, SiFs, from both buffered and clinical samples. The photostability of the Albumin Blue 580 is shown to be much more prolonged from the SiFs as compared to glass (a control sample), potentially allowing for longer detection times to further improve assay statistics. Our findings suggest the widespread use of nanoparticulate SiFs surfaces for the enhanced detection of HSA, particularly for Hypoproteinemia, where an enhanced assay performance at low protein abundance is required.

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

    PubMed

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

    2016-03-01

    Chemical cross-linking combined with mass spectrometry has proven useful for studying protein-protein interactions and protein structure, however the low density of cross-link data has so far precluded its use in determining structures de novo. Cross-linking density has been typically limited by the chemical selectivity of the standard cross-linking reagents that are commonly used for protein cross-linking. We have implemented the use of a heterobifunctional cross-linking reagent, sulfosuccinimidyl 4,4'-azipentanoate (sulfo-SDA), combining a traditional sulfo-N-hydroxysuccinimide (sulfo-NHS) ester and a UV photoactivatable diazirine group. This diazirine yields a highly reactive and promiscuous carbene species, the net result being a greatly increased number of cross-links compared with homobifunctional, NHS-based cross-linkers. We present a novel methodology that combines the use of this high density photo-cross-linking data with conformational space search to investigate the structure of human serum albumin domains, from purified samples, and in its native environment, human blood serum. Our approach is able to determine human serum albumin domain structures with good accuracy: root-mean-square deviation to crystal structure are 2.8/5.6/2.9 Å (purified samples) and 4.5/5.9/4.8Å (serum samples) for domains A/B/C for the first selected structure; 2.5/4.9/2.9 Å (purified samples) and 3.5/5.2/3.8 Å (serum samples) for the best out of top five selected structures. Our proof-of-concept study on human serum albumin demonstrates initial potential of our approach for determining the structures of more proteins in the complex biological contexts in which they function and which they may require for correct folding. Data are available via ProteomeXchange with identifier PXD001692. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. 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

  3. The expression and processing of two recombinant 2S albumins from soybean (Glycine max) in the yeast Pichia pastoris.

    PubMed

    Lin, Jing; Fido, Roger; Shewry, Peter; Archer, David B; Alcocer, Marcos J C

    2004-05-06

    Soybean seeds contain two 2S albumin storage proteins (AL1 and AL3) which may contribute to their industrial processing quality and allergenicity. We show that these proteins (AL1 and AL3) are well expressed by the methylotrophic yeast Pichia pastoris and that one of the secreted proteins (AL3) has a similar conformation and stability to that purified from soybean seeds. Further, we show that the subunits are post-translationally processed within the same loop region as the native protein but with some differences in the precise sites. This internal processing provides useful information on the endoproteolytic activity in P. pastoris. We also show that, similar to many plant allergens, the 2S albumins from soybean are stable to heat and chemical treatments.

  4. Evidence of recombination within human alpha-papillomavirus

    PubMed Central

    Angulo, Manuel; Carvajal-Rodríguez, Antonio

    2007-01-01

    Background Human papillomavirus (HPV) has a causal role in cervical cancer with almost half a million new cases occurring each year. Presence of the carcinogenic HPV is necessary for the development of the invasive carcinoma of the genital tract. Therefore, persistent infection with carcinogenic HPV causes virtually all cervical cancers. Some aspects of the molecular evolution of this virus, as the putative importance of recombination in its evolutionary history, are an opened current question. In addition, recombination could also be a significant issue nowadays since the frequency of co-infection with more than one HPV type is not a rare event and, thus, new recombinant types could be currently being generated. Results We have used human alpha-PV sequences from the public database at Los Alamos National Laboratory to report evidence that recombination may exist in this virus. A model-based population genetic approach was used to infer the recombination signal from the HPV DNA sequences grouped attending to phylogenetic and epidemiological information, as well as to clinical manifestations. Our results agree with recently published ones that use a different methodology to detect recombination associated to the gene L2. In addition, we have detected significant recombination signal in the genes E6, E7, L2 and L1 at different groups, and importantly within the high-risk type HPV16. The method used has recently been shown to be one of the most powerful and reliable procedures to detect the recombination signal. Conclusion We provide new support to the recent evidence of recombination in HPV. Additionally, we performed the recombination estimation assuming the best-fit model of nucleotide substitution and rate variation among sites, of the HPV DNA sequence sets. We found that the gene with recombination in most of the groups is L2 but the highest values were detected in L1 and E6. Gene E7 was recombinant only within the HPV16 type. The topic deserves further study

  5. Nanoparticle Albumin Bound Paclitaxel in the Treatment of Human Cancer: Nanodelivery Reaches Prime-Time?

    PubMed Central

    Cucinotto, Iole; Fiorillo, Lucia; Gualtieri, Simona; Arbitrio, Mariamena; Ciliberto, Domenico; Staropoli, Nicoletta; Grimaldi, Anna; Luce, Amalia; Tassone, Pierfrancesco; Caraglia, Michele; Tagliaferri, Pierosandro

    2013-01-01

    Nanoparticle albumin bound paclitaxel (nab-paclitaxel) represents the first nanotechnology-based drug in cancer treatment. We discuss the development of this innovative compound and report the recent changing-practice results in breast and pancreatic cancer. A ground-breaking finding is the demonstration that nab-paclitaxel can not only enhance the activity and reduce the toxicity of chromophore-diluted compound, but also exert activity in diseases considered refractory to taxane-based treatment. This is the first clinical demonstration of major activity of nanotechnologically modified drugs in the treatment of human neoplasms. PMID:23738077

  6. 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.

  7. 6-Nitro-L-tryptophan: a novel spectroscopic probe of trp aporepressor and human serum albumin.

    PubMed

    Phillips, R S; Marmorstein, R Q

    1988-04-01

    The binding of 6-nitro-L-tryptophan to trp aporepressor and human serum albumin has been examined by visible difference spectroscopy and circular dichroism. 6-Nitro-L-tryptophan, prepared by nitration of L-tryptophan with nitric acid in glacial acetic acid, exhibits a visible and near-uv absorption spectrum with lambda max at about 330 nm (epsilon = 7 X 10(3) M-1 cm-1) and a shoulder near 380 nm in H2O. In the presence of trp aporepressor, the visible absorption intensity is sharply diminished. Visible difference spectral titration data give KD = 1.27 X 10(-4) M and n = 0.95 per subunit at 25 degrees C. While 6-nitro-L-tryptophan exhibits no significant circular dichroism between 300 and 500 nm, the complex with trp aporepressor exhibits strong circular dichroism signals, with a negative maximum at 386 nm (delta epsilon = -7.5 M-1 cm-1) and a positive maximum at 310 nm (delta epsilon = +6 M-1 cm-1). Circular dichroism titration data give KD = 1.69 X 10(-4) M and n = 0.90 per subunit at 25 degrees C. The KD values determined spectroscopically are in excellent agreement with that determined by equilibrium dialysis, KD = 1.5 X 10(-4) M at 25 degrees C. In the presence of human serum albumin, the spectrum of 6-nitro-L-tryptophan exhibits a blue shift and an increase in absorption intensity; similar changes are observed in solvents of low dielectric contrast such as 80% aqueous dioxane. Visible difference spectral titration data give KD = 8.0 X 10(-5) M and n = 0.95 for human serum albumin. The complex of 6-nitro-L-tryptophan with human serum albumin exhibits a strong positive circular dichroism maximum at 380 nm (delta epsilon = +9.8 M-1 cm-1) with a shoulder at 310-320 nm. Circular dichroism titration data give KD = 6.4 X 10(-5) M and n = 0.83, in good agreement with the visible difference spectral results. Taken together, our results demonstrate the utility of 6-nitro-L-tryptophan as a spectroscopic probe for tryptophan-binding proteins.

  8. Radiation-induced chemical transformations of human serum albumin in aqueous solutions

    SciTech Connect

    Kondakova, N.V.; Ripa, N.V.; Kuznetsova, N.V.

    1994-11-01

    A rate constant of the reaction of human serum albumin (HSA) with OH radicals was determined using the method of competing scavengers. Changes in the amino acid composition and some biochemical properties of HSA on radiolysis in a borate buffer (pH 7.4) saturated with N{sub 2}O or O{sub 2} were correlated quantitatively. It was found that the yield of peptide bonds that lost the ability to be cleaved by trypsin is higher than the total yield of arginine and lysine residues in HSA. The possibility of accumulation of modified HSA in blood upon irradiation was considered.

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

    PubMed

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

    2016-02-08

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

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

    PubMed Central

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

    2016-01-01

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

  11. 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.

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

    PubMed Central

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

    2016-01-01

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

  13. 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.

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

    PubMed Central

    2013-01-01

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

  15. Extended in vivo half-life of human soluble complement receptor type 1 fused to a serum albumin-binding receptor.

    PubMed

    Makrides, S C; Nygren, P A; Andrews, B; Ford, P J; Evans, K S; Hayman, E G; Adari, H; Uhlén, M; Toth, C A

    1996-04-01

    A new approach has been used to extend the T(1/2) of human soluble complement receptor type 1 (sCR1) in rats. The albumin-binding domains B2A3 (BA) and B1A2B2A3 (BABA) from Streptococcal protein G were fused to the carboxyl terminus of sCR1, and the recombinant genes were expressed and amplified in Chinese hamster ovary cells. Western blot analysis and surface plasmon resonance measurements demonstrated the binding of rat serum albumin to both sCR1-BA and sCR1-BABA but not to sCR1. The in vitro complement inhibitory activity of the fusion proteins was shown to be similar to that of sCR1, indicating that neither the albumin-binding domains nor the presence of bovine serum albumin interfere with sCR1 function. Pharmacokinetic analysis showed that the T(1/2) of the distribution phase (T(1/2alpha)) was 3.3, 20.0 and 6.0 min for sCR1, sCR1-BA and sCR1-BABA, respectively. The T(1/2) of the elimination phase (T(1/2beta)) was 103, 297 and 170 min for sCR1, sCR1-BA and sCR1-BABA, respectively. The plasma elimination of sCR1-BA and sCR1-BABA was significantly (P < .05) prolonged as compared to sCR1. The proteins showed similar tissue distribution; at 4-hr postdosing, the highest levels of 125I-radioactivity per gram of tissue were localized in the urine, blood, liver, stomach, and small intestine.

  16. IgG autoantibody to human serum albumin studied by the ELISA-technique.

    PubMed

    Lindström, P; Wager, O

    1978-01-01

    ELISA was applied for analysis of the HSA-human IgG autoantibody system responsible for the immunoelectrophoretic 'Tailing Albumin' (TA) phenomenon induced in most of the TA patients by prolonged nitrofurantoin therapy. Both hyperimmune porcine anti-HSA and autoimmune human anti-HSA antibodies of the IgG class were detectable by ELISA. The presence of autologous or added HSA had some inhibitory effect upon the detectability of the anti-HSA antibodies. Partial elimination of the autologous HSA by sucrose gradient ultracentrifugation or salt precipitation increased or unmasked the anti-HSA activity of some TA sera. The sensitivity of the ELISA as detector of the anti-HSA autoantibodies of whole human sera was roughly equal to that of the immunoelectrophoretic TA phenomenon. The analogy of the anti-HSA autoantibodies and the rheumatoid factors and the theoretical interest of both of them is stressed.

  17. Preparation and analysis of peptide fragments produced by pepsin hydrolysis of human plasma albumin and their relationship to its structure

    PubMed Central

    Franglen, G.; Swaniker, G. R. E.

    1968-01-01

    Human plasma albumin was prepared and subjected to proteolysis by pepsin at pH2·45 at 25° for 10min. with albumin/pepsin ratio 3000:1. Five peptide fragments were detected in the proteolysate by means of zone electrophoresis and gel filtration; these were separated and purified. Molecular weights, amino acid composition and disulphide bond content of the purified fragments were determined. The results show that a high proportion of the polypeptide chain of albumin appears to have a low cystine content, and at low pH values the molecule would be expected to have a considerable degree of freedom in its structure in these regions of the chain. A tripartite model for the structure of plasma albumin is proposed. PMID:4876098

  18. Spectroscopic investigations of the binding interaction of a new indanedione derivative with human and bovine serum albumins.

    PubMed

    Stan, Dana; Matei, Iulia; Mihailescu, Carmen; Savin, Mihaela; Hillebrand, Mihaela; Baciu, Ion; Matache, Mihaela

    2009-04-24

    Binding of a newly synthesized indanedione derivative, 2-(2-hydroxy-3-ethoxybenzylidene)-1,3-indanedione (HEBID), to human and bovine serum albumins (HSA and BSA), under simulated physiological conditions was monitored by fluorescence spectroscopy. The binding parameters (binding constants and number of binding sites) and quenching constants were determined according to literature models. The quenching mechanism was assigned to a Förster non-radiative energy transfer due to the HEBID-SA complex formation. A slightly increased affinity of HEBID for HSA was found, while the number of binding sites is approximately one for both albumins. The molecular distance between donor (albumin) and acceptor (HEBID) and the energy transfer efficiency were estimated, in the view of Förster's theory. The effect of HEBID on the protein conformation was investigated using circular dichroism and synchronous fluorescence spectroscopies. The results revealed partial unfolding in the albumins upon interaction, as well as changes in the local polarity around the tryptophan residues.

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

    PubMed

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

    2006-01-01

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

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

    SciTech Connect

    Kielmas, Martyna; Szewczuk, Zbigniew; Stefanowicz, Piotr

    2013-09-13

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

  1. 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.

  2. Molten globule intermediates of human serum albumin in low concentration of urea.

    PubMed

    Muralidhara, B K; Prakash, V

    2002-10-01

    Interaction of non-electrolytes such as urea with proteins especially at lower concentrations is opening-up newer concepts in the understanding of protein stability and folding in proteomics. In this study, the secondary and tertiary structural characteristics and thermal stability of human serum albumin at lower concentrations of urea have been monitored. The protein attains a molten globule like structure at concentration urea below 2 M. This structural state also shows an increase in the alpha-helical content as compared to the native state. At concentrations of urea above 2 M, human serum albumin starts unfolding, resulting in a three-state transition with two mid points of transitions at around 4 M and 7 M urea concentrations. The characteristics of the partially folded intermediates are discussed with respect to the three component system analyses. Preferential hydration dominates over preferential interaction at lower concentration of urea (up to 2.5 M) and at higher concentration, the preferential interaction overtakes preferential hydration in a competitive manner. Formation of structural intermediates at lower concentration of urea is hypothesized as a general phenomenon in proteins and fits in with the observation with preferential interaction parameters by Timasheff and co-workers in the case of lysozyme and ribonuclease at different pH values.

  3. Solution structure and stability against digestion of rproBnIb, a recombinant 2S albumin from rapeseed: relationship to its allergenic properties.

    PubMed

    Pantoja-Uceda, David; Palomares, Oscar; Bruix, Marta; Villalba, Mayte; Rodríguez, Rosalía; Rico, Manuel; Santoro, Jorge

    2004-12-28

    NMR spectroscopy has been used to determine the solution structure of the precursor form of the recombinant napin BnIb, rproBnIb, a 2S albumin, 109-residue protein from the seeds of Brassica napus. More than 90% of the side-chain proton resonances were unambiguously assigned from the analysis of two-dimensional correlation (COSY), total correlation (TOCSY), and nuclear Overhauser effect (NOESY) spectra. The final structures were computed by using restrained molecular dynamics on the basis of 1316 upper-limit distance constraints derived from NOE cross-correlation intensities. The computed structures exhibited a root-mean-square deviation (RMSD) radius of 0.66 A for the backbone and 1.16 A for the side-chain heavy atoms of the structural core. The resulting structure consists of five amphipathic helices arranged in a right-handed super helix, a folding motif found in other proteins of the prolamin superfamily. As in the case of the mature protein, the recombinant precursor behaves as a plant food allergen. To trace out the origin and characteristics of its allergenic properties, rproBnIb was assayed against simulated gastric fluid and found to be very resistant to proteolysis. Also, heat treatment of the protein followed up to 85 degrees C by circular dichroism showed a very limited unfolding, which was recovered after cooling to 20 degrees C, indicating a high thermal stability. These results suggest that rproBnIb, as other 2S albumins, may be able to reach the gut immune system intact. A comparison of the putative epitopes against IgE antibodies of the three members of the prolamine family [2S albumins, nonspecific lipid transfer proteins (nsLTPs), and alpha-amylase/trypsin inhibitors] indicates that there are not common surfaces of interaction with IgE. Though the epitopes appear to be located in different regions of the proteins, they do comply with the requirements of being solvent-exposed and flexible.

  4. 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.

  5. Depletion of albumin and immunoglobulin G from human serum using epitope-imprinted polymers as artificial antibodies.

    PubMed

    Yang, Hsueh-Hui; Lu, Kuo-Hao; Lin, Yee-Fung; Tsai, Sheng-Hung; Chakraborty, Subrata; Zhai, Wei-Jun; Tai, Dar-Fu

    2013-07-01

    Serum is a readily available source for noninvasive studies in clinical research, but it contains abundant proteins such as albumin and immunoglobulin G that can hinder the presence of low-abundant proteins as well as decrease sample loading capacity of analytical methods. Therefore, depletion of these two proteins is required to observe low-abundance serum proteins. Molecularly imprinted polymers are template-induced artificial antibodies with the ability to recognize and selectively bind the target molecule. In this study, artificial albumin and immunoglobulin G antibodies were developed by using two epitopes of human serum albumin and immunoglobulin G as templates. Acrylic acid, acrylamide, and N-acryl tyramine were the corresponding monomers; N,N'-ethylene bisacrylamide served as a cross-linker, and cellulosic fibers were used as a supporting matrix. The adsorption capacity of these artificial antibodies was 15.2 mg, 10 mg, and 15 μL per gram for human serum albumin, immunoglobulin G, and human serum, respectively. The dissociation constant (Kd ) of these artificial antibodies toward the human serum albumin and immunoglobulin G was 1 μM and 0.6 μM, respectively. The biomimetic properties of these artificial antibodies, coupled with their economical and rapid production, high specificity and their reusability, make them attractive for protein separation and analysis.

  6. Spectrofluorimetric determination of human serum albumin using terbium-danofloxacin probe.

    PubMed

    Ramezani, Amir M; Manzoori, Jamshid L; Amjadi, Mohammad; Jouyban, Abolghasem

    2012-01-01

    A spectrofluorimetric method is proposed for the determination of human serum albumin (HSA) and bovine serum albumin (BSA) using terbium-danofloxacin (Tb(3+)-Dano) as a fluorescent probe. These proteins remarkably enhance the fluorescence intensity of the Tb(3+)-Dano complex at 545 nm, and the enhanced fluorescence intensity of Tb(3+)-Dano is proportional to the concentration of proteins (HSA and BSA). Optimum conditions for the determination of HSA were investigated and found that the maximum response was observed at: pH = 7.8, [Tb(3+)] = 8.5 × 10(-5) mol L(-1), [Dano] = 1.5 × 10(-4) mol L(-1). The calibration graphs for standard solutions of BSA, HSA, and plasma samples of HSA were linear in the range of 0.2 × 10(-6) - 1.3 × 10(-6) mol L(-1), 0.2 × 10(-6) - 1.4 × 10(-6) mol L(-1), and 0.2 × 10(-6) - 1 × 10(-6) mol L(-1), respectively. The detection limits (S/N = 3) for BSA, HSA, and plasma sample of HSA were 8.7 × 10(-8) mol L(-1), 6.2 × 10(-8) mol L(-1), and 8.1 × 10(-8) mol L(-1), respectively. The applicability of the method was checked using a number of real biological plasma samples and was compared with the UV spectrometric reference method. The results was showed that the method could be regarded as a simple, practical, and sensitive alternative method for determination of albumin in biological samples.

  7. Spectrofluorimetric Determination of Human Serum Albumin Using Terbium-Danofloxacin Probe

    PubMed Central

    Ramezani, Amir M.; Manzoori, Jamshid L.; Amjadi, Mohammad; Jouyban, Abolghasem

    2012-01-01

    A spectrofluorimetric method is proposed for the determination of human serum albumin (HSA) and bovine serum albumin (BSA) using terbium-danofloxacin (Tb3+-Dano) as a fluorescent probe. These proteins remarkably enhance the fluorescence intensity of the Tb3+-Dano complex at 545 nm, and the enhanced fluorescence intensity of Tb3+-Dano is proportional to the concentration of proteins (HSA and BSA). Optimum conditions for the determination of HSA were investigated and found that the maximum response was observed at: pH = 7.8, [Tb3+] = 8.5 × 10−5 mol L−1, [Dano] = 1.5 × 10−4 mol L−1. The calibration graphs for standard solutions of BSA, HSA, and plasma samples of HSA were linear in the range of 0.2 × 10−6 − 1.3 × 10−6 mol L−1, 0.2 × 10−6 − 1.4 × 10−6 mol L−1, and 0.2 × 10−6 − 1 × 10−6 mol L−1, respectively. The detection limits (S/N = 3) for BSA, HSA, and plasma sample of HSA were 8.7 × 10−8 mol L−1, 6.2 × 10−8 mol L−1, and 8.1 × 10−8 mol L−1, respectively. The applicability of the method was checked using a number of real biological plasma samples and was compared with the UV spectrometric reference method. The results was showed that the method could be regarded as a simple, practical, and sensitive alternative method for determination of albumin in biological samples. PMID:22645474

  8. Hexamethylene diisocyanate (HDI) vapor reactivity with glutathione and subsequent transfer to human albumin

    PubMed Central

    Wisnewski, Adam V.; Mhike, Morgen; Hettick, Justin M.; Liu, Jian; Siegel, Paul D.

    2012-01-01

    INTRODUCTION Airway fluid glutathione (GSH) reactivity with inhaled vapors of diisocyanate, a common occupational allergen, is postulated to be a key step in exposure-induced asthma pathogenesis. METHODS A mixed (vapor/liquid) phase exposure system was used to model the in vivo reactivity of inhaled HDI vapor with GSH in the airway fluid. HDI-GSH reaction products, and their capacity to transfer HDI to human albumin, were characterized through mass spectrometry and serologic assays, using HDI-specific polyclonal rabbit serum. RESULTS HDI vapor exposure of 10 mM GSH solutions resulted in primarily S-linked, bis(GSH)-HDI reaction products. In contrast, lower GSH concentrations (100 μM) resulted in mainly mono(GSH)-HDI conjugates, with varying degrees of HDI hydrolysis, dimerization and/or intra-molecular cyclization, depending upon the presence/absence of H2PO4-/HPO42- and Na+/Cl- ions. The ion composition and GSH concentration of the fluid phase, during HDI vapor exposure, strongly influenced the transfer of HDI from GSH to albumin, as did the pH and duration of the carbamoylating reaction. When carbamoylation was performed overnight at pH 7, twenty-five of albumin's lysines were identified as potential sites of conjugation with partially hydrolyzed HDI. When carbamoylation was performed at pH 9, more rapid (within 3 hours) and extensive modification was observed, including additional lysine sites, intra-molecular cross-linkage with HDI, and novel HDI-GSH conjugation. CONCLUSIONS The data define potential mechanisms by which the levels of GSH, H2PO4-4/HPO42-, and/or other ions (e.g. H+/OH-, Na+, Cl-) affect the reactivity of HDI vapor with self-molecules in solution (e.g. airway fluid), and thus, might influence the clinical response to HDI respiratory tract exposure. PMID:23178851

  9. 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.

  10. The effects of recombinant human growth hormone and intradialytic parenteral nutrition in malnourished hemodialysis patients.

    PubMed

    Schulman, G; Wingard, R L; Hutchison, R L; Lawrence, P; Hakim, R M

    1993-05-01

    Malnutrition in hemodialysis patients is associated with increased morbidity and mortality. The use of intradialytic parenteral nutrition (IDPN) to improve nutritional parameters has been shown to be of limited benefit in most studies. We studied the use of recombinant human growth hormone (rHuGH) in potentiating the effects of IDPN in seven hemodialysis patients dialyzed with a Kt/V of 1.03 +/- 0.11 (mean +/- SEM), but with evidence of malnutrition: albumin, 3.2 +/- 0.18 g/dL; transferrin, 215 +/- 30 mg/dL; insulin-like growth factor-1 (IGF-1), 115 +/- 19 ng/mL, protein catabolic rate (PCR), 0.70 +/- 0.05 g/kg/d; and weight, 12.3% +/- 4.0% below ideal body weight. During 6 weeks of IDPN, resulting in an additional 18 +/- 4 kcal and 0.69 +/- 0.03 g of protein/kg body weight per dialysis session, albumin concentration increased to 3.5 +/- 0.14 g/dL (compared with baseline, P = NS), transferrin increased to 279 +/- 36 mg/dL (P < 0.002), IGF-1 increased to 152 +/- 32 ng/mL (P = NS), and PCR increased to 0.81 +/- 0.04 g/kg/d (P = NS). During the next 6 weeks, IDPN administration was continued and rHuGH, at a dose of 5 mg subcutaneously during each dialysis, was added to the regimen. This resulted in an increase in albumin concentration to 3.8 +/- 0.08 g/dL (P < or = 0.04 compared with end of IDPN phase), an increase in transferrin to 298 +/- 41 mg/dL (P = NS compared with end of IDPN phase), and an increase in IGF-1 to 212 +/- 45 ng/mL (P = 0.05 compared with end of IDPN phase).(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Recombinant human gamma interferon inhibits simian malaria.

    PubMed Central

    Maheshwari, R K; Czarniecki, C W; Dutta, G P; Puri, S K; Dhawan, B N; Friedman, R M

    1986-01-01

    Prophylactic treatment with 0.1 mg of human gamma interferon per kg (body weight) per day completely suppressed experimental infection with Plasmodium cynomolgi B sporozoites in rhesus monkeys. Treatment with lower doses partially suppressed this infection. Prophylactic treatment with human gamma interferon, however, had no protective effect against trophozoite-induced infection, suggesting that the interferon effect was limited to the exoerythrocytic stage of parasitic development. PMID:3091507

  12. Human serum albumin adsorption on TiO2 from single protein solutions and from plasma.

    PubMed

    Sousa, S R; Moradas-Ferreira, P; Saramago, B; Melo, L Viseu; Barbosa, M A

    2004-10-26

    In the present work, the adsorption of human serum albumin (HSA) on commercially pure titanium with a titanium oxide layer formed in a H(2)O(2) solution (TiO(2) cp) and on TiO(2) sputtered on Si (TiO(2) sp) was analyzed. Adsorption isotherms, kinetic studies, and work of adhesion determinations were carried out. HSA exchangeability was also evaluated. Surface characterization was performed by atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and wettability studies. The two TiO(2) surfaces have very distinct roughnesses, the TiO(2) sp having a mean R(a) value 14 times smaller than the one of TiO(2) cp. XPS analysis revealed consistent peaks representative of TiO(2) on sputtered samples as well as on Ti cp substrate after 48 h of H(2)O(2) immersion. Nitrogen was observed as soon as protein was present, while sulfur, present in disulfide bonds in HSA, was observed for concentrations of protein higher than 0.30 mg/mL. The work of adhesion was determined from contact angle measurements. As expected from the surface free energy values, the work of adhesion of HSA solution is higher for the TiO(2) cp substrate, the more hydrophilic one, and lower for the TiO(2) sp substrate, the more hydrophobic one. The work of adhesion between plasma and the substrates assumed even higher values for the TiO(2) cp surface, indicating a greater interaction between the surface and the complex protein solutions. Adsorption studies by radiolabeling of albumin ((125)I-HSA) suggest that rapid HSA adsorption takes place on both surfaces, reaching a maximum value after approximately 60 min of incubation. For the higher HSA concentrations in solution, a multilayer coverage was observed on both substrates. After the adsorption step from single HSA solutions, the exchangeability of adsorbed HSA molecules by HSA in solution was evaluated. The HSA molecules adsorbed on TiO(2) sp seem to be more easily exchanged by HSA itself than those adsorbed on TiO(2) cp after 24 h. In

  13. Recombinant genetic libraries and human monoclonal antibodies.

    PubMed

    Adams, Jarrett J; Nelson, Bryce; Sidhu, Sachdev S

    2014-01-01

    In order to comprehensively manipulate the human proteome we require a vast repertoire of pharmacological reagents. To address these needs we have developed repertoires of synthetic antibodies by phage display, where diversified oligonucleotides are used to modify the complementarity-determining regions (CDRs) of a human antigen-binding fragment (Fab) scaffold. As diversity is produced outside the confines of the mammalian immune system, synthetic antibody libraries allow us to bypass several limitations of hybridoma technology while improving the experimental parameters under which pharmacological reagents are produced. Here we describe the methodologies used to produce synthetic antibody libraries from a single human framework with diversity restricted to four CDRs. These synthetic repertoires can be extremely functional as they produce highly selective, high affinity Fabs to the majority of soluble human antigens. Finally we describe selection methodologies that allow us to overcome immuno-dominance in our selections to target a variety of epitopes per antigen. Together these methodologies allow us to produce human monoclonal antibodies to manipulate the human proteome.

  14. Human serum albumin nanoparticles for efficient delivery of Cu, Zn superoxide dismutase gene

    PubMed Central

    Mo, Yun; Barnett, Micheal E.; Takemoto, Dolores; Davidson, Harriet

    2007-01-01

    Purpose To assess the potential of human serum albumin nanoparticles (HSA NP) as a nonviral vector for ocular delivery of Cu, Zn superoxide dismutase (SOD1) gene. Methods Cu, Zn superoxide dismutase (SOD1) gene-encapsulated nanoparticles (NP) were developed using human serum albumin (HSA), an endogenous protein, by a desolvation-crosslinking method. The pSOD-loaded HSA NP was evaluated for in vitro release characteristics, stability against DNase I and vitreous humor degradation, cytotoxicity, cellular uptake mechanisms, in vitro transfection efficiency, and in vivo gene expression. In vitro studies employed cultured human retinal pigment epithelial (ARPE-19) cells and in vivo studies employed a mouse model. For cell uptake analysis, fluorescein isothiocyanate (FITC)-labeled human serum albumin (HSA) was used. Results Plasmid containing SOD1 gene was encapsulated in HSA by a desolvation-crosslinking method. Gene-loaded HSA NP has a mean size of 120 nm, zeta potential of -44 mV, and plasmid encapsulation efficiency of 84%. At high crosslinking degree, HSA NP sustained the in vitro release of plasmid over 6 days, and stabilized plasmid DNA against DNase I and vitreous humor degradation. No cytotoxicity was observed in ARPE 19 cells treated with blank HSA NP at concentrations up to 5 mg/ml for 96 h. Cellular uptake of HSA NP was via receptor-mediated endocytosis that involves primarily caveolae-pathways. Confocal analysis indicated rapid endo/lysosomal escape of HSA NP. Further, confocal studies indicated that HSA readily enters the cell nucleus. In vitro, pSOD-HSA NP resulted in more than 80% transfection efficiency in ARPE-19 cells, which was 5 fold higher than Lipofectamine. HSA NP-transfected cells exhibited enhanced SOD1 activity that was 5 fold higher than untreated cells, indicating the overexpression of the functional gene. Intravitreal injection of HSA NP to the mouse eye at a dose of 130 ng of plasmid produced detectable level of fusion protein expression at

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

    SciTech Connect

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

    2011-03-04

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

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

    PubMed Central

    Traglia, German Matias; Quinn, Brettni; Schramm, Sareda T. J.; Soler-Bistue, Alfonso

    2016-01-01

    The increasing frequency of bacteria showing antimicrobial resistance (AMR) raises the menace of entering into a postantibiotic era. Horizontal gene transfer (HGT) is one of the prime reasons for AMR acquisition. Acinetobacter baumannii is a nosocomial pathogen with outstanding abilities to survive in the hospital environment and to acquire resistance determinants. Its capacity to incorporate exogenous DNA is a major source of AMR genes; however, few studies have addressed this subject. The transformation machinery as well as the factors that induce natural competence in A. baumannii are unknown. In this study, we demonstrate that naturally competent strain A118 increases its natural transformation frequency upon the addition of Ca2+or albumin. We show that comEA and pilQ are involved in this process since their expression levels are increased upon the addition of these compounds. An unspecific protein, like casein, does not reproduce this effect, showing that albumin's effect is specific. Our work describes the first specific inducers of natural competence in A. baumannii. Overall, our results suggest that the main protein in blood enhances HGT in A. baumannii, contributing to the increase of AMR in this threatening human pathogen. PMID:27270286

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

    PubMed

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

    2016-08-01

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

  18. Quantitation of Oxidative Modifications of Commercial Human Albumin for Clinical Use.

    PubMed

    Takahashi, Teppei; Terada, Tomoyoshi; Arikawa, Hajime; Kizaki, Kazuha; Terawaki, Hiroyuki; Imai, Hajime; Itoh, Yoshinori; Era, Seiichi

    2016-01-01

    We investigated the quantitation of oxidative chemical modifications, such as thiol oxidation and carbonylation, in medical-grade human serum albumin (HSA) preparations, in comparison with those of healthy and diseased subjects. Four kinds of HSA products were obtained from three major suppliers in Japan. Eight male collegiate students and six healthy male volunteers were recruited as the young (21.6 years) and older (57.2 years) groups, respectively. Four male stable patients (64.3 years) treated with regular hemodialysis (HD) also enrolled in this study. Quantitative analyses for thiol oxidation and carbonylation were performed using HPLC and spectroscopic methods, respectively. Structural characterization was further investigated by differential scanning calorimetry (DSC) and circular dichroism (CD) spectropolarimetry. Significantly larger amounts of thiol-oxidized and carbonylated HSA products were observed than HSA obtained from healthy subjects. In the structural characterization, the midpoint temperature of the denaturation curve (Tm) analyzed by DSC was relatively high, and may have been caused by the added albumin-specific stabilizers, and CD-resolved secondary structure showed that HSA products had a helical conformation. Commercial HSA products for clinical use have a more thermally stable state and remain in a helix-rich structure, even though their specific amino acids (mainly Cys and Lys residues) are oxidatively modified.

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

    PubMed

    Keswani, Neelam; Choudhary, Sinjan; Kishore, Nand

    2010-07-01

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

  20. Inhibitory effect of human serum albumin on Cu-induced Aβ(40) aggregation and toxicity.

    PubMed

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

    2015-11-15

    It has been suggested that the aggregation and cytotoxicity of amyloid-β (Aβ) peptide with transition-metal ions in neuronal cells is involved in the development and progression of Alzheimer's disease (AD). As the most abundant protein in blood plasma and in cerebrospinal fluid, human serum albumin (HSA) can bind Aβ in vivo and subsequently inhibit Aβ fibril growth. However, the roles of albumin in Cu-induced Aβ aggregation and toxicity, and its potential biological relevance to AD therapy, were not stressed enough. Here, we showed that HSA was capable of binding Cu (I) with much higher affinity than Aβ, competitively inhibiting the interaction of Aβ and Cu ions. In the presence of biological reducing agent ascorbate, HSA inhibited Cu (II)/Cu (I)-mediated Aβ40 aggregation, reactive oxygen species production, and neurotoxicity. However, in the absence of Cu (II)/Cu (I), HSA could not effectively inhibit Aβ40 aggregation and neurotoxicity at 24 h (or less) incubation time, but decreased Aβ40 aggregation at much longer incubation (120 h). Our data suggested that through competitively decreasing Cu-Aβ interaction, HSA could effectively inhibit Cu (II)/Cu (I)-induced Aβ40 aggregation and neurotoxicity, and play important roles in regulating redox balance as well as metal homeostasis in AD prevention and therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2015-10-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-09-01

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

  4. 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.

  5. Recombination in the Human Pseudoautosomal Region PAR1

    PubMed Central

    Hinch, Anjali G.; Altemose, Nicolas; Noor, Nudrat; Donnelly, Peter; Myers, Simon R.

    2014-01-01

    The pseudoautosomal region (PAR) is a short region of homology between the mammalian X and Y chromosomes, which has undergone rapid evolution. A crossover in the PAR is essential for the proper disjunction of X and Y chromosomes in male meiosis, and PAR deletion results in male sterility. This leads the human PAR with the obligatory crossover, PAR1, to having an exceptionally high male crossover rate, which is 17-fold higher than the genome-wide average. However, the mechanism by which this obligatory crossover occurs remains unknown, as does the fine-scale positioning of crossovers across this region. Recent research in mice has suggested that crossovers in PAR may be mediated independently of the protein PRDM9, which localises virtually all crossovers in the autosomes. To investigate recombination in this region, we construct the most fine-scale genetic map containing directly observed crossovers to date using African-American pedigrees. We leverage recombination rates inferred from the breakdown of linkage disequilibrium in human populations and investigate the signatures of DNA evolution due to recombination. Further, we identify direct PRDM9 binding sites using ChIP-seq in human cells. Using these independent lines of evidence, we show that, in contrast with mouse, PRDM9 does localise peaks of recombination in the human PAR1. We find that recombination is a far more rapid and intense driver of sequence evolution in PAR1 than it is on the autosomes. We also show that PAR1 hotspot activities differ significantly among human populations. Finally, we find evidence that PAR1 hotspot positions have changed between human and chimpanzee, with no evidence of sharing among the hottest hotspots. We anticipate that the genetic maps built and validated in this work will aid research on this vital and fascinating region of the genome. PMID:25033397

  6. Recombination in the human Pseudoautosomal region PAR1.

    PubMed

    Hinch, Anjali G; Altemose, Nicolas; Noor, Nudrat; Donnelly, Peter; Myers, Simon R

    2014-07-01

    The pseudoautosomal region (PAR) is a short region of homology between the mammalian X and Y chromosomes, which has undergone rapid evolution. A crossover in the PAR is essential for the proper disjunction of X and Y chromosomes in male meiosis, and PAR deletion results in male sterility. This leads the human PAR with the obligatory crossover, PAR1, to having an exceptionally high male crossover rate, which is 17-fold higher than the genome-wide average. However, the mechanism by which this obligatory crossover occurs remains unknown, as does the fine-scale positioning of crossovers across this region. Recent research in mice has suggested that crossovers in PAR may be mediated independently of the protein PRDM9, which localises virtually all crossovers in the autosomes. To investigate recombination in this region, we construct the most fine-scale genetic map containing directly observed crossovers to date using African-American pedigrees. We leverage recombination rates inferred from the breakdown of linkage disequilibrium in human populations and investigate the signatures of DNA evolution due to recombination. Further, we identify direct PRDM9 binding sites using ChIP-seq in human cells. Using these independent lines of evidence, we show that, in contrast with mouse, PRDM9 does localise peaks of recombination in the human PAR1. We find that recombination is a far more rapid and intense driver of sequence evolution in PAR1 than it is on the autosomes. We also show that PAR1 hotspot activities differ significantly among human populations. Finally, we find evidence that PAR1 hotspot positions have changed between human and chimpanzee, with no evidence of sharing among the hottest hotspots. We anticipate that the genetic maps built and validated in this work will aid research on this vital and fascinating region of the genome.

  7. Porphyrin conjugated with serum albumins and monoclonal antibodies boosts efficiency in targeted destruction of human bladder cancer cells.

    PubMed

    Pereira, Patrícia M R; Carvalho, José J; Silva, Sandrina; Cavaleiro, José A S; Schneider, Rudolf J; Fernandes, Rosa; Tomé, João P C

    2014-03-21

    The synthesis of a novel PS conjugated with bovine and human serum albumin (BSA and HSA) and a monoclonal antibody anti-CD104 is reported, as well as their biological potential against the human bladder cancer cell line UM-UC-3. No photodynamic effect was detected when the non-conjugated porphyrin was used. Yet, when it was coupled covalently with the mAb anti-CD104, BSA and HSA, the resulting photosensitizer conjugates demonstrated high efficacy in destroying the cancer cells, the mAb anti-CD104 efficacy overruling the albumins.

  8. Limited human infection due to recombinant raccoon pox virus

    USGS Publications Warehouse

    Rocke, T.E.; Dein, F.J.; Fuchsberger, M.; Fox, B.C.; Stinchcomb, D.T.; Osorio, J.G.

    2004-01-01

    A laboratory accident resulted in human exposure to a recombinant raccoon poxvirus (RCN) developed as a vaccine vector for antigens of Yersinia pestis for protection of wild rodents (and other animals) against plague. Within 9 days, the patient developed a small blister that healed within 4 weeks. Raccoon poxvirus was cultured from the lesion, and the patient developed antibody to plague antigen (F1) and RCN. This is the first documented case of human exposure to RCN.

  9. Molecular characteristics of methylglyoxal-modified bovine and human serum albumins. Comparison with glucose-derived advanced glycation endproduct-modified serum albumins.

    PubMed

    Westwood, M E; Thornalley, P J

    1995-07-01

    The amino acid modification, gel filtration chromatographic, and electrophoretic characteristics of bovine and human serum albumins irreversibly modified by methylglyoxal (MG-SA) and by glucose-derived advanced glycation endproducts (AGE-SA) were investigated. Methylglyoxal selectively modified arginine residues at low concentration (1 mM); at high methylglyoxal concentration (100 mM), the extent of arginine modification increased and lysine residues were also modified. Both arginine and lysine residues were modified in AGE-SA. Analytical gel filtration HPLC of serum albumin derivatives suggested that the proportion of dimers and oligomers increased with modification in both low and highly modified MG-SA and AGE-SA derivatives relative to unmodified serum albumins. In SDS-PAGE analysis, dimers and oligomers of low-modified MG-SA were dissociated into monomers, but not in highly modified MG-SA. MG-SA had increased anodic electrophoretic mobility under nondenaturing conditions at pH 8.6, indicating an increased net negative charge, which increased with extent of modification; highly modified MG-SA and AGE-SA had similar high electrophoretic mobilities. MG-SA derivatives were fluorescent: the fluorescence was characteristic of the arginine-derived imidazolone N delta-(5-methyl-4-imidazolon-2-yl)ornithine, but other fluorophores were also present. AGE-SA had similar fluorescence, attributed, in part, to glucose-derived imidazolones. AGE formed from glucose-modified proteins and AGE-like compounds formed from methylglyoxal-modified proteins may both be signals for recognition and degradation of senescent macromolecules.

  10. Disruption of human serum albumin fibrils by a static electric field

    NASA Astrophysics Data System (ADS)

    Pandey, Nitin K.; Mitra, Subhadeep; Chakraborty, Monojit; Ghosh, Sudeshna; Sen, Shubhatam; Dasgupta, Swagata; DasGupta, Sunando

    2014-07-01

    Protein self-association leads to toxic filamentous plaques believed to be the key reason behind several neurodegenerative diseases. Stabilization of the fibrillar architecture is guided by several forces; primarily electrostatic, hydrophobic, and the extent of hydrogen bonding. In this study, we experimentally demonstrate, for the first time, the effect of varying static electric fields on preformed fibrils of human serum albumin (HSA) in an electrowetting on dielectric type setup using spectroscopic and microscopic techniques. We have found appreciable and continuous reduction in the β-sheet content of HSA fibrils up to an applied field of ˜8 × 106 V m-1. The observed disruption of the fibrils has been attributed to the combined effects of electrostatic interactions and electrowetting phenomena. Kinetic studies indicate a prior intermediate formation followed by disruption of HSA fibrils after application of the electric field.

  11. 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.

  12. A Comparative Interaction between Copper Ions with Alzheimer's β Amyloid Peptide and Human Serum Albumin.

    PubMed

    Behbehani, G Rezaei; Barzegar, L; Mohebbian, M; Saboury, A A

    2012-01-01

    The interaction of Cu(2+) with the first 16 residues of the Alzheimer's amyliod β peptide, Aβ(1-16), and human serum albumin (HSA) were studied in vitro by isothermal titration calorimetry at pH 7.2 and 310 K in aqueous solution. The solvation parameters recovered from the extended solvation model indicate that HSA is involved in the transport of copper ion. Complexes between Aβ(1-16) and copper ions have been proposed to be an aberrant interaction in the development of Alzheimer's disease, where Cu(2+) is involved in Aβ(1-16) aggregation. The indexes of stability indicate that HSA removed Cu(2+) from Aβ(1-16), rapidly, decreased Cu-induced aggregation of Aβ(1-16), and reduced the toxicity of Aβ(1-16) + Cu(2+) significantly.

  13. A Comparative Interaction between Copper Ions with Alzheimer's β Amyloid Peptide and Human Serum Albumin

    PubMed Central

    Behbehani, G. Rezaei; Barzegar, L.; Mohebbian, M.; Saboury, A. A.

    2012-01-01

    The interaction of Cu2+ with the first 16 residues of the Alzheimer's amyliod β peptide, Aβ(1–16), and human serum albumin (HSA) were studied in vitro by isothermal titration calorimetry at pH 7.2 and 310 K in aqueous solution. The solvation parameters recovered from the extended solvation model indicate that HSA is involved in the transport of copper ion. Complexes between Aβ(1–16) and copper ions have been proposed to be an aberrant interaction in the development of Alzheimer's disease, where Cu2+ is involved in Aβ(1–16) aggregation. The indexes of stability indicate that HSA removed Cu2+ from Aβ(1–16), rapidly, decreased Cu-induced aggregation of Aβ(1–16), and reduced the toxicity of Aβ(1–16) + Cu2+ significantly. PMID:22844264

  14. DNA-duplex linker for AFM-SELEX of DNA aptamer against human serum albumin.

    PubMed

    Takenaka, Musashi; Okumura, Yuzo; Amino, Tomokazu; Miyachi, Yusuke; Ogino, Chiaki; Kondo, Akihiko

    2017-02-15

    DNA-duplex interactions in thymines and adenins are used as a linker for the novel methodology of Atomic Force Microscope-Systematic Evolution of Ligands by EXpotential enrichment (AFM-SELEX). This study used the hydrogen bonds in 10 mer of both thymines (T10) and adenines (A10). Initially, the interactive force in T10-A10 was measured by AFM, which returned an average interactive force of approximately 350pN. Based on this result, DNA aptamers against human serum albumin could be selected in the 4th round, and 15 different clones could be sequenced. The lowest dissociation constant of the selected aptamer was identified via surface plasmon resonance, and it proved to be identical to that of the commercial aptamer. Therefore, specific hydrogen bonds in DNA can be useful linkers for AFM-SELEX. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    PubMed

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

    2015-12-01

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

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

    NASA Astrophysics Data System (ADS)

    Toprak, Mahmut

    2016-02-01

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

  17. 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.

  18. 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.

  19. Sentinel lymph nodes fluorescence detection and imaging using Patent Blue V bound to human serum albumin

    PubMed Central

    Tellier, Franklin; Steibel, Jérôme; Chabrier, Renée; Blé, François Xavier; Tubaldo, Hervé; Rasata, Ravelo; Chambron, Jacques; Duportail, Guy; Simon, Hervé; Rodier, Jean-François; Poulet, Patrick

    2012-01-01

    Patent Blue V (PBV), a dye used clinically for sentinel lymph node detection, was mixed with human serum albumin (HSA). After binding to HSA, the fluorescence quantum yield increased from 5 × 10−4 to 1.7 × 10−2, which was enough to allow fluorescence detection and imaging of its distribution. A detection threshold, evaluated in scattering test objects, lower than 2.5 nmol × L−1 was obtained, using a single-probe setup with a 5-mW incident light power. The detection sensitivity using a fluorescence imaging device was in the µmol × L−1 range, with a noncooled CCD camera. Preclinical evaluation was performed on a rat model and permitted to observe inflamed nodes on all animals. PMID:23024922

  20. Sentinel lymph nodes fluorescence detection and imaging using Patent Blue V bound to human serum albumin.

    PubMed

    Tellier, Franklin; Steibel, Jérôme; Chabrier, Renée; Blé, François Xavier; Tubaldo, Hervé; Rasata, Ravelo; Chambron, Jacques; Duportail, Guy; Simon, Hervé; Rodier, Jean-François; Poulet, Patrick

    2012-09-01

    Patent Blue V (PBV), a dye used clinically for sentinel lymph node detection, was mixed with human serum albumin (HSA). After binding to HSA, the fluorescence quantum yield increased from 5 × 10(-4) to 1.7 × 10(-2), which was enough to allow fluorescence detection and imaging of its distribution. A detection threshold, evaluated in scattering test objects, lower than 2.5 nmol × L(-1) was obtained, using a single-probe setup with a 5-mW incident light power. The detection sensitivity using a fluorescence imaging device was in the µmol × L(-1) range, with a noncooled CCD camera. Preclinical evaluation was performed on a rat model and permitted to observe inflamed nodes on all animals.

  1. 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

    2017-04-12

    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.

  2. 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.

  3. Human serum albumin interaction with honokiol studied using optical spectroscopy and molecular modeling methods

    NASA Astrophysics Data System (ADS)

    Li, Jinhua; Ren, Cuiling; Zhang, Yaheng; Liu, Xiaoyan; Yao, Xiaojun; Hu, Zhide

    2008-06-01

    The interaction of honokiol with human serum albumin (HSA) was investigated for the first time using target protein as a probe by the methods of fluorescence anisotropy, circular dichroism (CD), Fourier transform infrared (FT-IR) and molecular modeling. Upon binding with HSA, the fluorescence intensity of honokiol decreased regularly with the gradual increasing concentration of HSA. In addition, the value of fluorescence anisotropy suggested that the drug was located in a restricted environment of protein. The FT-IR spectra and CD spectra measurements showed that the secondary structure of the protein was changed by the binding of honokiol to HSA. Furthermore, the study of molecular modeling indicated that honokiol could bind to the site I (subdomain IIA) of HSA and hydrophobic interaction was the major acting force.

  4. Behavior of human serum albumin on strong cation exchange resins: II. model analysis.

    PubMed

    Voitl, Agnes; Butté, Alessandro; Morbidelli, Massimo

    2010-08-20

    Experiments with human serum albumin on a strong cation exchange resin exhibit a peculiar elution pattern: the protein elutes with two peaks in a modifier gradient. This behavior is modeled with a general rate model, where the two elution peaks are modeled with two binding conformations, one of which is at equilibrium conditions, while for the other, the adsorption process is rate limited. Isocratic experiments under nonadsorbing conditions were used to characterize the mass transfer process. The isotherm of both adsorption conformations as well as the kinetic of adsorption and desorption for the second conformation are functions of the modifier concentration. They are evaluated with linear modifier gradient experiments and step experiments with various adsorption times. All experimental features are well reproduced by the proposed modified general rate model.

  5. 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.

  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. 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.

  8. 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.

  9. Weak interactive forces govern the interaction between a non-ionic surfactant with human serum albumin

    NASA Astrophysics Data System (ADS)

    Ghosh, Narayani; Mondal, Ramakanta; Deshmukh, Arundhati; Dutta, Sanjay; Mukherjee, Saptarshi

    2015-08-01

    The effect of the non-ionic surfactant Tween 40 (TW40) on Human Serum Albumin (HSA) has been studied by spectroscopic and isothermal titration calorimetric (ITC) methods. Our steady-state and time-resolved spectroscopic results reveal the perturbation of the native protein conformation upon interaction with TW40. The interaction of TW40 with HSA does not occur in a sequential manner unlike another anionic surfactant, sodium dodecyl sulfate (SDS). Our major conclusion is that the HSA-TW40 interaction is mainly driven by weak forces like van der Waal/hydrogen bonding interactions. This is also generalized from the results of interaction of HSA with another non-ionic surfactant TW80.

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

    PubMed

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

    2008-01-01

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

  11. 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).

  12. 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.

  13. Correlation of serum cytokine and acute phase reactant levels with alterations in weight and serum albumin in patients receiving immunotherapy with recombinant IL-2.

    PubMed Central

    Deehan, D J; Heys, S D; Simpson, W; Herriot, R; Broom, J; Eremin, O

    1994-01-01

    Recombinant IL-2 (rIL-2) has been used alone or in combination with other chemotherapeutic agents to enhance host defences against cancer. Prolonged administration of high doses, required for clinical efficacy, may precipitate serious dose-limiting toxicity. rIL-2-induced 'vascular leak syndrome' leads to hypotension, renal insufficiency, respiratory disturbances and other organ dysfunctions. Serial measurements of serum cytokines and the acute phase protein C-reactive protein (CRP) were performed on nine patients who received high-dose i.v. continuous therapy with rIL-2. The influence of these immunological parameters upon alterations in patients' weight and serum albumin, as indicators of toxicity, was assessed. All patients experienced weight increases during the cycle (3-11% of total body weight). The serum levels of tumour necrosis factor (TNF-alpha) and CRP were highly predictive of alterations in patients' weight (both P < 0.001), while no correlation was found with IL-6 and weight change. Serum albumin fell linearly throughout the infusion cycle, but this showed no correlation with variations in serum levels of IL-6, TNF-alpha, or CRP. The complement components C3 and C4 were significantly reduced at the end of the infusion, suggesting a possible role for this cascade system in mediating these clinical changes. The strong association between serum TNF-alpha and weight change, not previously documented, further supports the hypothesis that TNF-alpha is a key mediator in the pathogenesis of the 'vascular leak syndrome'. PMID:7511074

  14. Generation of hybrid human immunodeficiency virus by homologous recombination.

    PubMed Central

    Srinivasan, A; York, D; Jannoun-Nasr, R; Kalyanaraman, S; Swan, D; Benson, J; Bohan, C; Luciw, P A; Schnoll, S; Robinson, R A

    1989-01-01

    Human immunodeficiency virus (HIV) type 1, isolated from diverse sources, exhibits genomic diversity. The mechanisms by which the genomic diversity takes place in individuals exposed to multiple virus isolates is yet to be elucidated. Genetic variation, in general, might result from mutagenic events such as point mutations, rearrangements (insertions and deletions), and recombination. In an attempt to evaluate the process of genetic diversity, we designed experiments to analyze recombination between HIV DNAs by using DNA transfection in cell cultures. Here we report the successful recombination between truncated HIV proviral DNAs with an overlap homology of 53 base pairs that leads to the formation of viable hybrid virus. Recombination was also seen between exogenous DNA introduced into cells and homologous HIV sequences resident in the cells. These results indicate that recombination among various HIV isolates may play a significant role in the generation of genetic diversity of HIV. Further, the method used here enables the construction of hybrid HIV genomes to identify the viral determinants responsible for tropism, replication, and cytopathic effects. Images PMID:2474834

  15. Spectroscopic studies on the gemini surfactant mediated refolding of human serum albumin.

    PubMed

    Gull, Nuzhat; Khan, Javed Masood; Rukhsana; Khan, Rizwan Hassan

    2017-09-01

    Refolding of guanidinium hydrochloride (GdCl) denatured human serum albumin (HSA) using a combination of cationic gemini surfactants; pentanediyl-α,ω-bis(cetyldimethylammonium bromide) (C16H33(CH3)2N(+)-(CH2)5-N(+)(CH3)2C16H33)2Br(-) designated as G5 and methyl- β-cyclodextrin, is attempted in the present study. The studies were carried out in an aqueous medium (pH 7.4) using dynamic light scattering (DLS), circular dichroism (CD) and fluorescence spectroscopy. A careful study of the DLS data indicates that against the hydrodynamic radius (Rh) of 3.5nm in native human serum albumin (HSA), hydrodynamic radius after attempting refolding by simple dilution increases to 33.8nm. The large Rh values of the diluted protein sample is associated with the formation of aggregates as dilution is an aggregation prone pathway. Hydrodynamic radii equal to 5.4nm, that is very near to the native protein (3.5nm), is obtained on the sequential addition of G5 and methyl- β-cyclodextrin to the denatured protein. The results obtained from the multi-technique approach are associated with the presence of two charged head-groups and two hydrocarbon tails in the gemini surfactants resulting in very strong electrostatic and hydrophobic interactions.The present study suggests that gemini surfactants may be utilised in the protein refolding studies and may prove to be inexpensive and efficient folding agents. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Differential receptor targeting of liver cells using 99mTc-neoglycosylated human serum albumins.

    PubMed

    Kim, Sungeun; Jeong, Jae Min; Hong, Mee Kyung; Jang, Ja-June; Lee, Jaetae; Lee, Dong Soo; Chung, June-Key; Lee, Myung Chul

    2008-01-01

    Neolactosyl human serum albumin (LSA) targets asialoglycoprotein receptor and shows high liver uptake due to accumulation in hepatocytes. Although neomannosyl human serum albumin (MSA) also shows high liver uptake, it has been reported to be taken up by Kupffer cells and endothelial cells. We compared the biological properties of LSA and MSA. 99mTc-LSA and 99mTc-MSA biodistribution in mice were investigated after intravenous injection. In vivo localization of rhodaminisothiocyanate (RITC)-LSA and fluoresceineisothiocyanate (FITC)-MSA were investigated in mouse liver. Excretion routes of 99mTc-LSA and 99mTc-MSA metabolites were examined. Both 99mTc-LSA and 99mTc-MSA showed high liver uptakes. RITC-LSA was taken up by hepatocytes whereas FITC-MSA was taken up by Kupffer cells and endothelial cells. 99mTc-MSA showed higher spleen and kidney uptakes than 99mTc-LSA. 99mTc-LSA metabolites excreted in urine and feces accounted for 44.4 and 50.0% of 99mTc-LSA injected, respectively, while 99mTc-MSA metabolites accounted for 51.5 and 10.3%, respectively. In conclusion, LSA is specifically taken up by hepatcytes while MSA by Kupffer cells and endothelial cells. After taken up by the liver, LSA is metabolized by the hepatocytes and then excreted through both the hepatobiliary tract and kidney, whereas MSA is metabolized by Kupffer cells and endoghelial cells and then excreted mainly through the kidney.

  17. Experience of using human albumin solution 4.5% in 1195 therapeutic plasma exchange procedures.

    PubMed

    Pusey, C; Dash, C; Garrett, M; Gascoigne, E; Gesinde, M; Gillanders, K; Wallington, T

    2010-08-01

    The aim of the study was to document the incidence of adverse reactions (ADRs) in subjects undergoing therapeutic plasma exchange with human albumin 4.5% solution (Zenalb 4.5) and to explore whether there were any differences in tolerability with a change from UK to US plasma and a subsequent manufacturing modification. Zenalb 4.5 was initially manufactured from recovered plasma from UK blood donations and later from source plasma from US donors. The modification was a salt diafiltration step. A prospective survey was conducted at three UK aphaeresis units; data from 154 subjects undergoing 1195 plasma exchanges using Zenalb 4.5 were collected. Adverse events with at least a possible relationship to treatment were recorded. There were 20 ADRs per 1195 exchanges (1.7%), experienced by 14 subjects (9.1%). The most common reaction was rigours in 17 exchanges (1.4%) and 12 subjects (7.8%). ADRs occurred in 0.8% (2/250) of plasma exchanges with UK plasma, 0.2% (1/539) using US plasma/original manufacturing method, 4.3% (16/370) using US plasma/modified method and 12.5% (1/8) using US plasma/mixed original and modified methods. Data were incomplete for the remaining 28 exchanges, but no ADRs were reported. Moreover, 17 ADRs occurred over a 14-month period and involved 10 batches manufactured from US plasma (1 original, 9 by modified method). The incidence then returned to the previously lower level. There was no explanation for this cluster of events. Overall, there was no evidence that plasma source or manufacturing method affected tolerability and it was concluded that human albumin 4.5% solution (Zenalb 4.5) is well tolerated during plasma exchange therapy.

  18. Homologous recombination evidence in human and swine influenza A viruses.

    PubMed

    He, Cheng-Qiang; Han, Guan-Zhu; Wang, Dong; Liu, Wei; Li, Guo-Rong; Liu, Xi-Ping; Ding, Nai-Zheng

    2008-10-10

    Dynamic gene mutation and the reassortment of genes have been considered as the key factors responsible for influenza A virus virulence and host tropism change. This study reports several significant evidence demonstrating that homologous recombination also takes place between influenza A viruses in human and swine lineages. Moreover, in a mosaic descended from swine H1N1 subtype and human H2N2, we found that its minor putative parent might be a derivative from the human cold-adapted vaccine lineage, which suggests that live vaccine is capable of playing a role in genetic change of influenza A virus via recombination with circulating viruses. These results would be important for knowing the molecular mechanism of mammal influenza A virus heredity and evolution.

  19. Human serum albumin Cys34 oxidative modifications following infiltration in the carotid atherosclerotic plaque.

    PubMed

    Lepedda, Antonio Junior; Zinellu, Angelo; Nieddu, Gabriele; De Muro, Pierina; Carru, Ciriaco; Spirito, Rita; Guarino, Anna; Piredda, Franco; Formato, Marilena

    2014-01-01

    To evaluate if the prooxidant environment present in atherosclerotic plaque may oxidatively modify filtered albumin. Fluorescein-5-maleimide labelled plasma samples and plaque extracts from 27 patients who had undergone carotid endarterectomy were analysed through nonreducing SDS-PAGE for albumin-Cys(34) oxidation. Furthermore, degree and pattern of S-thiolation in both circulating and plaque-filtered albumin were assayed. Albumin filtered in the atherosclerotic plaque showed higher levels of Cys(34) oxidative modifications than the corresponding circulating form as well as different patterns of S-thiolation. Data indicate that the circulating albumin, once filtered in plaque, undergoes Cys(34) oxidative modifications and demonstrate for the first time that albumin is a homocysteine and cysteinylglycine vehicle inside the plaque environment.

  20. Human Serum Albumin Cys34 Oxidative Modifications following Infiltration in the Carotid Atherosclerotic Plaque

    PubMed Central

    Zinellu, Angelo; De Muro, Pierina; Carru, Ciriaco; Spirito, Rita; Guarino, Anna

    2014-01-01

    Objectives. To evaluate if the prooxidant environment present in atherosclerotic plaque may oxidatively modify filtered albumin. Methods. Fluorescein-5-maleimide labelled plasma samples and plaque extracts from 27 patients who had undergone carotid endarterectomy were analysed through nonreducing SDS-PAGE for albumin-Cys34 oxidation. Furthermore, degree and pattern of S-thiolation in both circulating and plaque-filtered albumin were assayed. Results. Albumin filtered in the atherosclerotic plaque showed higher levels of Cys34 oxidative modifications than the corresponding circulating form as well as different patterns of S-thiolation. Conclusions. Data indicate that the circulating albumin, once filtered in plaque, undergoes Cys34 oxidative modifications and demonstrate for the first time that albumin is a homocysteine and cysteinylglycine vehicle inside the plaque environment. PMID:24738021

  1. Optimization of human serum albumin production in methylotrophic yeast Pichia pastoris by repeated fed-batch fermentation.

    PubMed

    Ohya, Tomoshi; Ohyama, Masao; Kobayashi, Kaoru

    2005-06-30

    An optimization method for repeated fed-batch fermentation was established with the aim of improving the recombinant human serum albumin (rHSA) production in Pichia pastoris. A simulation model for fed-batch fermentation was formulated and the optimal methanol-feeding policy calculated by dynamic programming method using five different methanol-feeding periods. The necessary state variables were collected from the calculated results and used for further optimization of repeated fed-batch fermentation. The optimal operation policy was investigated using the pre-collected state variables by estimating the overall profit per total methanol-feeding time. The calculated results indicated that the initial cell mass from the 2nd fed-batch fermentation on should be set at 35 or 40 g and methanol-feeding time at 264 h. In repeated fed-batch fermentation using the optimal operation policy, actual culture volume was in good agreement with the values simulated by model equations, but some discrepancy was observed in rHSA production. Minimum experiments were therefore carried out to re-evaluate rHSA production levels, which were then applied in re-calculations to determine the optimal operation policy. The optimal policy for repeated fed-batch fermentation established in the present study (i.e., 4-times-repeated fed-batch fermentation) achieved a 47% increase in annual rHSA production. Optimization of the culture period also brought about a 28% increase in annual rHSA production even in simple (not repeated) fed-batch fermentation.

  2. [Study of the content of alpha-fetoprotein and serum albumin in the vitreous body of the eye of human embryos].

    PubMed

    Panova, I G; Tatikolov, A S

    2011-01-01

    The content of serum albumin and alpha-fetoprotein in the vitreous body of the eyes of human embryos from the 16th through the 24th week was investigated. It was detected that albumin and alpha-fetoprotein in the vitreous body of human eyes are presented in equal molar concentrations in the 16th week. There is 1.5-fold increased concentration of alpha-fetoprotein in comparison to albumin during the 17th week. Seventeen weeks later, there was a reduction in the concentration of both proteins. It was reported that cyanine dye, used for detection of albumin, does not interact with alpha-fetoprotein.

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2014-03-01

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

  5. Arabinosylation of recombinant human immunoglobulin-based protein therapeutics.

    PubMed

    Hossler, Patrick; Chumsae, Christopher; Racicot, Christopher; Ouellette, David; Ibraghimov, Alexander; Serna, Daniel; Mora, Alessandro; McDermott, Sean; Labkovsky, Boris; Scesney, Susanne; Grinnell, Christine; Preston, Gregory; Bose, Sahana; Carrillo, Ralf

    Protein glycosylation is arguably the paramount post-translational modification on recombinant glycoproteins, and highly cited in the literature for affecting the physiochemical properties and the efficacy of recombinant glycoprotein therapeutics. Glycosylation of human immunoglobulins follows a reasonably well-understood metabolic pathway, which gives rise to a diverse range of asparagine-linked (N-linked), or serine/threonine-linked (O-linked) glycans. In N-linked glycans, fucose levels have been shown to have an inverse relationship with the degree of antibody-dependent cell-mediated cytotoxicity, and high mannose levels have been implicated in potentially increasing immunogenicity and contributing to less favorable pharmacokinetic profiles. Here, we demonstrate a novel approach to potentially reduce the presence of high-mannose species in recombinant human immunoglobulin preparations, as well as facilitate an approximate 100% replacement of fucosylation with arabinosylation in Chinese hamster ovary cell culture through media supplementation with D-arabinose, an uncommonly used mammalian cell culture sugar substrate. The replacement of fucose with arabinose was very effective and practical to implement, since no cell line engineering or cellular adaptation strategies were required. Arabinosylated recombinant IgGs and the accompanying reduction in high mannose glycans, facilitated a reduction in dendritic cell uptake, increased FcγRIIIa signaling, and significantly increased the levels of ADCC. These aforementioned effects were without any adverse changes to various structural or functional attributes of multiple recombinant human antibodies and a bispecific DVD-Ig. Protein arabinosylation represents an expansion of the N-glycan code in mammalian expressed glycoproteins.

  6. Human Recombinant Insulin 1g - ug

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Proteins are the building blocks of our bodies and the living world around us. Within our bodies proteins make it possible for red blood cells to carry oxygen throughout the body. Others help transmit nerve impulses so we can hear, smell and feel the world around us. While others play a crucial role in preventing or causing disease. If the structure of a protein is known, then companies can develop new or improved drugs to fight the disease of which the protein is a part. To determine protein structure, researchers must grow near-perfect crystals of the protein. On Earth convection currents, sedimentation and other gravity-induced phenomena hamper crystal growth efforts. In microgravity researchers can grow near-perfect crystals in an environment free of these effects. Because of the enormous potential for new pharmaceutical products the Center for Macromolecular Crystallography--the NASA Commercial Space Center responsible for commercial protein crystal growth efforts has more than fifty major industry and academic partners. Research on crystals of human insulin could lead to improved treatments for diabetes.

  7. Human Recombinant Insulin 1g - ug

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Proteins are the building blocks of our bodies and the living world around us. Within our bodies proteins make it possible for red blood cells to carry oxygen throughout the body. Others help transmit nerve impulses so we can hear, smell and feel the world around us. While others play a crucial role in preventing or causing disease. If the structure of a protein is known, then companies can develop new or improved drugs to fight the disease of which the protein is a part. To determine protein structure, researchers must grow near-perfect crystals of the protein. On Earth convection currents, sedimentation and other gravity-induced phenomena hamper crystal growth efforts. In microgravity researchers can grow near-perfect crystals in an environment free of these effects. Because of the enormous potential for new pharmaceutical products the Center for Macromolecular Crystallography--the NASA Commercial Space Center responsible for commercial protein crystal growth efforts has more than fifty major industry and academic partners. Research on crystals of human insulin could lead to improved treatments for diabetes.

  8. Crystallization of recombinant human interleukin 1β

    NASA Astrophysics Data System (ADS)

    Einspahr, Howard; Clancy, L. L.; Muchmore, S. W.; Watenpaugh, K. D.; Harris, P. K. W.; Carter, D. B.; Curry, K. A.; Tomich, C.-S. C.; Yem, A. W.; Deibel, M. R.; Tracey, D. E.; Paslay, J. W.; Staite, N. D.; Carter, J. B.; Theriault, N. Y.; Reardon, I. M.; Zurcher-Neely, H. A.; Heinrikson, R. L.

    1988-07-01

    The gene for the fully processed form of human interleukin 1β was cloned from SK-hep-1 hepatoma cellular RNA and expressed at high levels in E. coli. The protein produced in E. coli. was purified to homogeneity by standard chromatographic methods, including adsorption and desorption from Procion Red Sepharose, sizing on a Superose 12 FPLC column, and anion exchange chromatography on QAE Sepharose. The result is a biologically active protein, rIL-1β, that migrates on two-dimensional gels as a single spot with a pI of 6.5 ± 0.2 and a molecular mass of 17, 500 daltons. Crystals of rIL-1β have been produced from concentrated solutions of the protein by ammonium sulfate precipitation. The crystals are tetragonal, have space group P41 or its enantiomer, have lattice constants of a = 58.46(1) Å and c = 77.02(3) Å, and scatter to at least 2 Å resolution. A structure determination ba these crystals is underway.

  9. Human DNA repair and recombination genes

    SciTech Connect

    Thompson, L.H.; Weber, C.A.; Jones, N.J.

    1988-09-01

    Several genes involved in mammalian DNA repair pathways were identified by complementation analysis and chromosomal mapping based on hybrid cells. Eight complementation groups of rodent mutants defective in the repair of uv radiation damage are now identified. At least seven of these genes are probably essential for repair and at least six of them control the incision step. The many genes required for repair of DNA cross-linking damage show overlap with those involved in the repair of uv damage, but some of these genes appear to be unique for cross-link repair. Two genes residing on human chromosome 19 were cloned from genomic transformants using a cosmid vector, and near full-length cDNA clones of each gene were isolated and sequenced. Gene ERCC2 efficiently corrects the defect in CHO UV5, a nucleotide excision repair mutant. Gene XRCC1 normalizes repair of strand breaks and the excessive sister chromatid exchange in CHO mutant EM9. ERCC2 shows a remarkable /approximately/52% overall homology at both the amino acid and nucleotide levels with the yeast RAD3 gene. Evidence based on mutation induction frequencies suggests that ERCC2, like RAD3, might also be an essential gene for viability. 100 refs., 4 tabs.

  10. Interaction of coomassie brilliant blue G250 with human serum albumin: Probing of the binding mechanism and binding site by spectroscopic and molecular modeling methods

    NASA Astrophysics Data System (ADS)

    Li, Yue-Sheng; Ge, Yu-Shu; Zhang, Yue; Zhang, Ai-Qing; Sun, Shao-Fa; Jiang, Feng-Lei; Liu, Yi

    2010-04-01

    The interaction between coomassie brilliant blue G250 and human serum albumin was investigated by spectroscopic methods such as fluorescence quenching, synchronous fluorescence , 3D fluorescence spectra, circular dichroism spectra and UV-vis absorption as well as molecular modeling. The fluorescence quenching of human serum albumin by coomassie brilliant blue G250 was attributed to static interaction. The binding reaction was mainly enthalpy-driven. Both van der Waals and hydrogen bonding forces played major roles in stabilizing the coomassie brilliant blue G250-human serum albumin complex. The Stern-Volmer quenching constant ( KSV) and corresponding thermodynamic parameters (Δ HΘ, Δ GΘ and Δ SΘ) were determined. Site marker competitive experiments indicated that coomassie brilliant blue G250 bound to site I (subdomain IIA) of human serum albumin. Molecular docking study further confirmed the binding mode obtained by experimental study. The conformational investigation demonstrated very minor micro-environmental and conformational changes in human serum albumin molecules.

  11. Mechanisms of fever induced by recombinant human interferon.

    PubMed Central

    Dinarello, C A; Bernheim, H A; Duff, G W; Le, H V; Nagabhushan, T L; Hamilton, N C; Coceani, F

    1984-01-01

    Since the early trials using human interferon (hIFN) derived from blood leukocytes or cell lines, fever has been a prominent component of IFN therapy. Human protein impurities might account for the fever to cell-derived hIFN, but recombinant hIFN, free of extraneous human proteins, has produced fever in nearly all recipients during clinical trials. Our present studies were carried out to determine the mechanisms of fever due to recombinant hIFN currently being used in humans. Because recombinant hIFN is produced in Escherichia coli, in these experiments we considered contaminating endotoxin as the cause of fever. Polymyxin B, which blocks endotoxin, had no effect on the pyrogenicity of hIFN in rabbits. In addition, hIFN injected into an endotoxin-resistant strain of mice produced fever. The pyrogenicity of hIFN does not appear to involve production of leukocytic pyrogen (LP), since no circulating LP was detected in rabbits during IFN fever. Furthermore, human mononuclear cells incubated with hIFN in vitro at 10(4)-10(6) U/ml did not release LP. However, hIFN stimulated prostaglandin E2 (PGE2) release from rabbit hypothalamic tissue in vitro. Intracerebroventricular injection of hIFN into the awake cat also produced fever and a rise in PGE2 levels in the cerebrospinal fluid; both effects were reversed by treatment with indomethacin. We conclude that the fever of recombinant hIFN is not due to endotoxin but that hIFN is intrinsically pyrogenic by inducing PGE2 in the hypothalamus. PMID:6590569

  12. Biphasic modulation of cell growth by recombinant human galectin-1.

    PubMed

    Adams, L; Scott, G K; Weinberg, C S

    1996-06-13

    Human soluble galactose-binding lectin (galectin-1) has been expressed as an Escherichia coli fusion protein, following the amplification by polymerase chain reaction of cDNA prepared from a human osteosarcoma cell line. The fusion protein is a functional beta-galactoside-binding lectin, as is the recombinant galectin when purified from the cleaved fusion protein. The recombinant galectin has a biphasic effect on cell proliferation. Unlike the fusion protein, it functions as a human cell growth inhibitor, confirming earlier findings with natural human galectin-1, though it is less effective than the natural galectin. This reaction is not significantly inhibited by lactose, and is thus largely independent of the beta-galactoside-binding site. At lower concentrations, recombinant galectin-1 is mitogenic, this activity being susceptible to inhibition by lactose, and thus attributable to the beta-galactoside-binding ability of the protein. Some tumour cells are susceptible to the growth-inhibitory effect, and the galectin-1 gene is expressed in both normal and tumour cells.

  13. Bioinformatic Analysis of the Human Recombinant Iduronate 2-Sulfate Sulfatase

    PubMed Central

    Morales-Álvarez, Edwin D.; Rivera-Hoyos, Claudia M.; Landázuri, Patricia; Poutou-Piñales, Raúl A.; Pedroza-Rodríguez, Aura M.

    2016-01-01

    Mucopolysaccharidosis type II is a human recessive disease linked to the X chromosome caused by deficiency of lysosomal enzyme Iduronate 2-Sulfate Sulfatase (IDS), which leads to accumulation of glycosaminoglycans in tissues and organs. The human enzyme has been expressed in Escherichia coli and Pichia pastoris in attempt to develop more successful expression systems that allow the production of recombinant IDS for Enzyme Replacement Therapy (ERT). However, the preservation of native signal peptide in the sequence has caused conflicts in processing and recognition in the past, which led to problems in expression and enzyme activity. With the main object being the improvement of the expression system, we eliminate the native signal peptide of human recombinant IDS. The resulting sequence showed two modified codons, thus, our study aimed to analyze computationally the nucleotide sequence of the IDSnh without signal peptide in order to determine the 3D structure and other biochemical properties to compare them with the native human IDS (IDSnh). Results showed that there are no significant differences between both molecules in spite of the two-codon modifications detected in the recombinant DNA sequence. PMID:27335624

  14. 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

  15. SMALL VOLUME RESUSCITATION FROM HEMORRHAGIC SHOCK WITH POLYMERIZED HUMAN SERUM ALBUMIN

    PubMed Central

    Messmer, Catalina; Yalcin, Ozlem; Palmer, Andre F.; Cabrales, Pedro

    2011-01-01

    Human serum albumin (HSA) is used as a plasma expander (PE); however, albumin is readily eliminated from the intravascular space. The objective of this study was to establish the effects of various sized polymerized HSAs (PolyHSAs) during small volume resuscitation from hemorrhagic shock on systemic parameters, microvascular hemodynamics and functional capillary density (FCD) in the hamster window chamber model. PolyHSA size was controlled by varying the cross-link density (i.e. molar ratio of glutaraldehyde to HSA). Hemorrhage was induced by controlled arterial bleeding of 50% of the animal’s blood volume (BV) and hypovolemic shock maintained for 1 h. Resuscitation was implemented in two phases, first by infusion of 3.5% of the BV of hypertonic saline (HTS, 7.5 wt% NaCl), followed by infusion of 10% of BV of each PolyHSAs. Resuscitation provided rapid recovery of blood pressure, blood gas parameters and microvascular perfusion. PolyHSA at a glutaraldehyde to HSA molar ratio of 60:1 (PolyHSA60:1) provided superior recovery of blood pressure, microvascular blood flow and FCD, and acid-base balance, with sustained volume expansion in relation to the volume infused. The high molecular weigh of PolyHSA60:1 increased hydrodynamic radius and solution viscosity. Pharmacokinetic analysis of PolyHSA60:1 indicates reduced clearance and increased circulatory half-life compared to monomeric HSA and other PolyHSA formulations. In conclusion, HSA molecular size and solution viscosity affects central hemodynamics, microvascular blood flow, volume expansion and circulation persistence during small volume resuscitation from hemorrhagic shock. Additionally, PolyHSA can be an alternative to HSA in situations with compromised vascular permeability. PMID:22100469

  16. Preparation and in vitro characterization of gallic acid-loaded human serum albumin nanoparticles

    NASA Astrophysics Data System (ADS)

    Mohammad-Beigi, Hossein; Shojaosadati, Seyed Abbas; Morshedi, Dina; Arpanaei, Ayyoob; Marvian, Amir Tayaranian

    2015-04-01

    Gallic acid (GA), as an antioxidant and antiparkinson agent, was loaded onto cationic human serum albumin nanoparticles (HSA NPs). Polyethylenimine (PEI)-coated HSA (PEI-HSA) NPs were prepared using three different methods: (I) coating negatively charged HSA NPs with positively charged PEI through attractive electrostatic interactions, (II) coating HSA NPs with PEI via covalent amide bond formation using N-(3-dimethylaminopropyl)- N-ethylcarbodiimide hydrochloride, and (III) coating HSA NPs with PEI via covalent bonding using glutaraldehyde for linking amine groups of PEI and amine groups of albumin NPs. Method II was selected since it resulted in a higher shift in the zeta potential value (mV) and less zeta potential value deviation, and also less size polydispersity. GA was loaded by adsorption onto the surface of PEI-HSA NPs of two different sizes: 117 ± 2.9 nm (PEI-P1) and 180 ± 3.1 nm (PEI-P2) NPs. Both GA-entrapment and GA-loading efficiencies increased slightly with the increasing size of NPs, and were affected intensely by the mass ratio of GA to PEI-HSA NPs. Free radical scavenging of GA was quantified based on the 2,2-diphenyl-1-picrylhydrazyl method. The obtained results showed that GA remains active during the preparation of GA-loaded PEI-HSA NPs. The cytotoxicities of HSA, PEI-HSA, and GA-loaded PEI-HSA NPs on the PC-12 cells, as the neuroendocrine cell line, were measured. Our results indicate that positively charged PEI-HSA NPs are good candidates for efficient and safe delivery of GA to the brain.

  17. 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.

  18. Biomonitoring Human Albumin Adducts: The Past, the Present, and the Future

    PubMed Central

    2016-01-01

    Serum albumin (Alb) is the most abundant protein in blood plasma. Alb reacts with many carcinogens and/or their electrophilic metabolites. Studies conducted over 20 years ago showed that Alb forms adducts with the human carcinogens aflatoxin B1 and benzene, which were successfully used as biomarkers in molecular epidemiology studies designed to address the role of these chemicals in cancer risk. Alb forms adducts with many therapeutic drugs or their reactive metabolites such as β-lactam antibiotics, acetylsalicylic acid, acetaminophen, nonsteroidal anti-inflammatory drugs, chemotherapeutic agents, and antiretroviral therapy drugs. The identification and characterization of the adduct structures formed with Alb have served to understand the generation of reactive metabolites and to predict idiosyncratic drug reactions and toxicities. The reaction of candidate drugs with Alb is now exploited as part of the battery of screening tools to assess the potential toxicities of drugs. The use of gas chromatography-mass spectrometry, liquid chromatography, or liquid chromatography-mass spectrometry (LC-MS) enabled the identification and quantification of multiple types of Alb xenobiotic adducts in animals and humans during the past three decades. In this perspective, we highlight the history of Alb as a target protein for adduction to environmental and dietary genotoxicants, pesticides, and herbicides, common classes of medicinal drugs, and endogenous electrophiles, and the emerging analytical mass spectrometry technologies to identify Alb-toxicant adducts in humans. PMID:27989119

  19. [Use of human recombinant erythropoietin in children with cancer].

    PubMed

    Guyot, D; Margueritte, G

    2005-09-01

    Eighty percent of children with cancer suffer from anemia at the time of diagnosis. The physiopathology of anemia is complex. Although anemia can be life threatening, its consequences on the physical, psychological and social state of the child are often minimized. Blood transfusion is the main treatment of anemia: its efficacy is immediate but shortlasting, and it involves infectious and hemolytic risks. The human recombinant erythropoietin has been used for more than 25-years, and is often prescribed to adults with cancer and anemia. The human recombinant erythropoietin rHuEPO is nowadays used when blood transfusion is contra-indicated because of religious or cultural considerations, although several promising studies have been conducted about rHuEPO and children with cancer since 1996: it might be soon the preferential alternative treatment to anemia in children with cancer.

  20. Plant-based biopharming of recombinant human lactoferrin.

    PubMed

    Yemets, Alla I; Tanasienko, Iryna V; Krasylenko, Yuliya A; Blume, Yaroslav B

    2014-09-01

    Recombinant proteins are currently recognized as pharmaceuticals, enzymes, food constituents, nutritional additives, antibodies and other valuable products for industry, healthcare, research, and everyday life. Lactoferrin (Lf), one of the promising human milk proteins, occupies the expanding biotechnological food market niche due to its important versatile properties. Lf shows antiviral, antimicrobial, antiprotozoal and antioxidant activities, modulates cell growth rate, binds glycosaminoglycans and lipopolysaccharides, and also inputs into the innate/specific immune responses. Development of highly efficient human recombinant Lf expression systems employing yeasts, filamentous fungi and undoubtedly higher plants as bioreactors for the large-scale Lf production is a biotechnological challenge. This review highlights the advantages and disadvantages of the existing non-animal Lf expression systems from the standpoint of protein yield and its biological activity. Special emphasis is put on the benefits of monocot plant system for Lf expression and the biosafety aspects of the transgenic Lf-expressing plants.

  1. Construction and expression of recombined human AFP eukaryotic expression vector

    PubMed Central

    Zhang, Li-Wang; Ren, Jun; Zhang, Liang; Zhang, Hong-Mei; Jin, Bin; Pan, Bo-Rong; Si, Xiao-Ming; Zhang, Yan-Jun; Wang, Zhong-Hua; Pan, Yang-Lin; Festein, Stephen M

    2003-01-01

    AIM: To construct a recombined human AFP eukaryotic expression vector for the purpose of gene therapy and target therapy of hepatocellular carcinoma (HCC). METHODS: The full length AFP-cDNA of prokaryotic vector was digested, and subcloned to the multi-clony sites of the eukaryotic vector. The constructed vector was confirmed by enzymes digestion and electrophoresis, and the product expressed was detected by electrochemiluminescence and immunofluorescence methods. RESULTS: The full length AFP-cDNA successfully cloned to the eukaryotic vector through electrophoresis, 0.9723 IU/mL AFP antigen was detected in the supernatant of AFP-CHO by electrochemiluminescence method. Compared with the control groups, the differences were significant (P < 0.05). AFP antigen molecule was observed in the plasma of AFP-CHO by immunofluorescence staining. CONCLUSION: The recombined human AFP eukaryotic expression vector can express in CHO cell line. It provides experimental data for gene therapy and target therapy of hepatocellular carcinoma. PMID:12854142

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

    PubMed

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

    2014-11-01

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

  3. Transient Expression of Tetrameric Recombinant Human Butyrylcholinesterase in Nicotiana benthamiana

    PubMed Central

    Alkanaimsh, Salem; Karuppanan, Kalimuthu; Guerrero, Andrés; Tu, Aye M.; Hashimoto, Bryce; Hwang, Min Sook; Phu, My L.; Arzola, Lucas; Lebrilla, Carlito B.; Dandekar, Abhaya M.; Falk, Bryce W.; Nandi, Somen; Rodriguez, Raymond L.; McDonald, Karen A.

    2016-01-01

    To optimize the expression, extraction and purification of plant-derived tetrameric recombinant human butyrylcholinesterase (prBChE), we describe the development and use of plant viral amplicon-based gene expression system; Tobacco Mosaic Virus (TMV) RNA-based overexpression vector (TRBO) to express enzymatically active FLAG-tagged plant made recombinant butyrylcholinesterase (rBChE) in Nicotiana benthamiana leaves using transient agroinfiltration. Two gene expression cassettes were designed to express the recombinant protein in either the ER or to the apoplastic compartment. Leaf homogenization was used to isolate ER-retained recombinant butyrylcholinesterase (prBChE-ER) while apoplast-targeted rBChE was isolated by either leaf homogenization (prBChE) or vacuum-extraction of apoplastic wash fluid (prBChE-AWF). rBChE from apoplast wash fluid had a higher specific activity but lower enzyme yield than leaf homogenate. To optimize the isolation and purification of total recombinant protein from leaf homogenates, an acidic extraction buffer was used. The acidic extraction buffer yielded >95% enzymatically active tetrameric rBChE as verified by Coomassie stained and native gel electrophoresis. Furthermore, when compared to human butyrylcholinesterase, the prBChE was found to be similar in terms of tetramerization and enzyme kinetics. The N-linked glycan profile of purified prBChE-ER was found to be mostly high mannose structures while the N-linked glycans on prBChE-AWF were primarily complex. The glycan profile of the prBChE leaf homogenates showed a mixture of high mannose, complex and paucimannose type N-glycans. These findings demonstrate the ability of plants to produce rBChE that is enzymatically active and whose oligomeric state is comparable to mammalian butyrylcholinesterase. The process of plant made rBChE tetramerization and strategies for improving its pharmacokinetics properties are also discussed. PMID:27379103

  4. Repeated intravenous injection of recombinant human hepatocyte growth factor ameliorates liver cirrhosis but causes albuminuria in rats.

    PubMed

    Kusumoto, Kazunori; Ido, Akio; Moriuchi, Akihiro; Katsura, Toshiya; Kim, Ildeok; Takahama, Yuka; Numata, Masatsugu; Kodama, Mayumi; Hasuike, Satoru; Nagata, Kenji; Uto, Hirofumi; Inui, Ken-Ichi; Tsubouchi, Hirohito

    2006-03-01

    Hepatocyte growth factor (HGF) is a promising agent for the treatment of liver cirrhosis because of its mitogenic and anti-fibrotic effects. We investigated the effect of recombinant human HGF (rh-HGF) on cirrhosis development; its pharmacokinetics and nephrotoxicity in rats with liver cirrhosis induced by 4-week treatment with dimethylnitrosamine (DMN). rh-HGF (0.3 mg/kg) was intravenously administered to rats once a day for 4 weeks in parallel with DMN treatment or twice a day for the last 2 weeks of DMN treatment. Repeated doses of rh-HGF increased the liver weight and serum albumin, and reduced serum ALT. The development of hepatic fibrosis was inhibited more efficiently by extended low-dose treatment with rh-HGF. In cirrhotic rats, serum levels of rh-HGF increased and clearance was decreased, leading to an increase in the area under the plasma-concentration time curve and a decrease in the steady-state volume of distribution. Repeated doses of rh-HGF led to increased urinary albumin excretion, but no rh-HGF-treated animals developed increased serum creatinine levels. Urinary albumin excretion returned to baseline after the cessation of rh-HGF. These results suggest that extended treatment with rh-HGF is required for the attenuation of cirrhosis, and repeated doses of rh-HGF cause adverse effects in extra-hepatic organs. These issues must be resolved before the widespread application of rh-HGF in the treatment of liver cirrhosis.

  5. Human recombinant type I collagen produced in plants.

    PubMed

    Shoseyov, Oded; Posen, Yehudit; Grynspan, Frida

    2013-07-01

    As a central element of the extracellular matrix, collagen is intimately involved in tissue development, remodeling, and repair and confers high tensile strength to tissues. Numerous medical applications, particularly, wound healing, cell therapy, bone reconstruction, and cosmetic technologies, rely on its supportive and healing qualities. Its synthesis and assembly require a multitude of genes and post-translational modifications, where even minor deviations can be deleterious or even fatal. Historically, collagen was always extracted from animal and human cadaver sources, but bare risk of contamination and allergenicity and was subjected to harsh purification conditions resulting in irreversible modifications impeding its biofunctionality. In parallel, the highly complex and stringent post-translational processing of collagen, prerequisite of its viability and proper functioning, sets significant limitations on recombinant expression systems. A tobacco plant expression platform has been recruited to effectively express human collagen, along with three modifying enzymes, critical to collagen maturation. The plant extracted recombinant human collagen type I forms thermally stable helical structures, fibrillates, and demonstrates bioactivity resembling that of native collagen. Deployment of the highly versatile plant-based biofactory can be leveraged toward mass, rapid, and low-cost production of a wide variety of recombinant proteins. As in the case of collagen, proper planning can bypass plant-related limitations, to yield products structurally and functionally identical to their native counterparts.

  6. Recombinant Human Peptidoglycan Recognition Proteins Reveal Antichlamydial Activity.

    PubMed

    Bobrovsky, Pavel; Manuvera, Valentin; Polina, Nadezhda; Podgorny, Oleg; Prusakov, Kirill; Govorun, Vadim; Lazarev, Vassili

    2016-07-01

    Peptidoglycan recognition proteins (PGLYRPs) are innate immune components that recognize the peptidoglycan and lipopolysaccharides of bacteria and exhibit antibacterial activity. Recently, the obligate intracellular parasite Chlamydia trachomatis was shown to have peptidoglycan. However, the antichlamydial activity of PGLYRPs has not yet been demonstrated. The aim of our study was to test whether PGLYRPs exhibit antibacterial activity against C. trachomatis Thus, we cloned the regions containing the human Pglyrp1, Pglyrp2, Pglyrp3, and Pglyrp4 genes for subsequent expression in human cell lines. We obtained stable HeLa cell lines that secrete recombinant human PGLYRPs into culture medium. We also generated purified recombinant PGLYRP1, -2, and -4 and confirmed their activities against Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria. Furthermore, we examined the activities of recombinant PGLYRPs against C. trachomatis and determined their MICs. We also observed a decrease in the infectious ability of chlamydial elementary bodies in the next generation after a single exposure to PGLYRPs. Finally, we demonstrated that PGLYRPs attach to C. trachomatis elementary bodies and activate the expression of the chlamydial two-component stress response system. Thus, PGLYRPs inhibit the development of chlamydial infection.

  7. Recombinant Human Peptidoglycan Recognition Proteins Reveal Antichlamydial Activity

    PubMed Central

    Manuvera, Valentin; Polina, Nadezhda; Podgorny, Oleg; Prusakov, Kirill; Govorun, Vadim; Lazarev, Vassili

    2016-01-01

    Peptidoglycan recognition proteins (PGLYRPs) are innate immune components that recognize the peptidoglycan and lipopolysaccharides of bacteria and exhibit antibacterial activity. Recently, the obligate intracellular parasite Chlamydia trachomatis was shown to have peptidoglycan. However, the antichlamydial activity of PGLYRPs has not yet been demonstrated. The aim of our study was to test whether PGLYRPs exhibit antibacterial activity against C. trachomatis. Thus, we cloned the regions containing the human Pglyrp1, Pglyrp2, Pglyrp3, and Pglyrp4 genes for subsequent expression in human cell lines. We obtained stable HeLa cell lines that secrete recombinant human PGLYRPs into culture medium. We also generated purified recombinant PGLYRP1, -2, and -4 and confirmed their activities against Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria. Furthermore, we examined the activities of recombinant PGLYRPs against C. trachomatis and determined their MICs. We also observed a decrease in the infectious ability of chlamydial elementary bodies in the next generation after a single exposure to PGLYRPs. Finally, we demonstrated that PGLYRPs attach to C. trachomatis elementary bodies and activate the expression of the chlamydial two-component stress response system. Thus, PGLYRPs inhibit the development of chlamydial infection. PMID:27160295

  8. Construction of human artificial chromosome vectors by recombineering.

    PubMed

    Kotzamanis, George; Cheung, Wing; Abdulrazzak, Hassan; Perez-Luz, Sara; Howe, Steven; Cooke, Howard; Huxley, Clare

    2005-05-23

    Human artificial chromosomes (HACs) can be formed de novo by transfection of large fragments of cloned alphoid DNA into human HT1080 cells in tissue culture. In order to generate HACs carrying a gene of interest, one can either co-transfect the alphoid DNA and the gene of interest, or one can clone both into a single vector prior to transfection. Here we describe linking approximately 70 kb of alphoid DNA onto a 156-kb BAC carrying the human HPRT gene using Red homologous recombination in the EL350 Escherichia coli host [Lee et al., Genomics 73 (2001) 56-65]. A selectable marker and EGFP marker were then added by loxP/Cre recombination using the arabinose inducible cre gene in the EL350 bacteria. The final construct generates minichromosomes in HT1080 cells and the HPRT gene is expressed. The retrofitting vector can be used to add the approximately 70 kb of alphoid DNA to any BAC carrying a gene of interest to generate a HAC vector. The method can also be used to link any unrelated BAC or PAC insert onto another BAC clone. The EL350 bacteria are an excellent host for building up complex vectors by a combination of homologous and loxP/Cre recombination.

  9. Economic and epidemiological modelling of full-length antihaemophilic factor (recombinant), plasma/albumin-free method, in previously treated patients with haemophilia A : comparison with B-domain deleted rFVIII, and value of potential viral transmission reduction due to plasma/albumin-free status.

    PubMed

    Sclar, David A; Evans, Marc A; Skaer, Tracy L; Robison, Linda M; Chung, Karen C; Poulios, Nick S

    2005-01-01

    To the extent that current recombinant clotting factor concentrates contain even trace amounts of human or animal protein, there is continuing potential for transmission of nonenveloped viruses, including hepatitis A, and parvovirus, and the theoretical potential for transmission of relatively unknown agents, such as prions (Creutzfeldt-Jakob disease, or its variant). Full-length antihaemophilic factor (recombinant), plasma/albumin-free method (rAHF-PFM; Advate), represents a novel pharmacotherapeutic option for the management of haemophilia A. This investigation was designed to discern: (i) the efficacy-based use pattern (International Units [IUs]) of rAHF-PFM versus B-domain deleted rFVIII (BDDrFVIII; ReFacto) required to resolve a bleeding episode (event) among previously treated patients with haemophilia A employing on-demand treatment; (ii) the health service expenditure pattern (percentage differential; payor's perspective) associated with use of rAHF-PFM versus BDDrFVIII among previously treated patients with haemophilia A employing on-demand treatment; and (iii) the fiscal utility attributable to the plasma/albumin-free status of rAHF-PFM under the assumed emergence of a novel and infectious blood (plasma)-borne virus. Data stemming from phase II/phase III clinical trials of rAHF-PFM, together with published literature on BDDrFVIII, afforded calculation of the probability of occurrence for specific endpoints of interest (e.g. non-response to first infusion). Monte Carlo simulation, a decision-analytical framework parameterised with stochastic (random) and deterministic (fixed) components (10 000 iterations per month [or year] of age examined [3, 6, 9 months; years 1 through 19; and years 20, 30, 40, 50, 60, 70 and 80]) was used to compare: (i) the efficacy-based use pattern by treatment option; and (ii) the health service utilisation-based expenditure pattern by treatment option, accounting for the need for subsequent infusion(s), and potential

  10. On the mechanisms of maternofetal transfer of human albumin and γG globulin in the mouse

    PubMed Central

    Gitlin, David; Koch, Christian

    1968-01-01

    Human serum albumin and human γG globulin were labeled with 131I, and the labeled proteins were then mixed with different amounts of the respective unlabeled protein. These mixtures were injected intravenously into pregnant mice near term, and the amounts of protein-bound radioactivity present in the fetuses and in maternal serum 24 hr later were determined. The concentration of human albumin found in the fetus was proportional to the maternal serum concentration of this protein over the maternal range studied, from 0.03 to 935 mg/100 ml. On the other hand, the fetal concentration of human γG first increased rapidly as the maternal concentration increased to approximately 200 mg/100 ml and then decreased as the maternal concentration continued to increase above this level; however, as the maternal human γG level increased above approximately 1100 mg/100 ml, the fetal concentration again increased and became proportional to the maternal concentration. The data suggest that maternofetal transfer of human γG in the mouse may be mediated by two processess; one of these, as with the transfer of human albumin, appears to be first order in relation to the maternal serum concentration, and the other appears to be consistent with a carrier or enzymatic process that is directly or indirectly inhibited at high maternal serum levels. PMID:4171453

  11. Enhanced resistance of mice to bacterial infection induced by recombinant human interleukin-1a.

    PubMed Central

    Ozaki, Y; Ohashi, T; Minami, A; Nakamura, S

    1987-01-01

    The effect of recombinant human interleukin-1a on the survival rate of Std-ddY male mice systemically infected with Pseudomonas aeruginosa 12 or Klebsiella pneumoniae P-5709 was evaluated. In P. aeruginosa infection, interleukin-1a given intramuscularly twice, 3 days and 1 day before inoculation of bacteria, most effectively protected animals from death due to infection. The effect was dose dependent, with a maximum survival rate of 92.5% at 10 micrograms per mouse, while only 8.3% of the control group survived until the end of the observation period. The 50% effective dose of interleukin-1a was 0.261 microgram per mouse. In K. pneumoniae infection, interleukin-1a given intramuscularly twice, simultaneously with and 1 day after the inoculation of bacteria, was most effective. The protective effect of interleukin-1a was again dose dependent and was generally more marked than in P. aeruginosa infection. The 50% effective dose was 0.034 microgram per mouse. In both infections, there was no significant increase in the survival rates of animals injected with human albumin or heat-inactivated interleukin-1a. These observations raise the possibility that human interleukin-1a could serve as a therapeutic tool for patients with bacterial infections. Images PMID:3553002

  12. Preparation of recombinant alpha-thrombin: high-level expression of recombinant human prethrombin-2 and its activation by recombinant ecarin.

    PubMed

    Yonemura, Hiroshi; Imamura, Takayuki; Soejima, Kenji; Nakahara, Yo; Morikawa, Wataru; Ushio, Yoshitaka; Kamachi, Yasuharu; Nakatake, Hiroshi; Sugawara, Keishin; Nakagaki, Tomohiro; Nozaki, Chikateru

    2004-05-01

    We have established a large-scale manufacturing system to produce recombinant human alpha-thrombin. In this system, a high yield of alpha-thrombin is prepared from prethrombin-2 activated by recombinant ecarin. We produced human prethrombin-2 using mouse myeloma cells and an expression plasmid carrying the chicken beta-actin promoter and mutant dihydrofolate reductase gene for gene amplification. To increase prethrombin-2 expression further, we performed fed-batch cultivation with the addition of vegetable peptone in 50 liters of suspension culture. After five feedings of vegetable peptone, the expression level of the recombinant prethrombin-2 reached 200 micro g/ml. Subsequently, the recombinant prethrombin-2 could be activated to alpha-thrombin by recombinant ecarin expressed in a similar manner. Finally, recombinant alpha-thrombin was purified to homogeneity by affinity chromatography using a benzamidine-Sepharose gel. The yield from prethrombin-2 in culture medium was approximately 70%. The activity of the purified recombinant alpha-thrombin, including hydrolysis of a chromogenic substrate, release of fibrinopeptide A, and activation of protein C, was indistinguishable from that of plasma-derived alpha-thrombin. Our system is suitable for the large-scale production of recombinant alpha-thrombin, which can be used in place of clinically available alpha-thrombin derived from human or bovine plasma.

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

    PubMed Central

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

    2014-01-01

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

  14. Enhancement mechanism of FCLA-1O II chemiluminescence by human serum albumin

    NASA Astrophysics Data System (ADS)

    Zhou, Jing; Xing, Da; Chen, Qun

    2006-09-01

    Fluoresceinyl Cypridina Lucifenn Analog (FCLA) is a reactive oxygen species (ROS) specific chemiluminescence (CL) probe. Its detection efficiency of singlet oxygen (10 II)couldbe significantly enhanced in the presence of human serum albumin (HSA). The enhancement mechanism of HSA-FCLA CL is studied in the current work by means ofdirect CL measurement and spectroscopy. The results show that, FCLA can combine with HSA. HSA is an effective 1O II quencher. It can react with 10 II and produce a protein carbonyl group with an elevated energy state. The HSA protein carbonyl group can transfer its energy to FCLA in the FCLA-HSA complex. Via this irradiative de-excitation pathway of the excited FCLA, luminescence production from FCLA is greatly enhanced, in addition to the chemiluminescence from the direct interaction of FCLA and 10 II FCLA has been reported for cancer diagnosis in vivo. Considering HSA is a natural protein that is present in all parts of a human body, the efficacy of FCLA used in vivo is expected to be enhanced through the coupling of FCLA and HSA.

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

    PubMed

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

    2014-01-01

    Human serum albumin (HSA) is the most abundant protein in plasma. Cys34, the only free Cys residue, is the predominant plasma thiol and a relevant sacrificial antioxidant. Both in vivo circulating HSA and pharmaceutical preparations are heterogeneous with respect to the oxidation state of Cys34. In this work, we developed an external pH gradient chromatofocusing procedure that allows the analysis of the oxidation status of HSA in human plasma and biopharmaceutical products based on the different apparent isoelectric points and chemical properties of the redox isoforms. Specifically, reduced-mercury blocked HSA (HSA-SHg(+)), HSA with Cys34 oxidized to sulfenic acid (HSA-SOH) and HSA oxidized to sulfinate anion (HSA-SO2(-)) can be separated with resolutions of 1.4 and 3.1 (first and last pair) and hence quantified and purified. In addition, an N-terminally degraded isoform (HSA3-585) in different redox states can be resolved as well. Confirmation of the identity of the chromatofocusing isolated isoforms was achieved by high resolution whole protein MS. It is proposed that the chromatofocusing procedure can be used to produce more exact and complete descriptions of the redox status of HSA in vivo and in vitro. Finally, the scalability capabilities of the chromatofocusing procedure allow for the preparation of highly pure standards of several redox isoforms of HSA. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. 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.

  17. 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.

  18. 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.

  19. Selective ex-vivo photothermal ablation of human pancreatic cancer with albumin functionalized multiwalled carbon nanotubes

    PubMed Central

    Mocan, Lucian; Tabaran, Flaviu A; Mocan, Teodora; Bele, Constantin; Orza, Anamaria Ioana; Lucan, Ciprian; Stiufiuc, Rares; Manaila, Ioana; Iulia, Ferencz; Dana, Iancu; Zaharie, Florin; Osian, Gelu; Vlad, Liviu; Iancu, Cornel

    2011-01-01

    The process of laser-mediated ablation of cancer cells marked with biofunctionalized carbon nanotubes is frequently called “nanophotothermolysis”. We herein present a method of selective nanophotothermolisys of pancreatic cancer (PC) using multiwalled carbon nanotubes (MWCNTs) functionalized with human serum albumin (HSA). With the purpose of testing the therapeutic value of these nanobioconjugates, we have developed an ex-vivo experimental platform. Surgically resected specimens from patients with PC were preserved in a cold medium and kept alive via intra-arterial perfusion. Additionally, the HSA-MWCNTs have been intra-arterially administered in the greater pancreatic artery under ultrasound guidance. Confocal and transmission electron microscopy combined with immunohistochemical staining have confirmed the selective accumulation of HSA-MWCNTs inside the human PC tissue. The external laser irradiation of the specimen has significantly produced extensive necrosis of the malign tissue after the intra-arterial administration of HSA-MWCNTs, without any harmful effects on the surrounding healthy parenchyma. We have obtained a selective photothermal ablation of the malign tissue based on the selective internalization of MWCNTs with HSA cargo inside the pancreatic adenocarcinoma after the ex-vivo intra-arterial perfusion. PMID:21720504

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

    PubMed

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

    2014-09-01

    Decabrominateddiphenyl ether (deca-BDE) and tetrabromobisphenol A (TBBPA) are known as brominated flame-retardants, which are commonly found in the environment. The binding mechanisms of deca-BDE and TBBPA with human serum albumin (HSA) are still unknown. In this report, the interactions of deca-BDE and TBBPA with HSA were investigated using different spectroscopic methods and molecular modeling. The experimental results indicated the formation of complexes between deca-BDE/TBBPA and HSA with different affinity. These interactions affected the secondary structure of HSA. Thermodynamic investigations revealed that hydrophobic forces mainly drove the binding interactions of deca-BDE/TBBPA with HSA. For TBBPA, hydrogen-bonding interactions were also involved in the binding process of TBBPA with HSA. According to the analysis of experimental and theoretical data, we concluded that the binding site of deca-BDE to HSA located in the subdomain IB, while TBBPA was near to subdomain IIA and Trp-214. The binding interactions of deca-BDE and TBBPA with the most prominent carrier protein in the human circulatory system could influence mechanisms of their biochemical processes. Thus, these binding interactions can play central roles in studying the distribution and toxicity mechanisms of brominated flame-retardants. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Exploring the copper(II)-aminotriazole complex-binding sites of human serum albumin.

    PubMed

    Yang, Lijun; He, Lian; Zhang, Jing; An, Shuai; Zhang, Lei

    2015-09-01

    The potential impact on human exposure to aminotriazole (ATA) and heavy metal in the environment becomes a concerning issue. In the current study, a water-soluble Cu(II)-aminotriazole complex [Cu(II)-ATA] was synthesized. To explore the binding mechanism of the complex with human serum albumin (HSA), their effects on conformation and activity of HSA by multispectroscopic approach and molecular modeling were investigated. Further fluorescent tests revealed that the quenching mechanism of HSA by Cu(II)-ATA was overall static. Meanwhile, the obtained binding constant and thermodynamic parameters on complex-HSA interaction showed that the types of interaction force of Cu(II)-ATA and HSA were hydrogen bonding, van der Waals and electrostatic. The analysis of three-dimensional fluorescence, circular dichroism and Fourier transform infrared spectroscopy showed that Cu(II)-ATA induced the changes in the secondary structure of HSA. Molecular docking simulation was performed and docking model suggested that the complex docked into HSA at subdomain IIA. Furthermore, amino group and attractive electrostatic interaction of Cu(II)-ATA greatly contributed to the hydrogen bonding, van der Waals and electrostatic interaction between Cu(II)-ATA and HSA, as confirmed by experimental data.

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

    PubMed Central

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

    2012-01-01

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

  3. 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.

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

    PubMed

    Wang, Yaping; Zhang, Guowen; Wang, Langhong

    2014-01-01

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

  5. 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.

  6. Binding of the anticancer drug BI-2536 to human serum albumin. A spectroscopic and theoretical study.

    PubMed

    Fernández-Sainz, Jesús; Pacheco-Liñán, Pedro J; Granadino-Roldán, José M; Bravo, Iván; Garzón, Andrés; Rubio-Martínez, Jaime; Albaladejo, José

    2017-07-01

    BI-2536 is a potent Polo-like kinase inhibitor which induces apoptosis in diverse human cancer cell lines. The binding affinity of BI-2536 for human serum albumin (HSA) protein may define its pharmacokinetic and pharmacodynamic profile. We have studied the binding of BI-2536 to HSA by means of different spectroscopic techniques and docking calculations. We have experimentally observed that the affinity of BI-2536 for HSA is higher than that of other common HSA binding drugs. Therefore, it can be postulated that the drug dose should be increased to achieve a certain concentration of free drug in plasma, although BI-2536 could also reach tumour tissues by uptaking HSA/BI-2536 complex. Only a single binding site on HSA has been observed for BI-2536 which seems to correspond to the subdomain IIA pocket. The formation of the HSA/BI-2536 complex is a spontaneous and entropy-driven process that does not cause a significant change of the secondary structure of the protein. Its endothermic character could be related to proton release. Thermodynamic analysis showed that the main protein-drug interactions are of the van der Waals type although the presence of amide and ether groups in BI-2536 could also allow H-bonding with some residues in the subdomain IIA pocket. Copyright © 2017. Published by Elsevier B.V.

  7. Resveratrol-loaded glycyrrhizic acid-conjugated human serum albumin nanoparticles wrapping resveratrol nanoparticles: Preparation, characterization, and targeting effect on liver tumors.

    PubMed

    Wu, Mingfang; Lian, Bolin; Deng, Yiping; Feng, Ziqi; Zhong, Chen; Wu, Weiwei; Huang, Yannian; Wang, Lingling; Zu, Chang; Zhao, Xiuhua

    2017-08-01

    In this study, glycyrrhizic acid-conjugated human serum albumin nanoparticles wrapping resveratrol nanoparticles were prepared to establish a tumor targeting nano-sized drug delivery system. Glycyrrhizic acid was coupled to human serum albumin, and resveratrol was encapsulated in glycyrrhizic acid-conjugated human serum albumin by high-pressure homogenization emulsification. The average particle size of sample nanoparticles prepared under the optimal conditions was 108.1 ± 5.3 nm with a polydispersity index (PDI) of 0.001, and the amount of glycyrrhizic acid coupled with human serum albumin was 112.56 µg/mg. The drug encapsulation efficiency and drug loading efficiency were 83.6 and 11.5%, respectively. The glycyrrhizic acid-conjugated human serum albumin nanoparticles wrapping resveratrol nanoparticles were characterized through laser light scattering, scanning electron microscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, differential scanning calorimetry, thermogravimetric analyses, and gas chromatography. The characterization results showed that resveratrol in glycyrrhizic acid-conjugated human serum albumin nanoparticles wrapping resveratrol nanoparticles existed in amorphous state and the residual amounts of chloroform and methanol in nanoparticles were separately less than the international conference on harmonization (ICH) limit. The in vitro drug-release study showed that the nanoparticles released the drug slowly and continuously. The inhibitory rate of glycyrrhizic acid-conjugated human serum albumin nanoparticles wrapping resveratrol nanoparticles was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 H-tetrazolium bromide method. The IC50 values of glycyrrhizic acid-conjugated human serum albumin nanoparticles wrapping resveratrol nanoparticles and resveratrol were 62.5 and 95.5 µg/ml, respectively. The target ability of glycyrrhizic acid-conjugated human serum albumin nanoparticles wrapping resveratrol nanoparticles

  8. 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

  9. Binding of antioxidant flavone isovitexin to human serum albumin investigated by experimental and computational assays.

    PubMed

    Caruso, Ícaro Putinhon; Vilegas, Wagner; de Souza, Fátima Perreira; Fossey, Marcelo Andrés; Cornélio, Marinônio Lopes

    2014-09-01

    The flavonoids are a large class of polyphenolic compounds which occur naturally in plants where they are widely distributed. Isovitexin (ISO) is a glycosylated flavonoid that exhibits a potential antioxidant activity. Some recent studies have shown the pharmacokinetic activity of isovitexin in rat blood plasma, however, without detailing the molecular target that is linked and what physicochemical forces govern the interaction. In mammalians, the most abundant protein in blood plasma is the albumin and is not unlike with human, which human serum albumin (HSA) is the major extracellular protein and functions as a carrier of various drugs. The interaction between HSA and ISO was investigated using fluorescence, UV-vis absorbance, circular dichroism (CD), Fourier transform infrared spectroscopy (FT-IR) together with, computational methods like ab initio and molecular modeling calculation. Fluorescence quenching indicated that ISO location is within the hydrophobic pocket in subdomain IIA (site 1) of HSA, close to the Trp214 residue. The Stern-Volmer quenching constants determined at 288, 298 and 308K and its dependence on temperature indicated that the quenching mechanism was static. From the analysis of binding equilibrium were determined; the binding site number and binding constants, with the correspondent thermodynamic parameters, ΔH, ΔG and ΔS for HSA-ISO complex. Also, a second binding analysis, binding density function (BDF) method, which is independent of any binding model pre-established obtained similar results. The fluorescence resonance energy transfer estimated the distance between the donor (HSA-Trp214) and acceptor (ISO), while FT-IR and CD spectroscopy measured possible changes of secondary structure at the formation of the HSA-ISO complex. The optimized geometry of isovitexin calculation performed with its ground state by using DFT/B3LYP/6-311+G(d,p) method. The HSA-ISO complex interactions determined by molecular modeling tool corroborated with

  10. Unique cyanide adduct in human serum albumin: potential as a surrogate exposure marker.

    PubMed

    Fasco, Michael J; Stack, Robert F; Lu, Shijun; Hauer, Charles R; Schneider, Erasmus; Dailey, Michael; Aldous, Kenneth M

    2011-04-18

    Cyanide (CN = HCN + CN(-)) is a renowned poison and neurotoxicant that is prevalent throughout the environment. Despite a plethora of studies conducted over the last half century, relatively little is known of its potential to cause adverse health outcomes at sublethal exposures. CN exposure is normally determined from blood, but because CN is rapidly metabolized and cleared from this compartment (t(1/2) < 1 h), it is common for several half-lives to have passed before blood samples are drawn for analysis. This variable, coupled with a very narrow toxic index and metabolic diversity within the human population, has rendered accurate assessment of CN exposure, and consequently any predictions of possible adverse health outcomes, highly problematic. Prior studies by us showed the potential of Cys-SCN adducts within human serum albumin (HSA) to act as retrospective surrogates of CN exposure. Here, we report the discovery of a stable, SCN adduct at Cys(567) formed by the reaction of CN with the C-terminal Cys(558)Cys(567) disulfide bond of HSA. Treatment of HSA purified from human serum with base in guanidine hydrochloride releases a readily detectable, uniquely modified, C-terminal-19-mer peptide from Cys(567)-SCN moieties in all the samples examined thus far. Inclusion of a HSA-Cys(567)-S(13)C(15)N labeled internal standard permits quantitation of the Cys(567)-SCN adduct by LC-MS/MS in selective reaction monitoring (SRM) of the surrogate peptide with high sensitivity and good precision. Reaction of CN in vitro with the Cys(558)Cys(567) disulfide bond in HSA is specific, rapid, and concentration dependent within a putative, physiologically relevant range. Data from various human sera demonstrate the potential usefulness of this adduct as a biomarker of CN exposure.

  11. Adductomics Pipeline for Untargeted Analysis of Modifications to Cys34 of Human Serum Albumin.

    PubMed

    Grigoryan, Hasmik; Edmands, William; Lu, Sixin S; Yano, Yukiko; Regazzoni, Luca; Iavarone, Anthony T; Williams, Evan R; Rappaport, Stephen M

    2016-11-01

    An important but understudied class of human exposures is comprised of reactive electrophiles that cannot be measured in vivo because they are short-lived. An avenue for assessing these meaningful exposures focuses on adducts from reactions with nucleophilic loci of blood proteins, particularly Cys34 of human serum albumin, which is the dominant scavenger of reactive electrophiles in serum. We developed an untargeted analytical scheme and bioinformatics pipeline for detecting, quantitating, and annotating Cys34 adducts in tryptic digests of human serum/plasma. The pipeline interrogates tandem mass spectra to find signatures of the Cys34-containing peptide, obtains accurate masses of putative adducts, quantitates adduct levels relative to a "housekeeping peptide", and annotates modifications based on a combination of retention time, accurate mass, elemental composition, and database searches. We used the adductomics pipeline to characterize 43 adduct features in archived plasma from healthy human subjects and found several that were highly associated with smoking status, race, and other covariates. Since smoking is a strong risk factor for cancer and cardiovascular disease, our ability to discover adducts that distinguish smokers from nonsmokers with untargeted adductomics indicates that the pipeline is suitable for use in epidemiologic studies. In fact, adduct features were both positively and negatively associated with smoking, indicating that some adducts arise from reactions between Cys34 and constituents of cigarette smoke (e.g., ethylene oxide and acrylonitrile) while others (Cys34 oxidation products and disulfides) appear to reflect alterations in the serum redox state that resulted in reduced adduct levels in smokers.

  12. In vivo potential of recombinant granulysin against human tumors

    PubMed Central

    Al-Wasaby, Sameer; de Miguel, Diego; Aporta, Adriana; Naval, Javier; Conde, Blanca; Martínez-Lostao, Luis; Anel, Alberto

    2015-01-01

    9 kDa granulysin is a protein present in the granules of human CTL and NK cells, with cytolytic activity against microbes and tumors. Previous work from our group demonstrated that this granulysin isoform induced apoptosis in vitro on hematological tumor cells and on primary tumor cells from B-CLL patients. In the present work, recombinant 9 kDa granulysin was used as an anti-tumoral agent to study its in vivo effect on tumor development in athymic “nude” mice models bearing human breast adenocarcinoma MDA-MB-231 or multiple myeloma NCI-H929–derived xenografts. Granulysin prevented the in vivo development of detectable MDA-MB-231-derived tumors. In addition, recombinant granulysin was able to completely eradicate NCI-H929-derived tumors. All granulysin-treated tumors exhibited signs of apoptosis induction and an increased NK cell infiltration inside the tumor tissue comparing to control ones. Moreover, no in vivo deleterious effects of the recombinant 9 kDa granulysin doses used in this study were observed on the skin or on the internal organs of the animals. In conclusion, granulysin was able to inhibit the progression of MDA-MB-231-derived xenografts and also to eradicate multiple myeloma NCI-H929-derived xenografts. This work opens the door to the initiation of preclinical and possibly clinical studies for the use of 9 kDa granulysin as a new anti-tumoral treatment. PMID:26405603

  13. Human recombinant soluble guanylyl cyclase: expression, purification, and regulation

    NASA Technical Reports Server (NTRS)

    Lee, Y. C.; Martin, E.; Murad, F.

    2000-01-01

    The alpha1- and beta1-subunits of human soluble guanylate cyclase (sGC) were coexpressed in the Sf9 cells/baculovirus system. In addition to the native enzyme, constructs with hexahistidine tag at the amino and carboxyl termini of each subunit were coexpressed. This permitted the rapid and efficient purification of active recombinant enzyme on a nickel-affinity column. The enzyme has one heme per heterodimer and was readily activated with the NO donor sodium nitroprusside or 3-(5'-hydroxymethyl-2'furyl)-1-benzyl-indazole (YC-1). Sodium nitroprusside and YC-1 treatment potentiated each other in combination and demonstrated a remarkable 2,200-fold stimulation of the human recombinant sGC. The effects were inhibited with 1H-(1,2, 4)oxadiazole(4,3-a)quinoxalin-1one (ODQ). The kinetics of the recombinant enzyme with respect to GTP was examined. The products of the reaction, cGMP and pyrophosphate, inhibited the enzyme. The extent of inhibition by cGMP depended on the activation state of the enzyme, whereas inhibition by pyrophosphate was not affected by the enzyme state. Both reaction products displayed independent binding and cooperativity with respect to enzyme inhibition. The expression of large quantities of active enzyme will facilitate structural characterization of the protein.

  14. Human recombinant soluble guanylyl cyclase: expression, purification, and regulation

    NASA Technical Reports Server (NTRS)

    Lee, Y. C.; Martin, E.; Murad, F.

    2000-01-01

    The alpha1- and beta1-subunits of human soluble guanylate cyclase (sGC) were coexpressed in the Sf9 cells/baculovirus system. In addition to the native enzyme, constructs with hexahistidine tag at the amino and carboxyl termini of each subunit were coexpressed. This permitted the rapid and efficient purification of active recombinant enzyme on a nickel-affinity column. The enzyme has one heme per heterodimer and was readily activated with the NO donor sodium nitroprusside or 3-(5'-hydroxymethyl-2'furyl)-1-benzyl-indazole (YC-1). Sodium nitroprusside and YC-1 treatment potentiated each other in combination and demonstrated a remarkable 2,200-fold stimulation of the human recombinant sGC. The effects were inhibited with 1H-(1,2, 4)oxadiazole(4,3-a)quinoxalin-1one (ODQ). The kinetics of the recombinant enzyme with respect to GTP was examined. The products of the reaction, cGMP and pyrophosphate, inhibited the enzyme. The extent of inhibition by cGMP depended on the activation state of the enzyme, whereas inhibition by pyrophosphate was not affected by the enzyme state. Both reaction products displayed independent binding and cooperativity with respect to enzyme inhibition. The expression of large quantities of active enzyme will facilitate structural characterization of the protein.

  15. Human recombinant soluble guanylyl cyclase: Expression, purification, and regulation

    PubMed Central

    Lee, Yu-Chen; Martin, Emil; Murad, Ferid

    2000-01-01

    The α1- and β1-subunits of human soluble guanylate cyclase (sGC) were coexpressed in the Sf9 cells/baculovirus system. In addition to the native enzyme, constructs with hexahistidine tag at the amino and carboxyl termini of each subunit were coexpressed. This permitted the rapid and efficient purification of active recombinant enzyme on a nickel-affinity column. The enzyme has one heme per heterodimer and was readily activated with the NO donor sodium nitroprusside or 3-(5′-hydroxymethyl-2′furyl)-1-benzyl-indazole (YC-1). Sodium nitroprusside and YC-1 treatment potentiated each other in combination and demonstrated a remarkable 2,200-fold stimulation of the human recombinant sGC. The effects were inhibited with 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1one (ODQ). The kinetics of the recombinant enzyme with respect to GTP was examined. The products of the reaction, cGMP and pyrophosphate, inhibited the enzyme. The extent of inhibition by cGMP depended on the activation state of the enzyme, whereas inhibition by pyrophosphate was not affected by the enzyme state. Both reaction products displayed independent binding and cooperativity with respect to enzyme inhibition. The expression of large quantities of active enzyme will facilitate structural characterization of the protein. PMID:10995472

  16. Albumin Test

    MedlinePlus

    ... may also be ordered to evaluate a person's nutritional status. ^ Back to top When is it ordered? An ... albumin test to check or monitor a person's nutritional status. However, since albumin concentrations respond to a variety ...

  17. Evaluation of capillary zone electrophoresis for the determination of protein composition in therapeutic immunoglobulins and human albumins.

    PubMed

    Christians, Stefan; van Treel, Nadine Denise; Bieniara, Gabriele; Eulig-Wien, Annika; Hanschmann, Kay-Martin; Giess, Siegfried

    2016-07-01

    Capillary zone electrophoresis (CZE) provides an alternative means of separating native proteins on the basis of their inherent electrophoretic mobilities. The major advantage of CZE is the quantification by UV detection, circumventing the drawbacks of staining and densitometry in the case of gel electrophoresis methods. The data of this validation study showed that CZE is a reliable assay for the determination of protein composition in therapeutic preparations of human albumin and human polyclonal immunoglobulins. Data obtained by CZE are in line with "historical" data obtained by the compendial method, provided that peak integration is performed without time correction. The focus here was to establish a rapid and reliable test to substitute the current gel based zone electrophoresis techniques for the control of protein composition of human immunoglobulins or albumins in the European Pharmacopoeia. We believe that the more advanced and modern CZE method described here is a very good alternative to the procedures currently described in the relevant monographs.

  18. Recombinant production of TEV cleaved human parathyroid hormone.

    PubMed

    Audu, Christopher O; Cochran, Jared C; Pellegrini, Maria; Mierke, Dale F

    2013-08-01

    The parathyroid hormone, PTH, is responsible for calcium and phosphate ion homeostasis in the body. The first 34 amino acids of the peptide maintain the biological activity of the hormone and is currently marketed for calcium imbalance disorders. Although several methods for the production of recombinant PTH(1-34) have been reported, most involve the use of cleavage conditions that result in a modified peptide or unfavorable side products. Herein, we detail the recombinant production of (15) N-enriched human parathyroid hormone, (15) N PTH(1-34), generated via a plasmid vector that gives reasonable yield, low-cost protease cleavage (leaving the native N-terminal serine in its amino form), and purification by affinity and size exclusion chromatography. We characterize the product by multidimensional, heteronuclear NMR, circular dichroism, and LC/MS.

  19. Albumin holograms

    NASA Astrophysics Data System (ADS)

    Ordóñez-Padilla, M. J.; Olivares-Pérez, A.; Vega-Criollo, R.; Berriel-Valdos, L. R.; Mejias-Brizuela, N. Y.

    2011-02-01

    A Characterization is made with performance analysis of new photosensitive films of albumin to certain conditions for holographic recording based on interferometric array. We carried out the photo-oxidation of gallus gallus albumin albumin chemically combining powdered sugar (Glass ®) to an aqueous solution of ammonium dichromate. It was the analysis of the behavior of diffraction efficiency parameter through the intensity diffraction pattern produced by the gratings made with albumin.

  20. [Biosynthesis and isolation of a recombinant protein for producing genetically-engineered human proinsulin].

    PubMed

    Ivankin, A N; Mitaleva, S I; Nekliudov, A D

    1998-01-01

    Isolation of the recombinant protein from a genetically engineered Escherichia coli 1854 producer for further chemical enzymatic transformation into human insulin through proinsulin was studied. Under optimal conditions, the recombinant protein formation was more than 35% of the total cell proteins. Structures of the polypeptides obtained and purified chromatographically were confirmed by amino acid analysis. Human proinsulin was derived from the recombinant protein isolated.

  1. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2012-09-01

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

  3. Characterization of interaction between esculin and human serum albumin in membrane mimetic environments

    NASA Astrophysics Data System (ADS)

    Zhang, Yaheng; Li, Jiazhong; Dong, Lijun; Li, Ying; Chen, Xingguo

    2008-10-01

    In this study the interaction between esculin and human serum albumin (HSA) in AOT/isooctane/water microemulsions was studied for the first time using fluorescence quenching technique in combination with UV absorption spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, circular dichroism (CD) spectroscopy and dynamic light scattering (DLS) technique. Fluorescence data in ω o 20 microemulsions revealed the presence of the binding site of esculin on HSA and its binding constants at four different temperatures were obtained. The affinities in microemulsions are similar to that in buffer solution. The alterations of protein secondary structure in the microemulsions in the absence and presence of esculin compared with the free form of HSA in buffer were qualitatively and quantitatively analyzed by the evidence from CD and FT-IR spectroscopes. The displacement experiments confirmed that esculin could bind to the site I of HSA, which was in agreement with the result of the molecular modeling study. Furthermore, the DLS data suggested that HSA may locate at the interface of the microemulsion and esculin could interact with them.

  4. Chiral recognition of metalaxyl enantiomers by human serum albumin: evidence from molecular modeling and photophysical approach.

    PubMed

    Ding, Fei; Li, Xiu-Nan; Diao, Jian-Xiong; Sun, Ye; Zhang, Li; Sun, Ying

    2012-06-01

    Metalaxyl is an acylamine fungicide, belonging to the most widely known member of the amide group. This task is aimed to scrutinize binding region and spatial structural change of principal vector human serum albumin (HSA) complex with (R)-/(S)-metalaxyl by exploiting molecular modeling, steady-state and time-resolved fluorescence, and circular dichroism (CD) approaches. According to molecular modeling, (R)-metalaxyl is situated within subdomains IIA and IIIA and the affinity of site I with (R)-metalaxyl is greater than site II, whereas (S)-metalaxyl is only located at subdomain IIA and the affinity of (S)-metalaxyl with site I is superior compared with that with (R)-metalaxyl. This coincides with the competitive ligand binding, guanidine hydrochloride-induced unfolding of protein, and hydrophobic 8-anilino-1-naphthalenesulfonic acid experiments; the acting forces between (R)-/(S)-metalaxyl and HSA are hydrophobic, π-π interactions, and hydrogen bonds, as derived from molecular modeling. Fluorescence emission manifested that the complex of (R)-/(S)-metalaxyl to HSA is the formation of adduct with an affinity of 10(4) M(-1), which corroborates the time-resolved fluorescence that the static type was operated. Furthermore, the changes of far-UV CD spectra evidence the polypeptide chain of HSA partially unfolded after conjugation with (R)-/(S)-metalaxyl. Through this work, we envisage that it can offer central clues on the biodistribution, absorption, and bioaccumulation of (R)-/(S)-metalaxyl. Copyright © 2012 Wiley Periodicals, Inc.

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

    NASA Astrophysics Data System (ADS)

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

    2016-01-01

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

  6. 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.

  7. 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.

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

    PubMed

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

    2015-06-21

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

  9. Mechanistic investigation of domain specific unfolding of human serum albumin and the effect of sucrose.

    PubMed

    Yadav, Rajeev; Sen, Pratik

    2013-11-01

    This study is devoted to understand the unfolding mechanism of a multidomain protein, human serum albumin (HSA), in absence and presence of the sucrose by steady-state and time-resolved fluorescence spectroscopy with domain specific marker molecules and is further being substantiated by molecular dynamics (MD) simulation. In water, the domain III of HSA found to unfold first followed by domains I and II as the concentration of GnHCl is increased in the medium. The sequential unfolding behavior of different domains of HSA remains same in presence of sucrose; however, a higher GnHCl concentration is required for unfolding, suggesting stabilizing effect of sucrose on HSA. Domain I is found to be most stabilized by sucrose. The stabilization of domain II is somewhat similar to domain I, but the effect of sucrose on domain III is found to be very small. MD simulation also predicted a similar behavior of sucrose on HSA. The stabilizing effect of sucrose is explained in terms of the entrapment of water molecules in between HSA surface and sucrose layer as well as direct interaction between HSA and sucrose.

  10. Targeted non-covalent self-assembled nanoparticles based on human serum albumin

    PubMed Central

    Bunschoten, Anton; Buckle, Tessa; Kuil, Joeri; Luker, Gary D.; Luker, Kathryn E.; Nieweg, Omgo; van Leeuwen, Fijs W. B.

    2015-01-01

    Human serum albumin (HSA) is a biological nanocarrier that forms non-covalent complexes with a number of synthetic and biomolecules. Previously we demonstrated radiolabeled HSA-based nanoparticles can form non-covalent complexes with fluorescent cyanine dyes yielding imaging agents for surgical guidance towards tumor draining lymph nodes. Here the self-assembly approach enabled rapid clinical translation. Based on this experience we reasoned it would be interesting to expand this non-covalent technology to a targeted approach. The ability of HSA to form non-covalent self-assembled complexes with peptides via near-infrared (NIR) cyanine dyes was explored. Föster resonance energy transfer (FRET) quenching interactions between HSA-Cy5 and the non-covalently bound fluorescent molecules indocyanine green (ICG), IR783-CO2H and three IR783-labeled targeting peptides were used to monitor complex assembly and disassembly. The host-guest interactions between HSA and IR783-labeled peptides enabled the formation of (bio)nanoparticles that are coated with peptides that may target αvβ3-integrins, the chemokine receptor 4 (CXCR4), and somatostatin receptors. The potential of CXCR4-targeted (bio)nanoparticles in sentinel lymph node procedures is demonstrated. By non-covalently binding NIR-dye labeled peptides to an already clinically approved HSA-scaffold, we have readily formed targeted bionanoparticles. PMID:22024362

  11. 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.

  12. Simple, fast preparation of gallium-68-labelled human serum albumin microspheres.

    PubMed

    Yvert, J P; Mazière, B; Verhas, M; Comar, D

    1979-04-01

    Following a study of the main factors involved in the 68-Ga labelling of human serum albumin microspheres (H.S.A.M.), especially methods of production and preparation of active solution and conditions of radioelement fixation on the protein support, the practical details of a fast technique (60 min) based on the process described by Hnatowich are presented. This method gives high labelling yields (93 +/- 3%), and after washing of the microspheres leads to a radiopharmaceutical product almost without free 68Ga (less than 2%). The spheres ready for use carry a total radioactivity corresponding to about 35%, including decay, of the activity originally recovered in the generator eluate and to more than 98% of that, found in the final suspension. The labelled product is sterile, non-pyrogenic and non-toxic. When it is injected in animals by left ventrical catheterization the uptake rates in the heart, lungs, spleen, left kidney and right kidney are similar to those observed with reference 85Sr-labelled carbonized microspheres. This radiopharmaceutical, easy to prepare and having excellent biological and nuclear properties, seems ideally suited for the scanning of organs by position emission tomoscintigraphy.

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

    PubMed

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

    2001-08-30

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

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

    PubMed

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

    2005-08-18

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

  15. Suppression of agglomeration of ciprofloxacin-loaded human serum albumin nanoparticles.

    PubMed

    Kumar, P Vijayaraj; Jain, Narendr K

    2007-03-02

    The present study is aimed at developing and exploring the use of pectin in suppression of agglomeration of ciprofloxacin-loaded human serum albumin (HSA) nanoparticles. The HSA-pectin nanoparticles loaded with ciprofloxacin were prepared by the pH-coacervation method, and various physicochemical parameters such as particle size, morphology, zeta-potential, electrolyte-induced flocculation, pH-dependent zeta-potential, drug loading, in vitro drug release, and stability of nanoparticles, were evaluated. The size of the HSA-pectin nanoparticles (F3) was found to be 180 to 290 nm. The HSA nanoparticles were modified with pectin when the critical flocculation concentration of nanoparticles in Na(2)SO(4) solution was increased from 0.3 M to 0.9 M. The isoelectric points of the formed nanoparticles were found to be relatively lower between pH values 3 and 6. Pectin may be used as a pharmaceutical additive for the suppression of particle agglomeration in HSA nanoparticles, and the effect may be attributed to the pectin segments present on the surface of nanoparticles.

  16. (99m)Tc-human serum albumin nanocolloids: particle sizing and radioactivity distribution.

    PubMed

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

    2015-07-01

    Several parameters affect the biodistribution of administered nanocolloids (NC) for Sentinel Lymph Node (SLN) detection: particle size distribution, number of Tc atoms per particle and specific activity (SA). Relatively few data are available with frequently conflicting results. (99m)Tc-NC-human serum albumin (HSA) Nanocoll®, Nanoalbumon® and Nanotop® were analysed for particles' dimensional and radioactivity distribution, and a mathematical model was elaborated to estimate the number of particles involved. Commercially available kits were reconstituted at maximal SA of 11 MBq/µg HSA. Particles size distribution was evaluated by Dynamic Light Scattering. These data were related to the radioactivity distribution analysis passing labelled NC through three polycarbonate filters (15-30-50-nm pore size) under vacuum. Highest radioactivity was carried by 30-50 nm particles. The smallest ones, even though most numerous, carried only the 10% of (99m)Tc atoms. Nanocoll and Nanotop are not significantly different, while Nanoalbumon is characterized by largest particles (>30 nm) that carried the most of radioactivity (80%). Smallest particles could saturate the clearing capacity of macrophages; therefore, if the tracer is used for SLN detection, more node tiers could be visualized, reducing accuracy of SLN mapping. Manufacturers could implement technical leaflets with particle size distribution and could improve the labelling protocol to provide clinicians useful information. Copyright © 2015 John Wiley & Sons, Ltd.

  17. Immune response to acetaldehyde-human serum albumin adduct among healthy subjects related to alcohol intake.

    PubMed

    Romanazzi, Valeria; Schilirò, Tiziana; Carraro, Elisabetta; Gilli, Giorgio

    2013-09-01

    Acetaldehyde (AA) is the main metabolic product in ethanol metabolism, although it can also derive from sources of airborne pollution. As a typical aldehyde, AA is able to react with a variety of molecular targets, including DNA and protein. This property justifies the hypothesis of a immune reaction against this kind of adduct, to be studied by a seroprevalence screening approach. In this study, the correlation between drinking habits and the amount of circulating AA-human serum albumin adduct (AA-HSA) was evaluated in a group of healthy subjects, non alcohol-addicted. Daily ethanol intake (grams) was inferred for each subject using the information collected through a questionnaire, and AA-HSA antibodies (AA-HSA ab) analyses were performed using the Displacement Assay on whole blood samples. The findings showed a correlation between ethanol intake and immune response to molecular adduct. These results underscore the evaluation of AA-HSA ab amount as a suitable molecular marker for alcohol intake that can be applied in future investigations on a large scale for prevention screening.

  18. Molecular interaction study of flavonoids with human serum albumin using native mass spectrometry and molecular modeling.

    PubMed

    Wang, Bohong; Qin, Qian; Chang, Mengmeng; Li, Shuyan; Shi, Xianzhe; Xu, Guowang

    2017-08-24

    Noncovalent interactions between proteins and small-molecule ligands widely exist in biological bodies and play significant roles in many physiological and pathological processes. Native mass spectrometry (MS) has emerged as a new powerful tool to study noncovalent interactions by directly analyzing the ligand-protein complexes. In this work, an ultrahigh-resolution native MS method based on a 15-T SolariX XR Fourier transform ion cyclotron resonance mass spectrometer was firstly used to investigate the interaction between human serum albumin (HSA) and flavonoids. Various flavonoids with similar structure were selected to unravel the relationship between the structure of flavonoids and their binding affinity for HSA. It was found that the position of the hydroxyl groups and double bond of flavonoids could influence the noncovalent interaction. Through a competitive experiment between HSA binding site markers and apigenin, the subdomain IIA (site 1) of HSA was determined as the binding site for flavonoids. Moreover, a cooperative allosteric interaction between apigenin and ibuprofen was found from their different HSA binding sites, which was further verified by circular dichroism spectroscopy and molecular docking studies. These results show that native MS is a useful tool to investigate the molecular interaction between a protein and its ligands. Graphical abstract Unravel the relationship between the structure of flavonoids and their binding affinity to HSA by native MS.

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

    PubMed

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

    2012-09-01

    The interaction between Daphnin with human serum albumin has been studied for the first time by spectroscopic methods including fluorescence quenching technology, circular dichroism (CD) spectroscopy and Fourier transform infrared (FT-IR) spectroscopy under simulative physiological conditions. The results of fluorescence titration revealed that Daphnin can quench the intrinsic fluorescence of HSA by static quenching and there is a single class of binding site on HSA. In addition, the studies of CD spectroscopy and FT-IR spectroscopy showed that the protein secondary structure changed with increases of α-helices at the drug to protein molar ratio of 2. Furthermore, the thermodynamic functions ΔH(0) and ΔS(0) for the reaction were calculated to be 11.626 kJ mol(-1) and 118.843 J mol(-1)K(-1) according to Van't Hoff equation. The thermodynamic parameters (ΔH(0) and ΔS(0)) and the molecular modeling study indicated that hydrophobic force played an important role to stabilize the Daphnin-HSA complex, and Daphnin could bind within the subdomain IIA of the HSA. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. 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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2016-01-05

    The interaction and binding behavior of the well-known drug sulfadiazine (SDZ) and psychoactive stimulant caffeine (CAF) with human serum albumin (HSA) was monitored by in vitro fluorescence titration and molecular docking calculations under physiological condition. The quenching of protein fluorescence on addition of CAF is due to the formation of protein-drug complex in the ground state; whereas in case of SDZ, the experimental results were explained on the basis of sphere of action model. Although both these compounds bind preferentially in Sudlow's site 1 of the protein, the association constant is approximately two fold higher in case of SDZ (∼4.0×10(4)M(-1)) in comparison with CAF (∼9.3×10(2)M(-1)) and correlates well with physico-chemical properties like pKa and lipophilicity of the drugs. Temperature dependent fluorescence study reveals that both SDZ and CAF bind spontaneously with HSA. However, the binding of SDZ with the protein is mainly governed by the hydrophobic forces in contrast with that of CAF; where, the interaction is best explained in terms of electrostatic mechanism. Molecular docking calculation predicts the binding of these drugs in different location of sub-domain IIA in the protein structure. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Enhanced Gene Silencing through Human Serum Albumin-Mediated Delivery of Polyethylenimine-siRNA Polyplexes

    PubMed Central

    Nicolì, Elena; Syga, Marie Isabel; Bosetti, Michela; Shastri, V. Prasad

    2015-01-01

    Small interfering RNA (siRNA) targeted therapeutics (STT) offers a compelling alternative to tradition medications for treatment of genetic diseases by providing a means to silence the expression of specific aberrant proteins, through interference at the expression level. The perceived advantage of siRNA therapy is its ability to target, through synthetic antisense oligonucleotides, any part of the genome. Although STT provides a high level of specificity, it is also hindered by poor intracellular uptake, limited blood stability, high degradability and non-specific immune stimulation. Since serum proteins has been considered as useful vehicles for targeting tumors, in this study we investigated the effect of incorporation of human serum albumin (HSA) in branched polyethylenimine (bPEI)-siRNA polyplexes in their internalization in epithelial and endothelial cells. We observed that introduction of HSA preserves the capacity of bPEI to complex with siRNA and protect it against extracellular endonucleases, while affording significantly improved internalization and silencing efficiency, compared to bPEI-siRNA polyplexes in endothelial and metastatic breast cancer epithelial cells. Furthermore, the uptake of the HSA-bPEI-siRNA ternary polyplexes occurred primarily through a caveolae-mediated endocytosis, thus providing evidence for a clear role for HSA in polyplex internalization. These results provide further impetus to explore the role of serum proteins in delivery of siRNA. PMID:25856158

  4. 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.

  5. Ruthenium (II) complexes interact with human serum albumin and induce apoptosis of tumor cells.

    PubMed

    Sun, Jing; Huang, Yongchao; Zheng, Chuping; Zhou, Yanhui; Liu, Ying; Liu, Jie

    2015-02-01

    The interaction of ruthenium (II) complex [Ru(bpy)2(mal)](2+) (RBM) and [Ru(phen)2(mal)](2+) (RPM) (bpy = 2, 2-bipyridine, phen = 1,10-phenanthroline, mal = malonyl carboxylate) with human serum albumin (HSA) has been investigated by using fluorescence, UV absorption and circular dichroism (CD) spectroscopy approaches. A strong fluorescence quenching reaction of complexes to HSA was observed and the quenching mechanism was suggested as static quenching according to the Stern-Volmer (S-V) equation. The number of binding sites n and observed binding constant Kb was measured by fluorescence quenching method. The thermodynamic parameters ΔH, ΔS, and ΔG at different temperatures were calculated and the results indicate the binding reaction is mainly entropy-driven and Vander Waals force played a major role in the reaction. The result of CD showed that the secondary structure of HSA molecules was changed in the presence of the ruthenium (II) complexes. Furthermore, the cell viability of ruthenium (II) complexes was evaluated by MTT and complex RPM has shown significant higher anticancer potency than RBM against all the cell lines screened. RPM showed a significant antitumor activity through induction of apoptosis in A549 cells.

  6. 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.

  7. 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.

  8. Effects of titania nanotubes with or without bovine serum albumin loaded on human gingival fibroblasts

    PubMed Central

    Liu, Xiangning; Zhou, Xiaosong; Li, Shaobing; Lai, Renfa; Zhou, Zhiying; Zhang, Ye; Zhou, Lei

    2014-01-01

    Modifying the surface of the transmucosal area is a key research area because this process positively affects the three functions of implants: attachment to soft tissue, inhibiting bacterial biofilm adhesion, and the preservation of the crestal bone. To exploit the potential of titania nanotube arrays (TNTs) with or without using bovine serum albumin (BSA) to modify the surface of a dental implant in contact with the transmucosal area, BSA was loaded into TNTs that were fabricated by anodizing Ti sheets; the physical characteristics of these arrays, including their morphology, chemical composition, surface roughness, contact angle, and surface free energy (SFE), were assessed. The effect of Ti surfaces with TNTs or TNTs-BSA on human gingival fibroblasts (HGFs) was determined by analyzing cell morphology, early adhesion, proliferation, type I collagen (COL-1) gene expression, and the extracellular secretion of COL-1. The results indicate that early HGF adhesion and spreading behavior is positively correlated with surface characteristics, including hydrophilicity, SFE, and surface roughness. Additionally, TNT surfaces not only promoted early HGF adhesion, but also promoted COL-1 secretion. BSA-loaded TNT surfaces promoted early HGF adhesion, while suppressing late proliferation and COL-1 secretion. Therefore, TNT-modified smooth surfaces are expected to be applicable for uses involving the transmucosal area. Further study is required to determine whether BSA-loaded TNT surfaces actually affect closed loop formation of connective tissue because BSA coating actions in vivo are very rapid. PMID:24623977

  9. 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

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

    PubMed

    Li, Jinyu; Jiang, Lizhi; Zhu, Xiaolei

    2015-07-01

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

  11. Noncovalent interactions between a trinuclear monofunctional platinum complex and human serum albumin.

    PubMed

    Wang, Yanqing; Wang, Xiaoyong; Wang, Jing; Zhao, Yongmei; He, Weijiang; Guo, Zijian

    2011-12-19

    Interactions between platinum complexes and human serum albumin (HSA) play crucial roles in the metabolism, distribution, and efficacy of platinum-based anticancer drugs. Polynuclear monofunctional platinum(II) complexes represent a new class of anticancer agents that display distinct molecular characters of pharmacological action from those of cisplatin. In this study, the interaction between a trinuclear monofunctional platinum(II) complex, [Pt(3)LCl(3)](ClO(4))(3) (L = N,N,N',N',N",N"-hexakis(2-pyridylmethyl)-1,3,5-tris(aminomethyl)benzene) (1), and HSA was investigated using ultraviolet-visible spectroscopy, Fourier transform infrared spectroscopy, circular dichroism spectroscopy, fluorescence spectroscopy, molecular docking, and inductively coupled plasma mass spectrometry. The spectroscopic and thermodynamic data show that the interaction is a spontaneous process with the estimated enthalpy and entropy changes being 14.6 kJ mol(-1) and 145.5 J mol(-1) K(-1), respectively. The reactive sites of HSA to complex 1 mainly locate within its hydrophobic cavity in domain II. Noncovalent actions such as π-π stacking and hydrophobic bonding are the primary contributors to the interaction between HSA and complex 1, which is different from the scenario for cisplatin in similar conditions. The results suggest that the connection between complex 1 and HSA is reversible, and therefore the cytotoxic activity of the complex could be preserved during blood circulation.

  12. Effect of (−)-Epigallocatechin-3-Gallate on Glucose-Induced Human Serum Albumin Glycation

    PubMed Central

    Li, Min; Hagerman, Ann E.

    2016-01-01

    (−)-Epigallocatechin-3-gallate (EGCg) is a naturally occurring polyphenol found in plant-based foods and beverages such as green tea. Although EGCg can eliminate carbonyl species produced by glucose autoxidation and thus can inhibit protein glycation, it is also reported to be a pro-oxidant that stimulates protein glycation in vitro. To better understand the balance between antioxidant and pro-oxidant features of EGCg, we evaluated EGCg-mediated bioactivities in a human serum albumin (HSA)/glucose model by varying three different parameters (glucose level, EGCg concentration, and time of exposure to EGCg). Measurements of glycation-induced fluorescence, protein carbonyls, and electrophoretic mobility showed that the level of HSA glycation was positively related to the glucose level over the range 10 to 100 mM during a 21-day incubation at 37 °C and pH 7.4. Under mild glycemic pressure (10 mM), long exposure to EGCg enhanced HSA glycation, while brief exposure to low concentrations of EGCg did not. Under high glycemic pressure (100 mM glucose), long exposure to EGCg inhibited glycation. For the first time we showed that brief exposure to EGCg reversed glycation-induced fluorescence, indicating a restorative effect. In conclusion, our research identified glucose level, EGCg concentration, and time of exposure as critical factors dictating EGCg bioactivities in HSA glycation. EGCg did not affect HSA glycation under normal physiological conditions but had a potential therapeutic effect on HSA severely damaged by glycation. PMID:25794449

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

    PubMed

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

    2008-08-01

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

  14. Comparative study of flavins binding with human serum albumin: a fluorometric, thermodynamic, and molecular dynamics approach.

    PubMed

    Sengupta, Abhigyan; Sasikala, Wilbee D; Mukherjee, Arnab; Hazra, Partha

    2012-06-04

    Flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) are derivatives of riboflavin (RF), a water-soluble vitamin, more commonly known as vitamin B(2). Flavins have attracted special attention in the last few years because of the recent discovery of a large number of flavoproteins. In this work, these flavins are used as extrinsic fluorescence markers for probing the microheterogeneous environment of a well-known transport protein, human serum albumin (HSA). Steady-state and time-resolved fluorescence experiments confirm that both FMN and FAD bind to the Sudlow's site-1 (SS1) binding pocket of HSA, where Trp214 resides. In the case of RF, a fraction of RF molecules binds at the SS1, whereas the major fraction of RF molecules remains unbound or surface bound to the protein. Moreover, flavin(s)-HSA interactions are monitored with the help of isothermal titration calorimetry, which provides free energy, enthalpy, and entropy changes of binding along with the binding constants. The molecular picture of binding interaction between flavins and HSA is well explored by docking and molecular dynamics studies. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. 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.

  16. Effects of titania nanotubes with or without bovine serum albumin loaded on human gingival fibroblasts.

    PubMed

    Liu, Xiangning; Zhou, Xiaosong; Li, Shaobing; Lai, Renfa; Zhou, Zhiying; Zhang, Ye; Zhou, Lei

    2014-01-01

    Modifying the surface of the transmucosal area is a key research area because this process positively affects the three functions of implants: attachment to soft tissue, inhibiting bacterial biofilm adhesion, and the preservation of the crestal bone. To exploit the potential of titania nanotube arrays (TNTs) with or without using bovine serum albumin (BSA) to modify the surface of a dental implant in contact with the transmucosal area, BSA was loaded into TNTs that were fabricated by anodizing Ti sheets; the physical characteristics of these arrays, including their morphology, chemical composition, surface roughness, contact angle, and surface free energy (SFE), were assessed. The effect of Ti surfaces with TNTs or TNTs-BSA on human gingival fibroblasts (HGFs) was determined by analyzing cell morphology, early adhesion, proliferation, type I collagen (COL-1) gene expression, and the extracellular secretion of COL-1. The results indicate that early HGF adhesion and spreading behavior is positively correlated with surface characteristics, including hydrophilicity, SFE, and surface roughness. Additionally, TNT surfaces not only promoted early HGF adhesion, but also promoted COL-1 secretion. BSA-loaded TNT surfaces promoted early HGF adhesion, while suppressing late proliferation and COL-1 secretion. Therefore, TNT-modified smooth surfaces are expected to be applicable for uses involving the transmucosal area. Further study is required to determine whether BSA-loaded TNT surfaces actually affect closed loop formation of connective tissue because BSA coating actions in vivo are very rapid.

  17. 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.

  18. Fluorescence study on site-specific binding of perfluoroalkyl acids to human serum albumin.

    PubMed

    Chen, Yan-Min; Guo, Liang-Hong

    2009-03-01

    Binding of five perfluoroalkyl acids with human serum albumin (HSA) was investigated by site-specific fluorescence. Intrinsic fluorescence of tryptophan-214 in HSA was monitored upon addition of the chemicals. Although perfluorobutyl acid (PFBA) and perfluorobutane sulfonate (PFBS) did not cause fluorescence change, perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), and perfluorododecanoic acid (PFDoA) induced fluorescence quenching, from which binding constant of 2.7 x 10(5) M(-1) for PFOA and 2.2 x 10(4) M(-1) for PFOS was calculated. Two fluorescent probes, dansylamide (DA) and dansyl-L: -proline (DP), were employed in fluorescence displacement measurements to study the interaction at two Sudlow's binding sites. At Site I, both PFBA and PFBS displaced DA with binding constants of 1.0 x 10(6) M(-1) and 2.2 x 10(6) M(-1). At Site II, PFBS and PFDoA displaced DP with binding constants of 6.5 x 10(6) M(-1) and 1.2 x 10(6) M(-1), whereas PFBA did not bind. The data were compared with fatty acids to evaluate the potential toxicological effect of these environmental chemicals.

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

    NASA Astrophysics Data System (ADS)

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

    2008-10-01

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

  20. Ibuprofen and propofol cobinding effect on human serum albumin unfolding in urea.

    PubMed

    Del Giudice, Alessandra; Leggio, Claudia; Balasco, Nicole; Galantini, Luciano; Pavel, Nicolae V

    2014-08-28

    The unfolding pathway of the defatted human serum albumin (HSA) binding ibuprofen and propofol has been studied by using small-angle X-ray scattering (SAXS) and the support of circular dichroism data. A set of HSA solutions with urea concentrations between 0.00 and 9.00 M was analyzed, and the singular value decomposition method applied to the complete SAXS data set allowed us to distinguish four different states in solution. Besides the native and unfolded forms, two intermediates I1 and I2 have been identified, and the low-resolution structures of these states were obtained by exploiting both ab initio and rigid body fitting methods. The I1 structure was characterized by only one open domain (domain I, which does not host a binding site for either of the ligands), whereas I2 presents only one closed domain (domain III). A direct comparison with the unfolding pathway of the HSA:Ibu complex (Galantini et al. Biophys. Chem. 2010, 147, 111-122) pointed out that the presence of propofol as a second ligand, located in subdomain IIIB, leads to the appearance of an intermediate with two closed domains (domains II and III), which are those that accommodate the ligands. Moreover, the equilibrium between I2 and the unfolded form is slightly shifted toward higher urea concentrations. These results suggest that the cobinding significantly hinders the unfolding process.

  1. Oxidation Enhances Human Serum Albumin Thermal Stability and Changes the Routes of Amyloid Fibril Formation

    PubMed Central

    Sancataldo, Giuseppe; Vetri, Valeria; Foderà, Vito; Di Cara, Gianluca; Militello, Valeria; Leone, Maurizio

    2014-01-01

    Oxidative damages are linked to several aging-related diseases and are among the chemical pathways determining protein degradation. Specifically, interplay of oxidative stress and protein aggregation is recognized to have a link to the loss of cellular function in pathologies like Alzheimer's and Parkinson's diseases. Interaction between protein and reactive oxygen species may indeed induce small changes in protein structure and lead to the inhibition/modification of protein aggregation process, potentially determining the formation of species with different inherent toxicity. Understanding the temperate relationship between these events can be of utmost importance in unraveling the molecular basis of neurodegeneration. In this work, we investigated the effect of hydrogen peroxide oxidation on Human Serum Albumin (HSA) structure, thermal stability and aggregation properties. In the selected conditions, HSA forms fibrillar aggregates, while the oxidized protein undergoes aggregation via new routes involving, in different extents, specific domains of the molecule. Minute variations due to oxidation of single residues affect HSA tertiary structure leading to protein compaction, increased thermal stability, and reduced association propensity. PMID:24416244

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

    PubMed

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

    2015-12-05

    Human serum albumin (HSA)-drug binding is an important factor to determine half life and bioavailability of drugs. In the present research, the interaction of sertraline (SER) to HSA was investigated using combination of spectroscopic and molecular modeling techniques. Changes in the UV-Vis, CD and FT-IR spectra as well as a significant degree of tryptophan fluorescence quenching were observed upon SER-HSA interaction. Data obtained by spectroscopic methods along with the computational studies suggest that SER binds to residues located in subdomain IIA of HSA. Analysis of spectroscopic data represented the formation of 1:1 complex, significant binding affinity, negative values of entropy and enthalpy changes and the essential role of hydrophobic interactions in binding of SER to HSA. The binding models were demonstrated in the aspects of SER's conformation, active site interactions, important amino acids and hydrogen bonding. Computational mapping of the possible binding site of SER confirmed that the ligand to be bound in a large hydrophobic cavity of HSA. In accordance with experimental data, computational analyses indicated that SER binding does not alter the secondary structure of the protein. The results not only lead to a better understanding of interaction between SER and HSA but also provide useful data about the influence of SER on the protein conformation. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  3. 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.

  4. [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.

  5. [Regularities of formation of chlorophyll-human serum albumin functionally active complexes in the aqueous medium].

    PubMed

    Semichaevskiĭ, V D

    1975-01-01

    In the system with constant content of the chlorophyll a and increasing amounts of human serum albumin, dependence of pigment incorporation into the complex upon interaction of its aqueous associates with protein solutions was studied by applying the gel filtration on Sephadex G-75 and by measuring light scattering and rate of sensitized photoreduction of the methyl red by ascorbic-acid. The curves were obtained after extraction of the chlorophyll by acetone from dry pigment-protein films formed after desiccation of the aqueous systems. Sigmoid character of the above dependences, their linearization in Hill's coordinates and the value of cooperativity coefficient close to 2 testifies in favour of the cooperative character of the complex formation, two pigment molecules reacting with a single protein molecule. Measurement of adsorption isotherms and their treatment with use of the Brunauer-Emmett-Teller theory of polymolecular adsorption make it possible to evaluate the maximum molar ratio of the pigment to the protein in the complex (close to 2). The pigment-pigment interaction suggests that the chlorophyll molecules adsorbed on the protein are in the state of loosely packed dimers. Deaggregation of aqueus pigment associates by the protein in the course of complex formation results in a considerable increase of the protosensitizing chlorophyll activity.

  6. Protective effects of curcumin against gamma ray induced conformational change of human serum albumin.

    PubMed

    Kar, Turban; Basak, Pijush; Ghosh, Rittik Kumar; Bhattacharyya, Maitree

    2017-06-01

    This study explores the possibility for protection by curcumin during the molecular and structural changes of human serum albumin (HSA) exposed to gamma irradiation. We used a combination of spectroscopic methods to probe the conformational ensemble of the irradiated HSA and finally evaluated the extent of restoration by curcumin. SDS-PAGE study unfolded the formation of cross linked aggregates as a consequence of increasing exposure of gamma radiation. CD and FTIR spectroscopy indicated significant decrease in the alpha helix content of HSA from 57% to 15% with increasing radiation doses. Steady state and time resolved fluorescence studies complemented the spectroscopic measurements, when lifetime decay was significantly reduced from 6.35ns to 0.37ns. Hydrophobic study showed the effectiveness of curcumin for protection at low dose of gamma irradiated HSA samples. We integrated these observations to investigate protein aggregation under increasing gamma radiation and estimated the same in presence of curcumin. It was elucidated, that when HSA is irradiated at low dose of gamma radiation in presence of curcumin, it is capable of retaining the characteristic properties to a higher extent indicating stabilization of molecular structure of HSA by curcumin. A model for curcumin based protection has been proposed utilizing ThT assay. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Encapsulating gold nanomaterials into size-controlled human serum albumin nanoparticles for cancer therapy platforms.

    PubMed

    Peralta, Donna V; He, Jibao; Wheeler, Damon A; Zhang, Jin Z; Tarr, Matthew A

    2014-01-01

    Progress has been made in using human serum albumin nanoparticles (HSAPs) as promising colloidal carrier systems for early detection and targeted treatment of cancer and other diseases. Despite this success, there is a current lack of multi-functional HSAP hybrids that offer combinational therapies. The size of the HSAPs has crucial importance on drug loading and in vivo performance and has previously been controlled via manipulation of pH and cross-linking parameters. Gold nanomaterials have also gained attention for medicinal use due to their ability to absorb near-infrared light, thus offering photothermal capabilities. In this study, the desolvation and cross-linking approach was employed to encapsulate gold nanorods, nanoparticles, and nanoshells into HSAPs. Incorporation of gold nanomaterials caused some changes in HSAP sizes, but the general size trends remained. This encasement strategy facilitated size-controlled HSAPs, in the range of 100-300 nm, loaded with gold nanostructures; providing composite particles which incorporate photothermally active components.

  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. 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).

  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. Structural characteristics of green tea catechins for formation of protein carbonyl in human serum albumin.

    PubMed

    Ishii, Takeshi; Mori, Taiki; Ichikawa, Tatsuya; Kaku, Maiko; Kusaka, Koji; Uekusa, Yoshinori; Akagawa, Mitsugu; Aihara, Yoshiyuki; Furuta, Takumi; Wakimoto, Toshiyuki; Kan, Toshiyuki; Nakayama, Tsutomu

    2010-07-15

    Catechins are polyphenolic antioxidants found in green tea leaves. Recent studies have reported that various polyphenolic compounds, including catechins, cause protein carbonyl formation in proteins via their pro-oxidant actions. In this study, we evaluate the formation of protein carbonyl in human serum albumin (HSA) by tea catechins and investigate the relationship between catechin chemical structure and its pro-oxidant property. To assess the formation of protein carbonyl in HSA, HSA was incubated with four individual catechins under physiological conditions to generate biotin-LC-hydrazide labeled protein carbonyls. Comparison of catechins using Western blotting revealed that the formation of protein carbonyl in HSA was higher for pyrogallol-type catechins than the corresponding catechol-type catechins. In addition, the formation of protein carbonyl was also found to be higher for the catechins having a galloyl group than the corresponding catechins lacking a galloyl group. The importance of the pyrogallol structural motif in the B-ring and the galloyl group was confirmed using methylated catechins and phenolic acids. These results indicate that the most important structural element contributing to the formation of protein carbonyl in HSA by tea catechins is the pyrogallol structural motif in the B-ring, followed by the galloyl group. The oxidation stability and binding affinity of tea catechins with proteins are responsible for the formation of protein carbonyl, and consequently the difference in these properties of each catechin may contribute to the magnitude of their biological activities.

  12. 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.

  13. 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.

  14. Comparison of interactions between human serum albumin and silver nanoparticles of different sizes using spectroscopic methods.

    PubMed

    Zhang, Wanju; Zhang, Qingbo; Wang, Fang; Yuan, Lian; Xu, Ziqiang; Jiang, Fenglei; Liu, Yi

    2015-06-01

    Three different sizes (15.9 ± 2.1 nm, 26.4 ± 3.2 nm and 39.8 ± 4.0 nm, respectively) of citrate-coated silver nanoparticles (SNPs) have been synthesized and characterized. The interactions of the synthesized SNPs with human serum albumin (HSA) at physiological pH have been systematically studied by UV-vis absorption spectroscopy, fluorescence spectroscopy, synchronous fluorescence spectroscopy, three-dimensional fluorescence spectroscopy and circular dichroism (CD) spectroscopy. The results indicate that the SNPs can bind to HSA with high affinity and quench the intrinsic fluorescence of HSA. The binding constants and quenching rate constants were calculated. The apparent association constants (Kapp ) values are 2.14 × 10(4) M(-1) for 15.9 nm SNP, 1.65 × 10(4) M(-1) for 26.4 nm SNP and 1.37 × 10(4) M(-1) for 39.8 nm SNP, respectively. The values of binding constant obtained from the fluorescence quenching data match well with that determined from the absorption spectral changes. These results suggest that the smaller SNPs have stronger interactions to HSA than the larger ones at the same concentrations. Synchronous fluorescence, three-dimensional fluorescence and CD spectroscopy studies show that the synthesized SNPs can induce slight conformational changes in HSA.

  15. Investigating the movement of intravitreal human serum albumin nanoparticles in the vitreous and retina.

    PubMed

    Kim, Hyuncheol; Robinson, Shaun B; Csaky, Karl G

    2009-02-01

    To investigate the movement of intravitreally injected human serum albumin nanoparticles (HSA-NP) with respect to nanoparticle surface charge and retinal injury. HSA-NPs were developed by a desolvation technique. HSA-NPs were cationized by covalent coupling of hexamethylenediamine on the particle surface. Either anionic or cationic HSA-NPs were injected to determine the effect of surface charge on intravitreal nanoparticle movement. HSA-NPs were injected intravitreally into both normal and laser photocoagulated eyes to examine the effect of the integrity of retinal tissue on the retinal penetration. The retinal penetration of fluorescence labeled anionic HSA-NPs was investigated by confocal microscopy. Anionic particles (-33.3+/-6.1 mV) more easily diffused through the 3-dimensional vitreal network of collagen fibrils than did their cationic counterparts (11.7+/-7.2 mV). In the laser photocoagulated retina, more HSA-NPs were detected in the choroidal space, compared to the normal retina. The immunohistochemical studies indicated that HSA-NPs were taken up into Müller cells. The movement of intravitreal nanoparticles depended on both nanoparticles surface charge and retinal injury. The Müller cells might play an important role in the retinal penetration of nanoparticles. The anionic HSA-NP is a promising drug or gene delivery carrier to the sub-retinal space and RPE.

  16. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2014-09-01

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

  18. Binding of doxyl stearic spin labels to human serum albumin: an EPR study.

    PubMed

    Pavićević, Aleksandra A; Popović-Bijelić, Ana D; Mojović, Miloš D; Šušnjar, Snežana V; Bačić, Goran G

    2014-09-18

    The binding of spin-labeled fatty acids (SLFAs) to the human serum albumin (HSA) examined by electron paramagnetic resonance (EPR) spectroscopy was studied to evaluate the potential of the HSA/SLFA/EPR technique as a biomarking tool for cancer. A comparative study was performed on two spin labels with nitroxide groups attached at opposite ends of the fatty acid (FA) chain, 5-doxyl stearic (5-DS) and 16-doxyl stearic (16-DS) acid. The effects of incubation time, different [SLFA]/[HSA] molar ratios, ethanol, and temperature showed that the position of the nitroxide group produces certain differences in binding between the two SLFAs. Spectra for different [SLFA]/[HSA] molar ratios were decomposed into two spectral components, which correspond to the weakly and strongly bound SLFAs. The reduction of SLFA with ascorbate showed the existence of a two component process, fast and slow, confirming the decomposition results. Warfarin has no effect on the binding of the two SLFAs, whereas ibuprofen significantly decreases the binding of 5-DS and has no effect on 16-DS. Together, the results of this study indicate that both SLFAs, 5-DS and 16-DS, should be used for the study of HSA conformational changes in blood induced by various medical conditions.

  19. Interaction of [V(IV) O(acac)2 ] with Human Serum Transferrin and Albumin.

    PubMed

    Correia, Isabel; Chorna, Ielyzaveta; Cavaco, Isabel; Roy, Somnath; Kuznetsov, Maxim L; Ribeiro, Nádia; Justino, Gonçalo; Marques, Fernanda; Santos-Silva, Teresa; Santos, Marino F A; Santos, Hugo M; Capelo, José L; Doutch, James; Pessoa, João Costa

    2017-08-17

    [VO(acac)2 ] is a remarkable vanadium compound and has potential as a therapeutic drug. It is important to clarify how it is transported in blood, but the reports addressing its binding to serum proteins have been contradictory. We use several spectroscopic and mass spectrometric techniques (ESI and MALDI-TOF), small-angle X-ray scattering and size exclusion chromatography (SEC) to characterize solutions containing [VO(acac)2 ] and either human serum apotransferrin (apoHTF) or albumin (HSA). DFT and modeling protein calculations are carried out to disclose the type of binding to apoHTF. The measured circular dichroism spectra, SEC and MALDI-TOF data clearly prove that at least two VO-acac moieties may bind to apoHTF, most probably forming [V(IV) O(acac)(apoHTF)] complexes with residues of the HTF binding sites. No indication of binding of [VO(acac)2 ] to HSA is obtained. We conclude that V(IV) O-acac species may be transported in blood by transferrin. At very low complex concentrations speciation calculations suggest that [(VO)(apoHTF)] species form. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Fibrillar disruption by AC electric field induced oscillation: A case study with human serum albumin.

    PubMed

    Sen, Shubhatam; Chakraborty, Monojit; Goley, Snigdha; Dasgupta, Swagata; DasGupta, Sunando

    2017-07-01

    The effect of oscillation induced by a frequency-dependent alternating current (AC) electric field to dissociate preformed amyloid fibrils has been investigated. An electrowetting-on-dielectric type setup has been used to apply the AC field of varying frequencies on preformed fibrils of human serum albumin (HSA). The disintegration potency has been monitored by a combination of spectroscopic and microscopic techniques. The experimental results suggest that the frequency of the applied AC field plays a crucial role in the disruption of preformed HSA fibrils. The extent of stress generated inside the droplet due to the application of the AC field at different frequencies has been monitored as a function of the input frequency of the applied AC voltage. This has been accomplished by assessing the morphology deformation of the oscillating HSA fibril droplets. The shape deformation of the oscillating droplets is characterized using image analysis by measuring the dynamic changes in the shape dependent parameters such as contact angle and droplet footprint radius and the amplitude. It is suggested that the cumulative effects of the stress generated inside the HSA fibril droplets due to the shape deformation induced hydrodynamic flows and the torque induced by the intrinsic electric dipoles of protein due to their continuous periodic realignment in presence of the AC electric field results in the destruction of the fibrillar species. Copyright © 2017. Published by Elsevier B.V.

  1. Quantification of specific glycation sites in human serum albumin as prospective type 2 diabetes mellitus biomarkers.

    PubMed

    Spiller, Sandro; Frolov, Andrej; Hoffmann, Ralf

    2017-02-02

    Type 2 diabetes mellitus (T2DM) is the most common lifestyle disease affecting all countries. Due to its asymptomatic onset, it is often diagnosed after irreversible vascular complications have been initiated. Therefore, specific markers characteristic for very early disease stages and suitable for early diagnostics are required. Glycation of plasma proteins, such as human serum albumin (HSA), has been often suggested as marker. However, the total glycation degree of HSA does not provide sufficient information about short-term fluctuations of blood glucose concentrations due to the large number of glycation sites. Analysis of individual modification sites might be more informative, but methods for reliable quantifications are still missing. Here, we present a precise and robust quantitative strategy based on boronic acid affinity chromatography (BAC) to enrich glycated peptides in tryptic digests of plasma samples and to quantify them by LC-MS/MS (multiple reaction monitoring, MRM) using internal calibration or standard isotope dilution (stable isotope-labeled peptides). The high selectivity and precision (relative standard deviations below 10%) of the overall approach appeared to be well suited for the identification of prospective biomarkers. Indeed, six glycated peptides corresponding to different glycation sites of HSA were present in plasma samples obtained from T2DM patients at significantly higher levels than in non-diabetic men matched for age. Additionally, each of the studied glycation site of HSA appeared to be affected at different degrees.

  2. 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.

  3. Mechanistic investigation of domain specific unfolding of human serum albumin and the effect of sucrose

    PubMed Central

    Yadav, Rajeev; Sen, Pratik

    2013-01-01

    This study is devoted to understand the unfolding mechanism of a multidomain protein, human serum albumin (HSA), in absence and presence of the sucrose by steady-state and time-resolved fluorescence spectroscopy with domain specific marker molecules and is further being substantiated by molecular dynamics (MD) simulation. In water, the domain III of HSA found to unfold first followed by domains I and II as the concentration of GnHCl is increased in the medium. The sequential unfolding behavior of different domains of HSA remains same in presence of sucrose; however, a higher GnHCl concentration is required for unfolding, suggesting stabilizing effect of sucrose on HSA. Domain I is found to be most stabilized by sucrose. The stabilization of domain II is somewhat similar to domain I, but the effect of sucrose on domain III is found to be very small. MD simulation also predicted a similar behavior of sucrose on HSA. The stabilizing effect of sucrose is explained in terms of the entrapment of water molecules in between HSA surface and sucrose layer as well as direct interaction between HSA and sucrose. PMID:24038622

  4. 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.

  5. 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.

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

    PubMed

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

    2016-09-10

    Non-esterified fatty acids bound to the human serum albumin (HSA) contribute to several HSAs properties of special concern in pathologies, for instance to the reactivity of the free HSA-Cys34 thiol group (important antioxidative thiol pool in plasma), and to the affinity for binding of molecules and ions (for example cobalt as a prominent biomarker in heart ischemia). Therefore, the method for determination of FAs bound to HSA was developed. FAs were released from HSA (previously isolated from serum by ammonium sulfate precipitation) using acidic copper(II) sulfate in phosphoric acid, extracted by n-heptane-chloroform (4:1, v/v) mixture, spotted on TL silica-gel and then developed with n-heptane-chloroform-acetic acid (5:3:0.3, v/v/v). Common office flatbed scanner and software solution for densitometric image analysis, developed in R, were used. The linearity of calibration curve in concentration range from 0.1 to 5.0mmol/L stearic acid was achieved. The method was proved to be precise (with RSD of 1.4-4.7%) and accurate. Accuracy was examined by standard addition method (recoveries 97.2-102.5%) and by comparison to results of GC. The method is sample saving, technically less demanding, and cheap, and therefore suitable for determination of FAs/HSA ratio when elevated concentrations of free FAs are reliable diagnostic/risk parameter of pathological states. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    NASA Astrophysics Data System (ADS)

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

    2004-11-01

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

  8. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2005-12-01

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

  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. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2005-07-01

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

  13. An approach to remove albumin for the proteomic analysis of low abundance biomarkers in human serum.

    PubMed

    Ahmed, Nuzhat; Barker, Gillian; Oliva, Karen; Garfin, David; Talmadge, Kenneth; Georgiou, Harry; Quinn, Michael; Rice, Greg

    2003-10-01

    Proteomic technologies are being used to discover and identify disease-associated biomarkers. The application of these technologies in the search for potential diagnostic/prognostic biomarkers in the serum of patients has been limited by the presence of highly abundant albumin and immunoglobulins that constitute approximately 60-97% of the total serum proteins. The purpose of the study was to evaluate whether treatment of human serum with Affi-Gel Blue alone or in combination with Protein A (Aurum serum protein mini kit, Bio-Rad) before two-dimensional gel electrophoresis (2-DE) analysis removed high abundance proteins to allow the visualization of low abundant proteins. Serum samples were treated with either Affi-Gel Blue or Aurum kit and then subjected to 2-DE using 11 cm, pH 4-7 isoelectric focussing strips for the first dimension and 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis for second dimension. Protein spots were visualized using a fluorescent protein dye (SYPRO Ruby, Bio-Rad). Comparison between treatment methods showed significant removal of albumin by both Affi-Gel Blue and Aurum kit and considerable differences in the protein profile of the gels after each treatment. Direct comparison between treatments revealed twenty-eight protein spots unique to Affi-Gel Blue while only two spots were unique after Aurum kit treatment. Unique spots in Affi-Gel Blue and Aurum kit treated serum were not visualized in untreated serum. Sixteen hours of Affi-Gel Blue treatment resulted in enhanced visualization of fifty-three protein spots by two-fold, thirty-one by five-fold, twelve by ten-fold and six by twenty-fold. In parallel after Aurum kit treatment two-, five-, ten- and twenty-fold enhancements of thirty, thirteen, eight and five protein spots, respectively, were observed. The pattern of increased visualization of protein spots with both treatment methods was similar. In conclusion, treatment of serum samples with Affi-Gel Blue or Aurum kit before

  14. 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.

  15. 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.

  16. Interaction of the antimalarial alpha-dibutylaminomethyl-2,6-bis(trifluoromethylphenyl)-4-pyridinemethanol with human serum albumin.

    PubMed

    Bouwsma, O J; Stewart, J T; Vallner, J J

    1979-01-01

    Binding of the antimalarial alpha-dibutylaminomethyl-2,6-bis(trifluoromethylphenyl)-4-pyridinemethanol with human serum albumin was studied using difference spectroscopy, fluorescence quenching, and equilibrium dialysis. Results indicated that the number of high affinity binding sites of the drug on protein is 0.45, with the total number of binding sites being 3.3--4.0. The binding constants were in the range of 0.57--4.00 x 10(6) M-1. The drug was bound more strongly to a nonionic detergent than to either a cationic or anionic detergent. Interpretation of these data and fluorescence quenching results indicated that the drug is possibly bound to a hydrophobic site on human serum albumin.

  17. A comparison of Coomassie blue dye with radioiodinated albumin as an indicator for plasma volume estimation in human subjects

    PubMed Central

    Menzies, Ian S.

    1966-01-01

    Plasma volume has been estimated in 10 human subjects using Coomassie blue and 131I radioiodinated human serum albumin dilution methods simultaneously. Three different methods of correction used by previous workers to overcome the error due to early dye loss were applied. Satisfactory agreement with the established radioiodinated albumin method was only obtained by extrapolation of the semilogarithmic plot of Coomassie blue plasma dye concentration between five and 10 minutes to the time of injection. The significance of the controversial Evans blue `mixing curve' is discussed. An analogous phase in the Coomassie blue disappearance slope is considered to be due to initial rapid loss of dye from the circulation rather than to the process of mixing. It is shown that Coomassie blue fulfils the criteria listed in the discussion for plasma volume estimation. PMID:4160095

  18. Long-acting recombinant coagulation factor IX albumin fusion protein (rIX-FP) in hemophilia B: results of a phase 3 trial.

    PubMed

    Santagostino, Elena; Martinowitz, Uri; Lissitchkov, Toshko; Pan-Petesch, Brigitte; Hanabusa, Hideji; Oldenburg, Johannes; Boggio, Lisa; Negrier, Claude; Pabinger, Ingrid; von Depka Prondzinski, Mario; Altisent, Carmen; Castaman, Giancarlo; Yamamoto, Koji; Álvarez-Roman, Maria-Teresa; Voigt, Christine; Blackman, Nicole; Jacobs, Iris

    2016-04-07

    A global phase 3 study evaluated the pharmacokinetics, efficacy, and safety of recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in 63 previously treated male patients (12-61 years) with severe hemophilia B (factor IX [FIX] activity ≤2%). The study included 2 groups: group 1 patients received routine prophylaxis once every 7 days for 26 weeks, followed by either 7-, 10-, or 14-day prophylaxis regimen for a mean of 50, 38, or 51 weeks, respectively; group 2 patients received on-demand treatment of bleeding episodes for 26 weeks and then switched to a 7-day prophylaxis regimen for a mean of 45 weeks. The mean terminal half-life of rIX-FP was 102 hours, 4.3-fold longer than previous FIX treatment. Patients maintained a mean trough of 20 and 12 IU/dL FIX activity on prophylaxis with rIX-FP 40 IU/kg weekly and 75 IU/kg every 2 weeks, respectively. There was 100% reduction in median annualized spontaneous bleeding rate (AsBR) and 100% resolution of target joints when subjects switched from on-demand to prophylaxis treatment with rIX-FP (P< .0001). The median AsBR was 0.00 for all prophylaxis regimens. Overall, 98.6% of bleeding episodes were treated successfully, including 93.6% that were treated with a single injection. No patient developed an inhibitor, and no safety concerns were identified. These results indicate rIX-FP is safe and effective for preventing and treating bleeding episodes in patients with hemophilia B at dosing regimens of 40 IU/kg weekly and 75 IU/kg every 2 weeks. This trial was registered at www.clinicaltrials.gov as #NCT0101496274.

  19. Long-acting recombinant coagulation factor IX albumin fusion protein (rIX-FP) in hemophilia B: results of a phase 3 trial

    PubMed Central

    Martinowitz, Uri; Lissitchkov, Toshko; Pan-Petesch, Brigitte; Hanabusa, Hideji; Oldenburg, Johannes; Boggio, Lisa; Negrier, Claude; Pabinger, Ingrid; von Depka Prondzinski, Mario; Altisent, Carmen; Castaman, Giancarlo; Yamamoto, Koji; Álvarez-Roman, Maria-Teresa; Voigt, Christine; Blackman, Nicole; Jacobs, Iris

    2016-01-01

    A global phase 3 study evaluated the pharmacokinetics, efficacy, and safety of recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in 63 previously treated male patients (12-61 years) with severe hemophilia B (factor IX [FIX] activity ≤2%). The study included 2 groups: group 1 patients received routine prophylaxis once every 7 days for 26 weeks, followed by either 7-, 10-, or 14-day prophylaxis regimen for a mean of 50, 38, or 51 weeks, respectively; group 2 patients received on-demand treatment of bleeding episodes for 26 weeks and then switched to a 7-day prophylaxis regimen for a mean of 45 weeks. The mean terminal half-life of rIX-FP was 102 hours, 4.3-fold longer than previous FIX treatment. Patients maintained a mean trough of 20 and 12 IU/dL FIX activity on prophylaxis with rIX-FP 40 IU/kg weekly and 75 IU/kg every 2 weeks, respectively. There was 100% reduction in median annualized spontaneous bleeding rate (AsBR) and 100% resolution of target joints when subjects switched from on-demand to prophylaxis treatment with rIX-FP (P < .0001). The median AsBR was 0.00 for all prophylaxis regimens. Overall, 98.6% of bleeding episodes were treated successfully, including 93.6% that were treated with a single injection. No patient developed an inhibitor, and no safety concerns were identified. These results indicate rIX-FP is safe and effective for preventing and treating bleeding episodes in patients with hemophilia B at dosing regimens of 40 IU/kg weekly and 75 IU/kg every 2 weeks. This trial was registered at www.clinicaltrials.gov as #NCT0101496274. PMID:26755710

  20. Preparation and immunogenicity of tag-free recombinant human eppin

    PubMed Central

    Zhang, Jie; Ding, Xin-Liang; Bian, Zeng-Hui; Xia, Yan-Kai; Wang, Shou-Lin; Song, Ling; Wang, Xin-Ru

    2011-01-01

    Human epididymal protease inhibitor (eppin) may be effective as a male contraceptive vaccine. In a number of studies, eppin with an engineered His6-tag has been produced using prokaryotic expression systems. For production of pharmaceutical-grade proteins for human use, however, the His6-tag must be removed. This study describes a method for producing recombinant human eppin without a His6-tag. We constructed plasmid pET28a (+)-His6-tobacco etch virus (TEV)-eppin for expression in Escherichia coli. After purification and refolding, the fusion protein His6-TEV-eppin was digested with TEV protease to remove the His6-tag and was further purified by NTA-Ni2+ affinity chromatography. Using this procedure, 2 mg of eppin without a His6-tag was isolated from 1 l of culture with a purity of >95%. The immunogenicity of the eppin was characterized using male Balb/c mice. PMID:21892195

  1. Human Serum Albumin and p53-Activating Peptide Fusion Protein Is Able to Promote Apoptosis and Deliver Fatty Acid-Modified Molecules

    PubMed Central

    Joshi, Michelle R.; Yao, Nianhuan; Myers, Kenneth A.; Li, Zhiyu

    2013-01-01

    Therapeutic peptides offer a high degree of specificity, potency, and low toxicity; making them promising candidates for cancer therapy. Despite these advantages, a number of hurdles, such as poor serum stability and inefficient cellular penetration, must be overcome. Fusing a therapeutic peptide to human serum albumin (HSA) is a common approach to extend the serum stability of a peptide that binds to extracellular receptors. However, no study has shown that this approach can be applied to target intracellular proteins. Here we demonstrate the feasibility of using a recombinant human serum albumin (rHSA) fusion protein to simultaneously deliver two types of molecules: a peptide capable of binding an intracellular target, as well as fatty acid (FA)-modified FITC (FA-FITC). Two peptides reported to disrupt the intracellular p53 and MDM2/MDMX interaction were fused to the C-terminal of HSA. Cellular and biochemical studies indicate that rHSA fusion proteins were efficiently taken up by SJSA-1 cells and retained MDM2- and MDMX-binding activity. By inducing the accumulation of p53, both fusion proteins promoted efficient cytotoxicity in SJSA-1 cells via caspase activation. Long chain fatty acid (LCFA) transportation is an essential endogenous function of HSA. This study also demonstrates that rHSA fusion proteins formed highly stable complexes with FA-FITC via non-covalent interactions. FA-FITC complexed with HSA could be internalized efficiently and rHSA-P53i and rHSA-PMI retained apoptotic activity as complex components. It is expected that such an approach can ultimately be used to facilitate intracellular delivery of two anticancer therapeutics, each with distinct but complimentary mechanisms, to achieve synergistic efficacy. PMID:24278348

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

    PubMed

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

    2016-09-01

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

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

    PubMed

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

    2016-08-01

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

  4. 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.

  5. Human Serum Albumin Nanoparticles for Use in Cancer Drug Delivery: Process Optimization and In Vitro Characterization

    PubMed Central

    Lomis, Nikita; Westfall, Susan; Farahdel, Leila; Malhotra, Meenakshi; Shum-Tim, Dominique; Prakash, Satya

    2016-01-01

    Human serum albumin nanoparticles (HSA-NPs) are widely-used drug delivery systems with applications in various diseases, like cancer. For intravenous administration of HSA-NPs, the particle size, surface charge, drug loading and in vitro release kinetics are important parameters for consideration. This study focuses on the development of stable HSA-NPs containing the anti-cancer drug paclitaxel (PTX) via the emulsion-solvent evaporation method using a high-pressure homogenizer. The key parameters for the preparation of PTX-HSA-NPs are: the starting concentrations of HSA, PTX and the organic solvent, including the homogenization pressure and its number cycles, were optimized. Results indicate a size of 143.4 ± 0.7 nm and 170.2 ± 1.4 nm with a surface charge of −5.6 ± 0.8 mV and −17.4 ± 0.5 mV for HSA-NPs and PTX-HSA-NPs (0.5 mg/mL of PTX), respectively. The yield of the PTX-HSA-NPs was ~93% with an encapsulation efficiency of ~82%. To investigate the safety and effectiveness of the PTX-HSA-NPs, an in vitro drug release and cytotoxicity assay was performed on human breast cancer cell line (MCF-7). The PTX-HSA-NPs showed dose-dependent toxicity on cells of 52%, 39.3% and 22.6% with increasing concentrations of PTX at 8, 20.2 and 31.4 μg/mL, respectively. In summary, all parameters involved in HSA-NPs’ preparation, its anticancer efficacy and scale-up are outlined in this research article. PMID:28335244

  6. Adsorption of human fibrinogen and albumin onto hydrophobic and hydrophilic Ti6Al4V powder

    NASA Astrophysics Data System (ADS)

    Rodríguez-Sánchez, Jesús; Gallardo-Moreno, Amparo M.; Bruque, José M.; González-Martín, M. Luisa

    2016-07-01

    Adsorption of proteins on solid surfaces has been widely studied because of its importance in various biotechnological, medical and technical applications, such as medical implants or biosensors. One of the main problems is the adsorption-induced conformational changes because they often modify the biological activity of the proteins, which is believed to be a key factor on the subsequent cellular adhesion. The aim of this work is the study of the adsorption of human fibrinogen (Fg) and human serum albumin (HSA) onto Ti6Al4V particles, commercially available on different size, that are used to elaborate scaffolds to provide structural support to cell proliferation, promoting tissue development and bone regeneration among others. The study was done through the analysis of the adsorption isotherms and the electrical characterization of surfaces after adsorption in terms of the zeta potential (ζ). From this analysis it seems that Fg adsorbs preferentially vertically oriented (end-on) and HSA moves sequentially over the surface of the Ti6Al4V particles through dimmer formation, allowing adsorption progress over this initial bilayer. The zeta potential values of both proteins remain constant when the monolayer is formed. The study also extends the analysis of both adsorption behaviour and ζ potential characterization factors to the influence of the substrate hydrophobicity as this property can be modified for the Ti6Al4V by irradiating it with ultraviolet light (UV-C) without changes on its chemical composition [1,2]. Differences at low protein concentrations were found for both isotherms and zeta-potential values.

  7. Vancouver Experience of Recombinant Human Platelet-Derived Growth Factor.

    PubMed

    Younger, Alistair; Penner, Murray; Montijo, Harvey E

    2016-12-01

    Joint arthrodesis utilizing autogenous bone graft remains the gold standard of treatment in fusion procedures of the foot and ankle. Graft harvest, however, has been associated with increased morbidity to patients as well as increased costs. With this in mind, multiple clinical studies have evaluated the efficacy of recombinant human platelet-derived growth factor (rh-PDGF-BB) with beta-tricalcium phosphate (B-TCP) to augment in foot and ankle arthrodesis with favorable results. These factors have led to the increased use of rh-PDGF-BB with B-TCP in Vancouver with good clinical results. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Polyethylene glycol enhanced refolding of the recombinant human tissue transglutaminase.

    PubMed

    Ambrus, A; Fésüs, L

    2001-02-01

    Tissue transglutaminase forms cross-links between lysine and glutamine side-chains of polypeptide chains in a Ca2+-dependent reaction; its structural basis is still not clarified. In this study, we demonstrate that the refolding of the human recombinant enzyme molecule to its catalytically active form from inclusion bodies needs the presence of a helper material with higher molecular mass, but only in the initiation phase. Ca2+ and nucleotides are ascribed as affector molecules also in the early phase of structural reconstitution. Two optimal concentrations of polyethylene glycol and a relatively long time scale for the evolution of the final structure were identified. The optimized refolding procedure is reported.

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

    PubMed Central

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

    2016-01-01

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

  10. Broadband measurements of the frequency dependence of attenuation coefficient and velocity in amniotic fluid, urine and human serum albumin solutions.

    PubMed

    Verma, Prashant K; Humphrey, Victor F; Duck, Francis A

    2005-10-01

    The frequency dependence of attenuation coefficient in amniotic fluid, urine and 4.5% and 20% human serum albumin solutions over the frequency range 5 MHz to 25 MHz was measured at both room temperature and physiological temperature using a variable path length technique. A 15 MHz (13 mm diameter) transducer was used to produce a broadband single-cycle pulse and a 4 mm diameter bilaminar polyvinylidene difluoride membrane hydrophone was used to detect the attenuated pulse. Standard time-of-flight measurement techniques were used to measure the acoustic velocity in the same fluid samples. At physiological temperature, the attenuation coefficients in amniotic fluid, urine and 4.5% and 20% human albumin solution were found to be 0.0053 f(1.65), 0.0047 f(1.67), 0.019 f(1.57) and 0.167 f(1.27) dB cm(-1), respectively, where f is in MHz. The velocities in amniotic fluid, urine and 4.5% human albumin solution at physiological temperature were found to be 1541.1 m s(-1) +/- 1.3 m s(-1), 1551.3 m s(-1) +/- 1.3 ms(-1) and 1547.3 m s(-1) +/- 1.0 m s(-1), respectively. The results provide unique data over the diagnostic and therapeutic ultrasonic frequency range that can be used as input data for theoretical models that attempt to simulate nonlinear pressure fields and temperature rises from medical ultrasonic transducers.

  11. Enhanced Proteolytic Processing of Recombinant Human Coagulation Factor VIII B-Domain Variants by Recombinant Furins.

    PubMed

    Demasi, Marcos A; de S Molina, Erika; Bowman-Colin, Christian; Lojudice, Fernando H; Muras, Angelita; Sogayar, Mari C

    2016-06-01

    Recombinant human factor VIII (rFVIII) is used in replacement therapy for hemophilia A. Current research efforts are focused on bioengineering rFVIII molecules to improve its secretion efficiency and stability, limiting factors for its efficient production. However, high expression yield in mammalian cells of these rFVIII variants is generally associated with limited proteolytic processing. Non-processed single-chain polypeptides constitute non-natural FVIII molecule configurations with unpredictable toxicity and/or antigenicity. Our main objective was to demonstrate the feasibility of promoting full-proteolytic processing of an rFVIII variant retaining a portion of the B-domain, converting it into the smallest natural activatable form of rFVIII, while keeping its main advantage, i.e., improved secretion efficiency. We generated and employed a CHO-DG44 cell clone producing an rFVIII variant retaining a portion of the B-domain and the FVIII native cleavage site between Arg(1648) and Glu(1649). By bioengineering CHO-DG44 cells to express stably the recombinant human endoproteases PACE, PACE-SOL, PCSK5, PCSK6, or PCKS7, we were able to achieve complete intra- or extracellular proteolytic processing of this rFVIII variant. Additionally, our quantitative data indicated that removal of the B-domain segment by intracellular proteolytic processing does not interfere with this rFVIII variant secretion efficiency. This work also provides the first direct evidence of (1) intracellular cleavage at the Arg(1648) FVIII processing site promoted by wild-type PACE and PCSK7 and (2) proteolytic processing at the Arg(1648) FVIII processing site by PCSK6.

  12. [Human serum albumin modified under oxidative/halogenative stress enhances luminol-dependent chemiluminescence of human neutrophils].

    PubMed

    Mikhal'chik, E V; Smolina, N V; Astamirova, T C; Gorudko, I V; Grigor'eva, D V; Ivanov, V A; Sokolov, A V; Kostevich, V A; Cherenkevich, S N; Panasenko, O M

    2013-01-01

    It is shown that human serum albumin, previously treated with HOCl (HSA-Cl), enhances luminol-dependent chemiluminescence of neutrophils activated by phorbol-12-myristate-13-acetate (PMA). The enzyme-linked immunosorbent assay revealed that addition of HSA-Cl to neutrophils promotes exocytosis of myeloperoxidase. Inhibitor of myeloperoxidase--4-aminobenzoic acid hydrazide, without any effect on lucigenin-dependent chemiluminescence of neutrophils stimulated with PMA, effectively suppressed luminol-dependent chemiluminescence (IC50 = 20 microM) under the same conditions. The transfer of the cells from medium with HSA-Cl and myeloperoxidase to fresh medium abolished an increase in PMA-induced luminol-dependent chemiluminescence, but not the ability of neutrophils to respond to re-addition of HSA-Cl. A direct and significant (r = 0.75, p) correlation was observed between the intensity of PMA stimulated neutrophil chemiluminescence response and myeloperoxidase activity in the cell-free media after chemiluminescence measurements. These results suggest the involvement of myeloperoxidase in the increase of neutrophil PMA-stimulated chemiluminescence response in the presence of HSA-Cl. A significant positive correlation was found between myeloperoxidase activity in blood plasma of children with severe burns and the enhancing effects of albumin fraction of the same plasma on luminol-dependent chemiluminescence of PMA-stimulated donor neutrophils. These results support a hypothesis that proteins modified in reactions involving myeloperoxidase under oxidative/halogenative stress, stimulate neutrophils, leading to exocytosis of myeloperoxidase, a key element of halogenative stress, and to closing a "vicious circle" of neutrophil activation at the inflammatory site.

  13. Recombinant human hemoglobin does not affect renal function in humans: analysis of safety and pharmacokinetics.

    PubMed

    Viele, M K; Weiskopf, R B; Fisher, D

    1997-04-01

    Recombinant human hemoglobin (OptroD; rHb1.1) is a genetically engineered protein produced in Escherichia coli. The two alpha-globin polypeptides are genetically joined, resulting in a stable tetramer that does not dissociate into dimers or monomers. Historically, infusion in humans of acellular hemoglobin preparations has resulted in renal toxicity. This study was performed to evaluate the safety and pharmacokinetics of rHb1.1 when infused in humans. After giving informed consent, 48 healthy male volunteers were randomly assigned to receive either 0.015-0.32 g/kg 5% rHb1.1 (n = 34) or an equivalent amount of 5% human serum albumin (HSA; n = 14) infused intravenously over 0.8-1.9 h. Serum creatinine, creatinine clearance, urine N-acetyl-beta-glucosaminidase, and serum rHb1.1 concentrations were measured before and at timed intervals after infusion. Postinfusion urine N-acetyl-beta-glucosaminidase activity did not exceed preinfusion values at any interval in either group. Serum creatinine did not differ from preinfusion values at 1 day, 2-3 days, or 7 days after infusion for either group. Creatinine clearance increased significantly for the HSA group 12 h after infusion (138 +/- 16 ml/min, means +/- SE) and in the rHb1.1 group 1 day after infusion (112 +/- 5 ml/min; P < 0.05). Values for creatinine clearance did not differ from preinfusion values for either group at any other postinfusion interval; serum creatinine and creatinine clearance did not differ between groups at any time. The amount of hemoglobin excreted in the urine did not exceed approximately 0.04% of the administered rHb1.1 dose in any volunteer. Plasma clearance of rHb1.1 decreased and half-life increased as a function of increasing plasma concentration (e.g., the half-life was 2.8 h at a plasma concentration of 0.5 mg/ml and 12 h at 5 mg/ml). The incidence of gastrointestinal symptoms, fever, and chills was greater after infusion of rHb1.1 than after HSA (P < 0.05). No evidence for rHb1.1-mediated

  14. Effects of angiotensin II type 1 receptor blocker on albumin-induced cell damage in human renal proximal tubular epithelial cells.

    PubMed

    Takao, Toshihiro; Horino, Taro; Kagawa, Toru; Matsumoto, Reiko; Inoue, Kousuke; Taguchi, Takafumi; Morita, Tatsuhito; Iwasaki, Yasumasa; Hashimoto, Kozo; Terada, Yoshio

    2009-01-01

    Proteinuria is not merely a marker of chronic nephropathies, but may also be involved in the progression to end-stage renal failure. We investigated the effect of angiotensin II type 1 receptor blockers (ARBs) on albumin-induced cell damage in human renal proximal tubular epithelial cells (RPTEC). The N-acetyl-beta-D-glucosaminidase (NAG) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in the medium after albumin treatment with ARBs were determined by commercially available kits. The levels of p22(phox) protein in RPTEC were measured using Western blotting after albumin treatment with ARBs. Angiotensin II concentrations in cell media and cell lysates were assayed with a commercially available kit. Human albumin (0.1-10 mg/ml) dose-dependently increased NAG release and olmesartan or valsartan (10(-9)-10(-7) mol/l) showed a significant reduction on albumin (1 mg/ml)-induced NAG release in RPTEC. Albumin treatment (1 mg/ml) showed significant increases in p22(phox) protein levels in RPTEC and ARBs significantly decreased albumin-induced p22(phox) protein levels. Significant increases in 8-OHdG levels were observed in the albumin (1 mg/ml)-treated group and ARBs markedly reduced albumin-induced 8-OHdG levels in RPTEC. Human albumin dose-dependently increased angiotensin II concentrations in both cell media and lysates. These observations suggest renal tubular cell-protective properties of ARBs related to decreased oxidative stress during proteinuria. Copyright (c) 2008 S. Karger AG, Basel.

  15. 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