Showing papers on "Human serum albumin published in 2003"
TL;DR: The structure of the HSA-hemin-myristate complex (PDB ID 1o9x) reveals the key polar and hydrophobic interactions that determine the hemin-binding specificity of HSA.
Abstract: Human serum albumin (HSA) is an abundant plasma protein that binds a wide variety of hydrophobic ligands including fatty acids, bilirubin, thyroxine and hemin. Although HSA-heme complexes do not bind oxygen reversibly, it may be possible to develop modified HSA proteins or heme groups that will confer this ability on the complex. We present here the crystal structure of a ternary HSA-hemin-myristate complex, formed at a 1:1:4 molar ratio, that contains a single hemin group bound to subdomain IB and myristate bound at six sites. The complex displays a conformation that is intermediate between defatted HSA and HSA-fatty acid complexes; this is likely to be due to low myristate occupancy in the fatty acid binding sites that drive the conformational change. The hemin group is bound within a narrow D-shaped hydrophobic cavity which usually accommodates fatty acid; the hemin propionate groups are coordinated by a triad of basic residues at the pocket entrance. The iron atom in the centre of the hemin is coordinated by Tyr161. The structure of the HSA-hemin-myristate complex (PDB ID 1o9x) reveals the key polar and hydrophobic interactions that determine the hemin-binding specificity of HSA. The details of the hemin-binding environment of HSA provide a structural foundation for efforts to modify the protein and/or the heme molecule in order to engineer complexes that have favourable oxygen-binding properties.
458 citations
TL;DR: PFOA binding to rat and human plasma proteins was investigated and it was found that greater than 90% of PFOA would be bound to serum albumin in bothRat and human blood.
Abstract: Perfluorooctanoic acid (PFOA) is a commercially important organic fluorochemical and is considered to have a long half-life in human blood. In this paper, PFOA binding to rat and human plasma proteins was investigated. On the basis of results from size-exclusion chromatography and ligand blotting, most PFOA was in protein-bound form in male and female rat plasma, and the primary PFOA binding protein in plasma was serum albumin. PFOA binding to rat serum albumin (RSA) in the gas phase was observed by electrospray ionization MS. 19F NMR experiments revealed that binding to RSA caused peak broadening and chemical shift changes of PFOA resonances, and on the basis of this observation, the dissociation constant was determined to be ∼0.3 mM. The dissociation constants for PFOA binding to RSA and human serum albumin (HSA) and the numbers of PFOA binding sites on RSA and HSA were also determined by a separation method using microdesalting columns. No significant difference was found between PFOA binding to RSA an...
339 citations
TL;DR: It was found that quercetin shows extrinsic optical activity on interaction with HSA, and the molecule was found to be bound in the large hydrophobic cavity of subdomain IIA, which is able to help to stabilize the negatively charged ligand bound in non-planar conformation.
318 citations
TL;DR: Human serum albumin (HSA), the most abundant protein in plasma, has been proposed to have an antioxidant role and HSA-SOH is proposed to serve as an intermediate in the formation ofLow molecular weight disulfides, which are the predominant plasma form of low molecular weight thiols, and in theformation of mixed HSA disulfide, which is present in approximately 25% of circulating HSA.
Abstract: Human serum albumin (HSA), the most abundant protein in plasma, has been proposed to have an antioxidant role. The main feature responsible for this property is its only thiol, Cys34, which comprises approximately 80% of the total free thiols in plasma and reacts preferentially with reactive oxygen and nitrogen species. Herein, we show that the thiol in HSA reacted with hydrogen peroxide with a second-order rate constant of 2.26 M(-1) s(-1) at pH 7.4 and 37 degrees C and a 1:1 stoichiometry. The formation of intermolecular disulfide dimers was not observed, suggesting that the thiol was being oxidized beyond the disulfide. With the reagent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl), we were able to detect the formation of sulfenic acid (HSA-SOH) from the UV-vis spectra of its adduct. The formation of sulfenic acid in Cys34 was confirmed by mass spectrometry using 5,5-dimethyl-1,3-cyclohexanedione (dimedone). Sulfenic acid was also formed from exposure of HSA to peroxynitrite, the product of the reaction between nitric oxide and superoxide radicals, in the absence or in the presence of carbon dioxide. The latter suggests that sulfenic acid can also be formed through free radical pathways since following reaction with carbon dioxide, peroxynitrite yields carbonate radical anion and nitrogen dioxide. Sulfenic acid in HSA was remarkably stable, with approximately 15% decaying after 2 h at 37 degrees C under aerobic conditions. The formation of glutathione disulfide and mixed HSA-glutathione disulfide was determined upon reaction of hydrogen peroxide-treated HSA with glutathione. Thus, HSA-SOH is proposed to serve as an intermediate in the formation of low molecular weight disulfides, which are the predominant plasma form of low molecular weight thiols, and in the formation of mixed HSA disulfides, which are present in approximately 25% of circulating HSA.
317 citations
TL;DR: Several compounds showed stronger HSA binding than can be expected from their lipophilicity alone, and comparison between predicted and experimental binding affinity allows the identification of compounds that have good complementarities with any of the known binding sites.
285 citations
TL;DR: This article reviews the field of drug binding to Human Serum Albumin, as well as models to predict drug‐binding affinities to HSA from drug structure, and describes the different QSAR models for HSA binding of restricted families of drugs.
Abstract: Drug binding to Human Serum Albumin (HSA) is an area of intense research. The pharmacokinetics and pharmacodynamics of drugs are strongly affected by their binding to this protein. In this article, the field is reviewed, as well as our models to predict drug-binding affinities to HSA from drug structure. The physiological role of HSA is described, as well as its influence in drug action. The crystal structures of this protein are discussed, emphasizing the two drug-binding sites and the fatty acids binding sites observed therein. The advantages of using high-performance affinity chromatography to rapidly screen drugs for HSA binding are explained. The different QSAR models for HSA binding of restricted families of drugs (both from other groups and our group) are enumerated. Finally, a detailed description of our general models to predict drug-binding strengths to HSA from structure is given. It is expected for these models to be useful in drug design and pharmaceutical research.
246 citations
TL;DR: In this article, the pharmacokinetics of albumin and methotrexate were examined in a mouse model of rheumatoid arthritis and the efficacy of MTX and MTX-HSA in arthritic mice were studied.
Abstract: We reported recently that albumin is a suitable drug carrier for targeted delivery of methotrexate (MTX) to tumors. Due to pathophysiological conditions in neoplastic tissue, high amounts of albumin accumulate in tumors and are metabolized by malignant cells. MTX, covalently coupled to human serum albumin (MTX-HSA) for cancer treatment, is currently being evaluated in phase II clinical trials. Because synovium of patients with rheumatoid arthritis (RA) shares various features observed also in tumors, albumin-based drug targeting of inflamed joints might be an attractive therapeutic approach. Therefore, the pharmacokinetics of albumin and MTX in a mouse model of arthritis was examined. Additionally, uptake of albumin by synovial fibroblasts of RA patients and the efficacy of MTX and MTX-HSA in arthritic mice were studied. The results show that when compared with MTX, significantly higher amounts of albumin accumulate in inflamed paws, and significantly lower amounts of albumin are found in the liver and the kidneys. The protein is metabolized by human synovial fibroblasts in vitro and in vivo. MTX-HSA was significantly more effective in suppression of the onset of arthritis in mice than was MTX. In conclusion, albumin appears to be a suitable drug carrier in RA, most likely due to effects on synovial fibroblasts, which might increase therapeutic efficacy and reduce side effects of MTX.
226 citations
TL;DR: Using monoclonal antibodies against human serum albumin (HSA) to develop an immunoaffinity resin that is effective in the removal of both full-length HSA and many of the HSA fragments present in serum shows markedly better performance than dye-based resins in terms of both the efficiency and specificity of albumin removal.
222 citations
TL;DR: In this article, an instant invention overcomes this by producing human serum albumin in plant plastids at high levels, which has not been successful in prokaryotic systems because HSA is highly susceptible to proteolytic degradation.
Abstract: Production of human serum albumin (HSA) in prokaryotic systems has not been successful to date because HSA is highly susceptible to proteolytic degradation. Production in plants has not yielded enough protein to be cost-effective. The instant invention overcomes this by producing HSA in plant plastids at high levels.
212 citations
TL;DR: Fractal silver structures formed on, or near, silver electrodes by passage of electric currents are tested for metal-enhanced fluorescence and photostability measurements indicate 160-fold more photons are detectable from FITC-HSA on the fractal silver surface.
Abstract: Recent reports have shown enhanced fluorescence for fluorophores in close proximity to chemically deposited silver islands or colloids. To expand the usefulness of metal-enhanced fluorescence we tested fractal silver structures formed on, or near, silver electrodes by passage of electric currents. The emission intensity of fluorescein-labeled human serum albumin (FITC-HSA) was enhanced over 100-fold when adsorbed to the fractal silver structures as compared to glass. The amplitude-weighted lifetime is dramatically reduced to near 3 ps. Enhanced fluorescence was shown to result in selective observation of FITC-HSA over a fluorophore not attached to the silver surface. And finally, photostability measurements indicate 160-fold more photons are detectable from FITC-HSA on the fractal silver surface. These results suggest the use of in situ generated silver structures for metal-enhanced fluorescence.
179 citations
TL;DR: In this article, a nonviral gene delivery vehicle consisting of DNA, human serum albumin (HSA) and polyethylenimine (PEI) nanoparticles containing the pGL3 vector coding for luciferase as a reporter gene were formed by charge neutralization.
TL;DR: The present findings indicate that the effect of glycation can be partly explained by blockage of the positive charges of lysine at positions 199, 439 and 525.
TL;DR: Significant inhibition of phagocytosis was observed, even when HSA was combined with strong opsonins such as alpha2GP or IgG or in mixtures of all three proteins, indicating the importance of studying the influence of protein adsorption in protein mixtures.
Journal Article•
TL;DR: MTX-HSA is effectively taken up by the cells via albumin receptor- or folate receptor-mediated endocytosis and time-dependently released as an active compound into the cytosol to exert an inhibiting effect on TS and to induce cell cycle alterations.
Abstract: To avoid systemic toxicity of the cytotoxic drug methotrexate (MTX) and to improve tumor selectivity, MTX was bound to human serum albumin (HSA) as a drug carrier. To understand more about the mechanism of action of MTX conjugated to HSA (MTX-HSA), the uptake of MTX-HSA into the cell was determined as well as the effect of MTX-HSA on thymidylate synthase (TS), cell cycle distribution, and cell proliferation. Different uptake kinetics were observed for [(3)H]MTX and [(3)H]MTX-HSA. However, similar uptake kinetics were measured for (125)I-HSA and (125)I-MTX-HSA (2.1 and 1.8 pmol/10(7) cells/h when cells were treated with 10 micro M (125)I-HSA and (125)I-MTX-HSA, respectively), suggesting that MTX-HSA enters the cells by albumin-mediated endocytosis. We observed no effect of MTX-HSA on TS when folate receptor-expressing KB cells were treated for 4 h (IC(50), >50 micro M). However, 24 h after incubation, MTX-HSA inhibited TS with an IC(50) of 6.9 micro M. In addition, we found that MTX-HSA had a delayed effect on the cell cycle compared with MTX and that this effect could be inhibited with the lysosomal inhibitor methylamine, suggesting that MTX-HSA activity is dependent on lysosomal processes. The proliferation of different wild-type and MTX-resistant tumor cell lines was inhibited at IC(50) concentrations between 2 and 78 micro M, respectively. MTX-HSA accumulates in vivo in the tumor tissue. Local concentrations of 18-29 micro M were measured, which are effective antiproliferative concentrations as determined in vitro. We also investigated the antitumor activity of MTX-HSA in vivo in different human tumor xenografts grown s.c. in nude mice. Fourteen tumors from eight different tissues were tested. Nine of 14 tumors (64%) showed a clear response with tumor inhibition, stasis, or regression; 5 of 14 (36%) gave a moderate response with tumor growth delay or no response. In conclusion, MTX-HSA is effectively taken up by the cells via albumin receptor- or folate receptor-mediated endocytosis and time-dependently released as an active compound into the cytosol to exert an inhibiting effect on TS and to induce cell cycle alterations. In vivo, effective concentrations of MTX-HSA were reached in tumor tissue to exhibit antitumor activity.
TL;DR: The optimum conditions of fluorometric determination of daunorubicin were studied and the developed method was successfully applied to the determination ofdaunorubsicin in serum samples.
TL;DR: Adsorption reactions are discussed, taking into account acid-base functional groups of the protein and the surface oxide in different pH ranges, considering various types of interactions.
TL;DR: It was possible to correlate the degree of electron blocking with the amount of adsorbed protein to show that the greater the adsorption, the larger the blocking effect, and HSA proved to be more efficient at blocking the electron transfer.
TL;DR: Results indicate that the C3-H in leflunomide is essential for ring opening, and a mechanism for the p450-mediated ring scission is proposed in which the isoxazole ring nitrogen or oxygen coordinates to the reduced form of the heme followed by charge transfer from p450Fe(II) to the C=N bond or deprotonation of the C4-H, which results in a cleavage of the N-O bond.
Abstract: The 3-unsubstituted isoxazole ring in the anti-inflammatory drug leflunomide undergoes a unique N-O bond cleavage to the active alpha-cyanoenol metabolite A771726, which resides in the same oxidation state as the parent. In vitro studies were conducted to characterize drug-metabolizing enzyme(s) responsible for ring opening and to gain insight into the mechanism of ring opening. Under physiological conditions, leflunomide was converted to A771726 in rat and human plasma (rat plasma,t1/2 = 36 min; human plasma, t1/2 = 12 min) and whole blood (rat blood, t1/2 = 59 min; human blood, t1/2 = 43 min). Human serum albumin also catalyzed A771726 formation, albeit at a much slower rate (t1/2 = 110 min). Rat and human liver microsomes also demonstrated NADPH-dependent A771726 formation (human liver microsomes, Vmax = 1797 pmol/min/mg and Km = 274 microM). Leflunomide metabolism in microsomes was sensitive to furafylline treatment, suggesting p4501A2 involvement. 3-Methylleflunomide, which contained a 3-methyl substituent on the isoxazole ring, was resistant to ring opening in base, plasma, blood, and liver microsomes. In microsomes, two monohydroxylated metabolites were formed, and metabolite identification studies established the 3- and the 5-methyl groups on the isoxazole ring as sites of hydroxylation. These results indicate that the C3-H in leflunomide is essential for ring opening. Although A771726 formation in human liver microsomes or recombinant p4501A2 required NADPH, its formation was greatly reduced by oxygen or carbon monoxide, suggesting that the isoxazole ring opening was catalyzed by the p450Fe(II) form of the enzyme. A mechanism for the p450-mediated ring scission is proposed in which the isoxazole ring nitrogen or oxygen coordinates to the reduced form of the heme followed by charge transfer from p450Fe(II) to the C=N bond or deprotonation of the C3-H, which results in a cleavage of the N-O bond.
TL;DR: It is established that the phenolic OH group of curcumin is the most acidic and that its dissociation is responsible for both the large red-shift of the main absorption band and the binding ofCurcumin to HSA in a right-handed chiral conformation.
Abstract: Curcumin binding to human serum albumin (HSA) has been found recently to induce bisignate CD curves due to intramolecular exciton coupling between the two feruloyl chromophoric parts The present study reports further results on this interaction UV–vis and chiroptical properties of HSA-bound curcumin were analyzed in detail by comparison with bilirubin–albumin complexes Data obtained by UV–vis and fluorescence spectroscopy, CD displacement experiments and molecular modelling methods suggested the primary binding site of curcumin to be located in site I of HSA Since acid–base dissociation of the polyphenol type curcumin molecule plays a fundamental role in albumin binding, light absorption spectra of curcumin and half-curcumin (dehydrozingerone) were studied in ethanol and in water at different pH values It is established that the phenolic OH group of curcumin is the most acidic and that its dissociation is responsible for both the large red-shift of the main absorption band and the binding of curcumin to HSA in a right-handed chiral conformation Additionally, it is demonstrated that pH dependent induced CD spectra can be utilized to determine the acid–base dissociation constant; from chiroptical data the first pKa value of curcumin was calculated (828)
TL;DR: The steady-state emission data suggest that quercetin binds to two distinct sites in HSA from which the emissions from the normal tautomer and complex species take place, and the preliminary results of studies on emission decay kinetics are reported herein.
Abstract: Flavonols are plant pigments that are ubiquitous in nature. Quercetin (3,3',4',5,7-pentahydroxyflavone) and other related plant flavonols have come into recent prominence because of their usefulness as anticancer, antitumor, anti-AIDS, and other important therapeutic activities of significant potency and low systemic toxicity. Quercetin is intrinsically weakly fluorescent in aqueous solution, showing an emission maximum at approximately 538 nm. Upon binding to human serum albumin (HSA), quercetin undergoes dramatic enhancement in its fluorescence emission intensity, along with the appearance of dual emission behavior, consisting of normal and excited-state proton transfer (ESPT) fluorescence. In addition, the occurrence of a third emitting species has been noted for the first time. This is attributed to a electronic ground-state complex formed in the protein environment. High values of the fluorescence anisotropy (r) are obtained in the presence of HSA for the ESPT tautomer (r = 0.18), as well as the complex species (r = 0.37) of quercetin, indicating that the precursor ground-state molecules for both these emitting species of quercetin molecules are located in the motionally constrained sites of HSA. The steady-state emission data suggest that quercetin binds to two distinct sites in HSA from which the emissions from the normal tautomer and complex species take place. The preliminary results of studies on emission decay kinetics are also reported herein. Studies by far-UV circular dichroism spectroscopy reveal that binding of quercetin induces no significant perturbation in the secondary structure of HSA.
TL;DR: A method that combines specific isolation of nitrated proteins with mass spectrometric determination of the amino acid sequence and the site of nitration of individual proteins is described and its identity is confirmed by peptide mass fingerprinting and MASCOT.
TL;DR: It is concluded that fatty acids regulate anti-/prooxidant properties of Cu-albumin via controlling redox status of Cys-34, which is a transition metal capable of undergoing one-electron oxidation-reduction conversions.
TL;DR: The results showed that TPNS combined with HSA through C=O and C-N groups of polypeptide chain through Fourier transformed infrared spectrometry and fluorescence methods showed the significant loss of alpha-helix structure and the microenvironment changes of the tyrosine residues in protein at higher drug concentration.
TL;DR: NN127 shows promise as a reagent capable of fluorescently labeling analyte proteins for analysis by CE-LIF without itself being significantly fluorescent under the aqueous solution conditions studied herein.
TL;DR: In rhesus monkeys administered a dose of 50 microg/kg intravenously (i.v.) or subcutaneously (s.c.), Albuferon beta induced sustained increases in serum neopterin levels and 2',5' mRNA expression, suggesting a clinical opportunity for improved IFN-beta therapy.
Abstract: The long half-life and stability of human serum albumin (HSA) make it an attractive candidate for fusion to short-lived therapeutic proteins. Albuferon™ (Human Genome Sciences [HGS], Inc., Rockville, MD) beta is a novel recombinant protein derived from a gene fusion of interferon-β (IFN-β ) and HSA. In vitro, Albuferon beta displays antiviral and antiproliferative activities and triggers the IFN-stimulated response element (ISRE) signal transduction pathway. Array analysis of 5694 independent genes in Daudi-treated cells revealed that Albuferon beta and IFN-β induce the expression of an identical set of 30 genes, including 9 previously not identified. In rhesus monkeys administered a dose of 50 μg/kg intravenously (i.v.) or subcutaneously (s.c.) or 300 μg/kg s.c., Albuferon beta demonstrated favorable pharmacokinetic properties. Subcutaneous bioavailability was 87%, plasma clearance at 4.7-5.7 ml/h/kg was approximately 140-fold lower than that of IFN-β, and the terminal half-life was 36-40 h compared with...
TL;DR: Time-of-flight secondary ion mass spectroscopy (ToF-SIMS) was used in this study to characterize human serum albumin (HSA) adsorption on two different polyearbonate surfaces: a native membrane and a hydrophilic treated one.
Abstract: The adsorption of proteins at biomaterial surfaces depends on the properties of the substrate and can cause changes in protein conformation. Time-of-flight secondary ion mass spectroscopy (ToF-SIMS) was used in this study to characterize human serum albumin (HSA) adsorption on two different polyearbonate surfaces: a native membrane and a hydrophilic treated one. The amount adsorbed as a function of HSA concentration in solution was compared for the two substrates. The treated membrane was found to have a lower affinity for albumin than the native one. Principal component analysis was used to reveal changes in albumin conformation as a function of albumin concentration in solution and to compare the conformations adopted on the two substrates. The albumin conformation was different on the two substrates, and in every case, the protein lost its native structure. A correlation was found between the amount adsorbed on the hydrophilic surface and the albumin conformation on this surface.
TL;DR: The results suggest that longer spacer binding to heparin can increase the opportunity of anti-coagulation on contacting blood and improve the hydrophilicity and blood compatibility of PSF membrane could be improved by chitosan andHeparin conjugate.
Abstract: Polysulfone (PSF) membranes were treated with ozone to introduce peroxides, and then grafted with either acrylic acid or chitosan, followed by the immobilization of heparin. The effect of spacer arm on blood compatibility was investigated using three chitosans of different molecular weight [1170 (water soluble), 160 000, and 400 000] and similar degrees of deacetylation (75%). The hydrophilicity was evaluated by measuring the contact angle of water. Blood compatibility was evaluated using the activated partial thromboplastin time (APTT) as well as the adhesion of platelets. The protein affinity was determined by the absorption of human serum albumin (HSA) and human plasma fibrinogen (HPF). The results show that by the coupling of chitosan, the amount of heparin immobilized can be increased by four times. Water contact angle (from 78 ° to 41 °) decreased with the increase of the amount of heparin immobilized, showing increased wettability. The heparinized PSF membrane showed longer APTT and decreasing platelet adhesion, compared to that of unmodified PSF membrane. The adsorption of HSA and HPF were reduced to 17 and 6%, respectively. This suggests that longer spacer binding to heparin can increase the opportunity of anti-coagulation on contacting blood. These results demonstrated that the hydrophilicity and blood compatibility of PSF membrane could be improved by chitosan and heparin conjugate. Copyright © 2003 John Wiley & Sons, Ltd.
TL;DR: In this article, the synthesis of a new reagent, saturated fatty hydrocarbon substituting group compound N-n-hexyl-N-′-(sodium p-aminobenzenesulfonate) thiourea (HXPT), is described.
Abstract: The synthesis of a new reagent, saturated fatty hydrocarbon substituting group compound N-n-hexyl-N ′-(sodium p-aminobenzenesulfonate) thiourea (HXPT), is described. The interactions between HXPT and bovine serum albumin or human serum albumin were studied by fluorescence spectroscopy. The binding constants of HXPT with BSA or HSA were determined at different temperatures under the optimum conditions. The binding sites were obtained and the acting force were suggested to be mainly hydrophobic. The effect of common ions on the binding constants was also investigated. A practical method was proposed for the determination of HXPT in bovine serum or human serum samples.
TL;DR: Bismuth complexes have been widely used in clinical treatment as antiulcer drugs, and it was found that over 70% of bismuth binds to transferrin even in the presence of a large excess of albumin at pH 7.4.
01 Oct 2003
TL;DR: Human serum albumin was adsorbed into porous silicon layers with thickness up to 3 microm and with different mean pore radius in the range 4.5-10 nm and the results show that albumin penetrated into the pores, but could not totally occupy the available surface area when the layer thickness was larger than 1 microm.
Abstract: Human serum albumin was adsorbed into porous silicon layers with thickness up to 3 μm and with different mean pore radius in the range 4.5–10 nm. The adsorbed amount of protein was quantified by I 125 radioactive labeling techniques and ellipsometry. The results show that albumin penetrated into the pores when the mean pore radius was larger than 5.5 nm, but could not totally occupy the available surface area when the layer thickness was larger than 1 μm. Loading of albumin both into porous layers and onto plane silicon as a function of albumin concentration was also investigated. These measurements show that loading of protein increased with protein concentration at least up to 10 mg/ml for porous silicon and up to 1 mg/ml for plane silicon. The maximum deposition into the type of porous layers used here was 28 μg/cm 2 , compared to 0.36 μg/cm 2 for plane silicon.