Showing papers on "Human serum albumin published in 1995"
TL;DR: Analysis of the kinetic data indicated that the rate-limiting step was the reaction of NO with oxygen, and the reaction rate of the nitrosating species N2O3 with thiols is competitive with its rate of hydrolysis.
357 citations
TL;DR: Data from this study support the hypothesis that the pharmacological activity of MPA is a function of unbound drug concentration.
Abstract: Mycophenolate mofetil, the prodrug form of the immunosuppressive agent mycophenolic acid (MPA), is currently in clinical trials evaluating its effectiveness in transplant recipients. In this study, we validated an ultrafiltration system for the reliable measurement of free MPA. Using this technique, we evaluated factors that might be important in modulating the free fraction of this drug. Human serum albumin (HSA), high concentrations of the primary glucuronide metabolite of MPA, and sodium salicylate significantly affected MPA binding. For HSA the mean +/- SE binding capacity (Bmax) and the dissociation constant (Kd) were 1095 +/- 34 mumol/L and 12.98 +/- 0.93 mumol/L, respectively. The dose for 50% inhibition (IC50) of inosine monophosphate dehydrogenase isoform II by MPA increased 5.4-fold as the concentration of HSA added to the enzyme reaction mixture increased from 0 to 50 g/L (0-724 mumol/L). Furthermore, the IC50 MPA concentration for phytohemagglutinin A-stimulated human peripheral blood mononuclear cells increased 4.8-fold when incubations were performed in the presence of 10 g/L (145 mumol/L) HSA vs no added HSA. These data support the hypothesis that the pharmacological activity of MPA is a function of unbound drug concentration.
320 citations
TL;DR: PLAPEG nanoparticles can be considered as a sustained release parenteral (intravenous) dosage form and significantly increases the plasma half-life of the colloidal carrier (‘stealth nanoparticles’).
218 citations
TL;DR: In healthy elderly subjects, f−(HMA) was significantly lower than in healthy young male subjects, indicating that HSA in the elderly becomes more oxidized than in the young subjects, thus securing a certain redox buffer capacity with age.
152 citations
TL;DR: Nonlinear least-squares analysis of the decrease in fluorescence accompanying the displacement yields the stoichiometry and the dissociation constants and is eminently suitable for large-scale screening.
138 citations
TL;DR: The use of proteins as chiral selectors in CE is reviewed in this article, where the performance of packed-capillary electrochromatography with protein phases is compared with free-solution systems.
138 citations
TL;DR: Zonal elution and high-performance affinity chromatography studies indicated that R- and S-ibuprofen had one common binding site on the immobilized HSA column and at least one other major binding region.
131 citations
TL;DR: Human serum albumin prepared by blood fractionation for clinical purposes was found to degrade when stored at or above 30 degree C, and an intact aspartyl-alanyl dipeptide, purified from degraded albumin solutions, differed substantially from a synthetic dipeptic on amino acid analysis, N-terminal sequencing and NMR.
Abstract: Human serum albumin prepared by blood fractionation for clinical purposes was found to degrade when stored at or above 30 degree C. Mass spectrometry and N-terminal sequencing of the protein identified degradation corresponding to the loss of the first two residues, aspartic acid and alanine. The reaction was shown to be dependent upon temperature and the N-terminal alpha-amino group. In addition, comparison with serum albumins derived from other species showed that the instability of the N-terminus was specific to the human albumin sequence. An intact aspartyl-alanyl dipeptide, purified from degraded albumin solutions, differed substantially from a synthetic dipeptide on amino acid analysis, N-terminal sequencing and NMR. It is suggested that the released dipeptide may be cyclic, implying a novel cleavage mechanism.
129 citations
TL;DR: The present limited data indicated that the four uremic toxins caused inhibition to any endo- or exogenous substances on HSA, and the binding parameter of CMPF was found to have the strongest affinity (10(7)M-1) among all the uremi toxins studied.
Abstract: The interaction of uremic toxins including indole-3-acetic acid (IA), indoxyl sulfate (IS), hippuric acid (HA) and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) with human serum albumin (HSA) has been investigated by three methods of fluorescent probe displacement, ultrafiltration and equilibrium dialysis. The binding parameter of CMPF was found to have the strongest affinity (10(7)M-1) among all the uremic toxins studied. Competitive experiment based on the method of Kragh-Hansen suggested that IA, IS and HA bind to site II, whereas CMPF binds to site I. The present limited data indicated that the four uremic toxins caused inhibition to any endo- or exogenous substances on HSA.
118 citations
TL;DR: S-ODNs are likely to interact and displace other therapeutic agents that bind to albumin, particularly those binding at site I, and suggest cooperative binding and/or the presence of secondary binding sites on the S-ODN.
Abstract: Phosphorothioate oligonucleotides (S-ODNs) have the ability to modulate gene expression selectively and thus have potential therapeutic capabilities. This potential led us to investigate the protein binding characteristics of selected S-ODNs. We evaluated S-ODN interactions with bovine serum albumin (BSA) and human serum albumin (HSA) in vitro. The equilibrium dissociation constants Km for the binding of a 20 mer S-ODN with BSA and HSA range between 1.1-5.2 x 10(-5) and 2.4-3.1 x 10(-4) M, respectively. The Km for an unrelated 15 mer S-ODN binding with HSA ranges between 3.7 and 4.8 x 10(-5) M. Studies with a fluorescently labeled 27 mer S-ODN suggest cooperative binding (Hill slope = 1.67) and/or the presence of secondary binding sites on the S-ODN. HSA or BSA linked to Sepharose was incubated with a 15, 20, or 24 mer S-ODN followed by the addition of selected drugs known to be highly protein bound (nifedipine, warfarin, midazolam, probenecid, indomethacin, and mitoxantrone). Up to 30% of S-ODN was displaced by warfarin in competition binding assays. Conversely, HSA-bound warfarin was incubated with a variety of oligonucleotides, including RNA and genomic dsDNA. Maximum displacement of warfarin-bound HSA was observed following incubation with 5'-cholesterol-conjugated 20 mer S-ODN. In summary, S-ODNs are likely to interact and displace other therapeutic agents that bind to albumin, particularly those binding at site I.
111 citations
TL;DR: In this paper, the corrected fluorescence maxima of the proton-transfer (PT) tautomer of a novel protein-binding site probe, 4-hydroxy-5-azaphenanthrene (HAP), correlate with the static polarity of the environment.
Abstract: The corrected fluorescence maxima of the proton-transfer (PT) tautomer of a novel protein-binding site probe, 4-hydroxy-5-azaphenanthrene (HAP), correlate with the static polarity of the environment. The PT fluorescence of HAP exhibits a maximum in water at 586 nm and at 623 nm in cyclohexane. This PT fluorescence is perturbed by either a strong base or acid, and the emission of the anion or cation shows up in the spectra, respectively. The fluorescence lifetime of the PT tautomer of HAP increases with solvent polarity, being 0.36 ns in cyclohexane and 0.50 ns in methanol. The steady-state and time-resolved data demonstrate the insensitivity of PT tautomer fluorescence of HAP to dipole-dipole relaxation of the environment and the high static polarity of the so-called hydrophobic binding site of human serum albumin (HSA). The overlap of the fluorescence spectra of HAP observed in water solution and in a complex with HSA suggests the similarity of the static polarities in these two environments. 22 refs., 10 figs., 1 tab.
TL;DR: In this paper, the adsorption of human serum albumin, immunoglobulin G, fibronectin, and fibrinogen at phospholipid surfaces was studied with in situ ellipsometry.
Journal Article•
TL;DR: In this article, a method to predict quantitatively peptide binding to HLA DRB1*0401 has been developed using a data set of the relative contributions of each of the naturally occurring amino acids in the context of a simplified peptide back-bone.
Abstract: A method to predict quantitatively peptide binding to HLA DRB1*0401 has been developed using a data set of the relative contributions of each of the naturally occurring amino acids in the context of a simplified peptide back-bone. The prediction assumed that the relative role of each of the peptide side chains could be treated independently and could be measured by assaying each of the 20 naturally occurring amino acids at the central 11 positions of a 13-residue peptide previously shown to contain the minimal requirements for high-affinity binding to HLA-DR proteins. The resultant database was shown to have predictive value when tested on a set of 13 unrelated peptides known to bind DRB1*0401 with a wide range of apparent affinity. The database was tested further by analyzing myelin basic protein. All 13 amino acid peptides containing a hydrophobic amino acid at the third position were synthesized and assayed for binding purified DRB1*0401. In every case, the measured affinity correlated with the predictive values within the experimental error of the assays. Finally, the ability to predict peptide binding to MHC class II molecules was shown to help in identifying T cell determinants. The specificity of DRB1*0401-restricted T cell hybridomas against human serum albumin corresponded to two peptides, predicted and shown to bind the class II protein with high affinity.
Journal Article•
TL;DR: The results establish that the binding of these reactive metabolites to nucleophilic sites of proteins occur via two different mechanisms: one involving imine (Schiff base) formation and the other involvingucleophilic displacement of glucuronic acid.
Abstract: The structures of adducts formed from in vitro incubation of a drug (tolmetin) glucuronide (TG) and human serum albumin (HSA), and the preferred binding sites on this protein were determined by mass spectrometry. In addition, the concentration dependence of covalent modification of HSA by TG was studied at three different concentration ratios of TG to HSA. Protein adducts were enzymatically digested and peptide fragments were separated by HPLC. Tolmetin-containing peptides (indicated by absorbance at 313 nm) were analyzed by liquid secondary-ion mass spectrometry, continuous flow-fast atom bombardment mass spectrometry, and collision-induced dissociation using a four-sector tandem mass spectrometer, matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry, and in selected cases by Edman sequencing. The identified peptides contained tolmetin linked covalently via a glucuronic acid to a protein lysine group (lysine 199 and to a lesser extent lysines 195 and 525) or tolmetin directly linked to lysines (lysines 199 and 541), serines (serines 220, 232, and 480), or arginines (arginine 222). In addition, there was indirect evidence for binding of TG to lysine 541, and binding of tolmetin to arginine 521. Our results establish that the binding of these reactive metabolites to nucleophilic sites of proteins occur via two different mechanisms: one involving imine (Schiff base) formation and the other involving nucleophilic displacement of glucuronic acid. Our data suggest, however, that the former, in which the glucuronic acid moiety of the acyl glucuronide is retained within the adducts, is favored at lower (closer to physiological) metabolite concentrations.
TL;DR: It was concluded that only a subset of urinary proteins is present in stone matrix, the same proteins are found in all types of stones, HSA shows significant affinity for several proteins of the matrix, but not for proteins absent from stones and HSA also displays significant affinity from calcium oxalate monohydrate crystals in a solution similar to urine.
Abstract: Our aims were to analyze the protein composition of the organic matrix of urinary stones and to investigate the role of albumin in its constitution. Five different morphological types of stones were studied. Proteins extracted from the stone were submitted to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by immunoblotting with antibodies to 13 urinary proteins. Nine of the 13 proteins were found in all types of stone: human serum albumin (HSA), alpha 1-acid glycoprotein (alpha 1-GP), alpha 1-microglobulin (alpha 1-M), immunoglobulins (Igs), apolipoprotein A1 (apo-A1), transferrin (Tr), alpha 1-antitrypsin (alpha 1-T), retinol-binding protein (RBP) and renal lithostathine (RL). The beta 2-microglobulin (beta 2-M) was present only in calcium oxalate and uric acid stones. In contrast, ceruloplasmin, haptoglobin and Tamm-Horsfall protein (THP) were detected in none of them. Because HSA appeared as the major protein component in all stones, we wondered whether it might play a specific role in the constitution of the stone matrix. Association of HSA with urinary proteins that were present in stones was demonstrated by showing that proteins present in the matrix comigrated with HSA on gel filtration, whereas proteins that were absent did not. Moreover, HSA induced the binding of stone matrix proteins to an albumin-specific affinity column. Finally, we evidenced HSA binding to calcium oxalate monohydrate (COM) crystals in a solution similar to urine.(ABSTRACT TRUNCATED AT 250 WORDS)
Patent•
30 Aug 1995
TL;DR: In this article, a process for purifying recombinant human serum albumin (rHSA) by heating a culture medium containing rHSA and the recombinant HSA-producing host cells was proposed.
Abstract: The invention provides a process for purifying recombinant human serum albumin (rHSA) by heating a culture medium containing rHSA and the rHSA-producing host cells, feeding said heated solution upwardly into a fluidized bed in which adsorbent particles are suspended to effect contacting with the adsorbent particles and then recovering the adsorbed fraction containing the rHSA, and a composition comprising rHSA which shows a A350/A280 ratio of below 0.015, when formulated into a 25 % solution of said albumin.
TL;DR: In this paper, the authors demonstrate the reliability of the use of FT-IR spectroscopy to monitor conformational changes when a protein (HSA) is adsorbed under chromatographic conditions on the silica material RP-C6.
Abstract: The aim of this study is to demonstrate the reliability of the use of FT-IR spectroscopy to monitor conformational changes when a protein (HSA) is adsorbed under chromatographic conditions on the silica material RP-C6. In the aqueous eluent (D2O), the amount of protein retained on the phase is minimal for 30% CH3CN, whereas no protein is retained for 40%. Structural results are deduced from quantitative analyses of the infrared Amide I' absorptions and from measurements of the peptide NH-ND exchange. Both are performed for HSA in solution and for HSA adsorbed on RP-C6 in suspension in equivalent eluents. For the solutions, 30% CH3CN in the solvent weakly unfolds some structured loops of the protein. Hydration and aggregation are enhanced in 40% CH3CN, which significantly denatures α- and β-domains. When the protein is adsorbed with 0-30% CH3CN in the solvent, about one-tenth of HSA backbone unfolds. In the adsorbed state, the protein is more hydrated and self-associated than in the corresponding solutions. The larger contacts between HSA and RP-C6 are observed when the retention amount is the weakest (30% CH3CN). Results show that retention should have a hydrophobic origin. The irreversibility of the retention is supposed to be dependent on the protein structural and solvation changes observed from the solutions to the adsorbed states. To explain the elution with organic solvent > ~38%, a competition between acetonitrile and the solid phase in solvating hydrophobic domains of the protein is suggested.
TL;DR: Simultaneous binding of two drugs to human serum albumin was investigated by difference circular dichroism (delta CD) spectroscopy and the results indicate two different interaction mechanisms: a direct competition one and an indirect competition one.
TL;DR: The molecular weights of plasma proteins from healthy subjects and from patients with well-or badly-controlled diabetes mellitus have been determined by use of a matrix-assisted laser desorption ionization method, representing a highly accurate technique for the determination of the molecular weight of large biomolecules.
Abstract: The molecular weights of plasma proteins from healthy subjects and from patients with well-or badly-controlled diabetes mellitus have been determined by use of a matrix-assisted laser desorption ionization method, representing a highly accurate technique for the determination of the molecular weight of large biomolecules. Using this approach, different molecular weights of human serum albumin have been found for healthy (66,572–66,694 dalton) and diabetic (66,785–68,959 dalton) subjects. Such differences can be rationalized as being due to the different number of glucose molecules condensed on the protein and/or their further oxidation products; in the case of our diabetic patients this number is in the range of 1.4–14.8. The data show the high validity and specificity of the technique, which allows us to evaluate, without any protein degradation procedure, the number of glucose molecules condensed on a specific protein and ascertain the relationship of this number to the physiopathogenetic conditions of the subjects studied.
TL;DR: Examination of binding of thyroid hormones and related compounds at the warfarin and indole sites of human serum albumin indicated that additional factors, such as interactions through the thyronine backbone or terminal amine and carboxyl groups of these compounds, could also be involved in the binding of Thyroid hormones to HSA.
TL;DR: Evidence was obtained that chemical acetylation of Lys199 results in a decreased apparent binding affinity constant (nK) for FU, which is induced under physiological conditions by aspirin.
TL;DR: Recombinant human granulocyte-colony stimulating factor was covalently conjugated to both rat and human serum albumin to increases the circulating half life (t1/2) of rhG-CSF and decreases plasma clearance in vivo, causes increased WBC response, and increases serum stability as compared to free rhG -CSF.
Abstract: Purpose. Recombinant human granulocyte-colony stimulating factor (rhG-CSF) was covalently conjugated to both rat and human serum albumin (RSA and HSA respectively) to increases the circulating half life (t1/2) of rhG-CSF.
TL;DR: An on-line frontal analysis HPLC system was developed for the determination of the unbound concentration of troglitazone (CS-045), a new oral antidiabetic agent, in human serum albumin (HSA) solution and in human plasma, and it was found that CS-045 strongly binds with HSA.
TL;DR: Denaturation measurements of free HSA and HSA in the presence of dirhodium(II) acetate complex with 8-M urea followed by CD spectroscopy, suggest that rhodium affects the secondary protein structure and might stabilize HSA against denaturing agents.
Journal Article•
TL;DR: The aim of this study was to investigate the HSA-mediated mechanism involved in the in vitro hydrolysis by albumin of the acyl glucuronides of (R)- and (S)-ketoprofen (a nonsteroidal anti-inflammatory drug), as model compounds, and to support the existence of distinct sites on HSA for reversible binding and Hydrolysis of ( R)-ketobrofen glucuronide.
Abstract: Many carboxylic acid-containing drugs undergo conjugation with D-glucuronic acid in humans, leading to the formation of acyl glucuronides, which are excreted into urine However, these metabolites can be hydrolyzed back to the parent aglycon; this reaction can be accelerated by human serum albumin (HSA) Although this phenomenon of interaction between the acyl glucuronide and HSA has been described for various drugs, the kinetics of the protein have not been characterized The aim of this study was to investigate the HSA-mediated mechanism involved in the in vitro hydrolysis by albumin of the acyl glucuronides of (R)- and (S)-ketoprofen (a nonsteroidal anti-inflammatory drug), as model compounds The conjugates of both ketoprofen enantiomers were incubated, separately or together, with increasing concentrations of albumin (145-145 microM) at pH 74 and 37 degrees The reaction followed Michaelis-Menten kinetics and was stereoselective; the (R)-ketoprofen glucuronide was a better substrate than the S-conjugate To identify the HSA domain involved in the hydrolysis reaction, specific probes of HSA binding sites were used as potential inhibitors These probes, added at an equimolar probe/glucuronide ratio (145 microM), slightly decreased the hydrolysis (by up to 30%) They affected the reversible binding of (R)-ketoprofen glucuronide to HSA, as shown by CD studies Because iodoacetic acid did not modify the single free cysteine residue on HSA, this amino acid residue cannot be the reactive one In addition, the chemical modification of a single tyrosine residue (probably Tyr-411) on HSA by diisopropyl fluorophosphate significantly but weakly affected the hydrolysis of (R)-ketoprofen glucuronide, suggesting that this residue also is not involved in the catalysis In contrast, the R-conjugate was not bound to modified albumin, as revealed in CD experiments These results support the existence of distinct sites on HSA for reversible binding and hydrolysis of (R)-ketoprofen glucuronide
TL;DR: In this article, a sensitive and selective spectrophotometric determination of human serum albumin (HSA) was proposed by using the ternary complex-formation reaction among 2-(5bromo-2-pyridylazo)-5-(N-phenyl-N-sulfopropylamino)phenol (5-Br.PAPS)-cobalt(II)-HSA.
Abstract: A sensitive and selective spectrophotometric determination of human serum albumin(HSA) was proposed by using the ternary complex-formation reaction among 2-(5-bromo-2-pyridylazo)-5-(N-phenyl-N-sulfopropylamino)phenol (5-Br.PAPS) as a pyridylazo derivative, cobalt(II) and HSA in the presence of poly(N-vinylpyrrolidone) (PVA) as a dispersion agent. The calibration curve was linear in the range of 0 - 7.0 μg/ml HSA by measuring the difference of absorbances at 636 nm between ((5-Br.PAPS)-cobalt(II)-HSA) and (5-Br.PAPS-cobalt(II)) solutions. The sensitivity was > 6-fold over the (Pyrogallol Red(PR)-molybdenum(VI)) method, and the recovery test in urine was satisfactory (97.5 ± 2.8%).
TL;DR: Preliminary biological characterisation of this conjugate indicates that it could be a good candidate to inject as a bolus, compared with the infusion regimen needed with pro-urokinase.
Abstract: Pro-urokinase is a natural plasminogen activator that displays a clot-lysis activity through a fibrin-dependent mechanism. It seems to be a promising agent for the treatment of coronary thrombosis. Like tissue-type plasminogen activator and two-chain urokinase-type plasminogen activator, pro-urokinase has a very short half-life in circulation. It has been described that conjugation of serum albumin with pro-urokinase in plasma may occur that could protect this protein from degradation. In this study we describe the insertion of an extra cysteine residue in the N-terminal end of des-(C11-K135)-pro-urokinase (delta 125-proUK), a pro-urokinase deletion mutant lacking amino acids 11-135. We have expressed and purified the new mutein [H5K, S9C, N10T] des-(C11-K135)-pro-urokinase (Cys-delta 125-pro-urokinase) and chemically conjugated it with serum albumin via the extra cysteine of Cys-delta-pro-urokinase. The purified conjugate obtained has a lower specific amidolytic activity (72,000 U/mg) than unconjugated Cys-delta 125-pro-urikinase (240,000 U/mg) due to its higher molecular mass and has a similar fibrinolytic activity in a clot lysis test to that of delta 125-pro-urokinase. We established an ELISA to measure the concentration of the conjugate in plasma and to follow the pharmacokinetics of the conjugate in monkeys after bolus injection. The conjugate displays significant lysis of human plasma clots in vivo and a dramatic increase of the half-life in the circulation, with respect to pro-urokinase and delta 125-pro-urokinase. Therefore, preliminary biological characterisation of this conjugate indicates that it could be a good candidate to inject as a bolus, compared with the infusion regimen needed with pro-urokinase.
Journal Article•
TL;DR: Copper-62-PtsM exhibits substantial interspecies variability in the strength of its binding to serum albumin, which appears to explain the problems encountered in using animal data to predict 62Cu-PTSM behavior in humans.
Abstract: Copper-62-labeled pyruvaldehyde bis(N4-methylthiosemicarbazonato)-copper(II) (Cu-PTSM) is a generator-based PET radiopharmaceutical under investigation for use in evaluation of tissue perfusion. Despite promising results from animals, problems have been encountered in the use of 62Cu-PTSM to quantitate myocardial perfusion in humans at high flow rates, possibly due to species-dependent interactions of the tracer with serum albumin. Methods: UltrafiltratiOn and plasma/erythrocyte partitioning studies were performed to assess the protein binding of 67Cu-labeled Cu-PTSM and six related copper(II) bis(thiosemicarbazone) complexes. Results: These studies reveal significant interspecies variability in the strength of Cu-PTSM binding to serum albumin, with 67Cu-PTSM binding much more strongly to human albumin than to dog albumin. Most of the related Cu(II)-bis(thiosemicarbazone) complexes examined exhibit interspecies variability of albumin binding similar to that observed with Cu-PTSM. Two such complexes, Cu-ETS and Cu-n-PrTS, however, were identified that exhibit no preferential association with human serum albumin. Conclusion: Copper-62-PTSM exhibits substantial interspecies variability in the strength of its binding to serum albumin, which appears to explain the problems encountered in using animal data to predict 62Cu-PTSM behavior in humans. The 62Cu-ETS and 62Cu-n-PrTS complexes may be viable alternatives to 62Cu-PTSM for PET studies to evaluate quantitatively myocardial blood flow in humans.
TL;DR: In this paper, a tapping mode (intermittent contact) scanning force microscopy technique was employed to image the molecules on the surface and observed clusters of molecules and features which were interpreted as single molecules adsorbed on the mica.