Showing papers on "Human serum albumin published in 1984"

Characterization of the copper(II)- and nickel(II)-transport site of human serum albumin. Studies of copper(II) and nickel(II) binding to peptide 1-24 of human serum albumin by 13C and 1H NMR spectroscopy

Abstract: As a basis for understanding the role of albumin in the transport of metal ions, detailed investigations have been carried out to elucidate the structure of Ni(II)- and Cu(II)-binding site of the peptide residue corresponding to the NH2-terminal peptide fragment 1-24 of human serum albumin by 1H and 13C NMR spectroscopy. These studies have been conducted in aqueous medium at different pH values and at different ligand/metal ratios. The results show the following: (i) Diamagnetic Ni(II) complex and paramagnetic Cu(II) complex are in slow exchange NMR time scale. (ii) Titration results of Ni(II)-bound form of peptide 1-24 show the presence of a 1:1 complex in the wide pH range (6.0-11.0), and the same stoichiometry is proposed for Cu(II) as well. (iii) Analysis of the spectra suggests that both Ni(II) and Cu(II) have one specific binding site at the NH2-terminal tripeptide segment (Asp-Ala-His...) involving the Asp alpha-NH2, His N(1) imidazole, two deprotonated peptide nitrogens (Ala NH and His NH), and the Asp COO- group. (iv) Complexation of Ni(II) and Cu(II) causes conformational change near the metal-binding site of the polypeptide chain, but there is no other binding group involved besides those in the first three residues.

9-Deoxy-delta 9, delta 12-13,14-dihydroprostaglandin D2, a metabolite of prostaglandin D2 formed in human plasma

Abstract: Incubation of prostaglandin D2 (PGD2) with human plasma yielded a product that has been identified as 9-deoxy-9,10-didehydro-12,13-didehydro-13,14-dihydro-PGD2 (9-deoxy-delta 9, delta 12-13,14-dihydro-PGD2). The identification was based on mass spectrometry, UV spectrometry, mobilities and retention time on TLC and HPLC, and NMR. The conversion of PGD2 to this product was dependent on the incubation time and the amount of plasma added to a reaction mixture and was abolished by prior boiling. The conversion rate of PGD2 to this metabolite was 0.03 nmol/min per mg of protein of whole plasma at pH 8.0 at 37 degrees C. Similar conversion was also found by incubating PGD2 with human serum albumin added at the concentration found in plasma. These results suggest that the conversion of PGD2 to this product is catalyzed by the enzymatic action of a plasma protein, probably serum albumin. The biological activities of this compound were examined in several systems. It showed negligible activity in inhibition of human platelet aggregation and relaxation of rabbit stomach strip. On the other hand, it exhibited a three times stronger inhibitory activity (IC50, 1.8 microM) than PGD2 (IC50, 5 microM) on the growth of L-1210 cultured cells.

Isolation of a somatomedin-binding protein from preterm amniotic fluid. Development of a radioimmunoassay.

Abstract: Amniotic fluid binding protein (AFBP) is a heat and acid stable somatomedin (Sm)-binding protein with a mol wt of 35-40,000 and an isoelectric point of +/- 4.7. It is reactive in RRAs for Sm and inhibits Sm activity in Sm bioassays. AFBP was purified from midgestational human amniotic fluid (AF) using acid-ethanol extraction, Sephadex G-150 chromatography, high speed gel filtration chromatography, and disc gel-electrophoresis. Specific binding activity (microgram equivalents per mg protein) was quantitated by incubation with 125I-insulin-like growth factor II and dextran-coated charcoal separation. Protein recovery was less than 1%. AFBP antiserum was produced by immunizing rabbits with purified AFBP. The antiserum was cleared of human serum albumin antibodies by affinity chromatography. Immunoelectrophoresis of 20x concentrated preterm AF and fetal serum resulted in one precipitin line. AFBP was labeled by the chloramine-T method. The AFBP antiserum specifically bound +/- 35% of added 125I-AFBP at a final dilution of 1:5000. A double antibody RIA was developed. The AFBP level measured by RIA in midgestation AF (n = 30) was 148 +/- 18 (SEM) and in term AF (n = 12) 72 +/- 36 mu geq/ml. Insulin-like growth factor I/Sm-C values (determined by RIA) in the same samples were uniformly very low (less than 0.10 U/ml). When serum was chromatographed on Sephadex G-200 at pH 2.2, AFBP-RIA activity eluted in one peak corresponding to a mol wt of 35-40,000. Highest activity was found in fetal serum (gestational age +/- 20 weeks) and lowest in serum from adults. The development of the AFBP-RIA may contribute to further elucidation of the physiological importance of Sm and the Sm-binding proteins in pre- and postnatal growth.

Nonenzymatic glucosylation of lysine residues in albumin.

Abstract: Publisher Summary This chapter describes the preparation and characterization of radiolabeled glycosylamine (GA) and N-substituted-l-amino-l-deoxyketopyranose (KP) derivatives of albumin. It also illustrates procedures for determining kinetic constants involved in their formation and dissociation and for detecting glucose adducts to lysine residues in human serum albumin (HSA) and other proteins. Nonenzymatic glucosylation is a common posttranslational modification of body protein, in which glucose reacts directly with primary amino groups on protein to yield stable covalent adducts. The sugar reacts primarily with the ɛ-amino groups of lysine residues in albumin and other proteins, although reaction at the α-amino terminus also occurs. Since, the rate of hydrolysis of glycosylamines is relatively slow at neutral pH and low temperature, the kinetics of dissociation of GA adducts can be measured by Sephadex G-25 chromatography. The Amadori adduct to the protein (after acid discharge of GA) is stable to extended incubation of the protein at 37° and has an estimated minimum half-life of about 3 weeks.

Effect of serum albumin on siderophore-mediated utilization of transferrin iron.

Abstract: The effect of serum and serum proteins on enterobactin- and aerobactin-mediated utilization of transferrin iron has been investigated. Serum was found to impede transfer of iron from iron transferrin to enterobactin and from [55Fe]ferric enterobactin to cells of Escherichia coli BN3040 Na 1R iuc . In contrast, serum had essentially no effect on the rate of these reactions mediated by aerobactin. Three purified serum proteins, human serum albumin, bovine serum albumin, and human immunoglobulin, were comparable to human serum in their selective ability to interfere with the transfer of 55Fe from [55Fe]ferric enterobactin to E. coli BN3040 Na 1R iuc . The inhibitory effect of human serum albumin on the enterobactin-mediated transfer of iron from [55Fe]transferrin was enhanced by preincubation of the protein with the siderophore. Pretreatment of the bacterial cells with human serum albumin did not affect the rate of utilization of siderophore iron. A linear, reciprocal relationship was found to hold for human albumin concentration vs. the first-order rate constant ( kobsd ) for the velocity of iron transfer from iron transferrin to enterobactin. Binding of serum albumin to enterobactin increased the intensity of the near-ultraviolet absorption band of the siderophore and shifted it to longer wavelengths. The stoichiometry of binding to human and bovine serum albumins was established as 1:1, and the binding constant for both enterobactin and ferric enterobactin was estimated to be in the range 1 X 10(4)-1.2 X 10(5) M-1. These results indicate that serum albumin may act synergistically with other factors in the serum, such as transferrin, to limit iron supply and in this way restrict the growth of invading microorganisms.

Albumin binding of anti-inflammatory drugs. Utility of a site-oriented versus a stoichiometric analysis.

Abstract: Binding equilibria of 12 nonsteroidal, anti-inflammatory substances, salicylic acid, diflunisal, phenylbutazone, azapropazone, fenbufen, biphenylacetic acid, naproxen, flurbiprofen, ibuprofin, diclofenac, indomethacin, and benoxaprofen, to defatted human serum albumin has been investigated at 37 degrees, pH 7.4, in a sodium phosphate buffer, 66 mM, by means of equilibrium dialysis and, in case of salicylic acid, by dialysis rate determinations. Cobinding of each of these drugs with monoacetyl-4,4'-diaminodiphenyl sulfone, warfarin, and diazepam has been studied by measuring dialysis rates of the last-mentioned ligands. Cobinding of each drug with bilirubin was investigated by two techniques, equilibrium dialysis against albumin with and without bilirubin, and by measuring rates of oxidation of free bilirubin with hydrogen peroxide and peroxidase. Results were analyzed in quantitative terms. The use of a site-oriented description versus a stoichiometric analysis is discussed. The stoichiometric description is preferred for the following reasons: (a) Simple relations exist between the percentage of bound drug at low drug concentrations and the first stoichiometric binding constant. (b) The stoichiometric description does not imply that preformed binding sites are present in the albumin molecule. (c) A quantitative, stoichiometric analysis of multiple cobinding of two ligands is possible.

Origin of mammalian biliprotein and rearrangement of bilirubin glucuronides in vivo in the rat.

Abstract: In hepatobiliary disease bilirubin becomes bound covalently to serum albumin, producing a nondissociable bile pigment-protein complex (biliprotein). To elucidate the mechanism of biliprotein formation we studied the bile pigment composition of blood from animals with experimental cholestasis and carried out comparative studies on the rate of biliprotein formation in vivo and in vitro during incubation of bilirubin glucuronides with albumin. Bile duct ligation in the rat and guinea pig led to rapid accumulation in the circulation of bilirubin, heterogeneous bilirubin esters of glucuronic acid, and a biliprotein that migrated along with albumin on high performance liquid chromatography. When the obstruction was removed, biliprotein remained longer in the circulation than did the other bile pigment species. Biliprotein and heterogeneous bilirubin esters of glucuronic acid were not formed in bile duct-ligated homozygous Gunn rats but they were formed when bilirubin glucuronides were incubated with Sprague-Dawley rat serum or human serum albumin at 37 degrees C in vitro. Bilirubin glucuronide rearrangement in vitro was accompanied by nonenzymic hydrolysis. We conclude that the formation of biliprotein in vivo is probably nonenzymic and suggest that mammalian biliprotein is formed by acyl migration of bilirubin from a bilirubin-glucuronic acid ester to a nucleophilic site on albumin.

Terbium chelate for use as a label in fluorescent immunoassays

Abstract: Human serum albumin has been labelled with a terbium complex by means of a reagent prepared from the bis-cyclic anhydride of diethylenetriamine pentaacetic acid and p-aminosalicylic acid. This reagent combines high fluorescent intensity with stability at high dilution (10–9M). The fluorescent conjugate so produced has been used in a simple fluoroimmunoassay, using human serum albumin as a model analyte.

Clearance of circulating IgA immune complexes is mediated by a specific receptor on Kupffer cells in mice.

Abstract: To characterize the physiology of circulating IgA immune complexes (IgA-IC), the dynamics of IgA-IC removal by the liver were examined. After intravenous injection, covalently cross-linked IgA antibodies to the dinitrophenyl determinant were rapidly removed from the circulation by the liver. Immunofluorescence microscopy and light and electron microscope autoradiography showed that the IgA-IC were associated with Kupffer cells. With increasing doses of injected IgA-IC the clearance velocity approached a maximum, thus prolonging the circulation of IgA-IC. All these observations indicated a receptor-mediated process. Saturating doses of various potential receptor-blocking agents, heat-aggregated mouse IgG, microaggregated human serum albumin, and purified dimeric IgA did not influence the clearance pattern and hepatic uptake of radiolabeled IgA-IC. Mouse livers were also perfused via the portal vein with 1 microgram of IgA-IC. In the presence or absence of serum proteins, 43% of the perfused IgA-IC were removed in a single passage. This liver uptake was not reduced with simultaneous perfusion of large doses of aggregated mouse IgG, aggregated human serum albumin, or purified free dimeric mouse IgA. In contrast, the liver uptake of radiolabeled IgA-IC was decreased by 88% with the addition of 1 mg unlabeled IgA-IC. These observations support the conclusion that removal of IgA-IC from circulation is mediated by a specific IgA receptor on Kupffer cells.

Alpha1‐acid glycoprotein and albumin in human serum bupivacaine binding

Abstract: Bupivacaine protein binding was studied with the use of human serum, isolated human serum albumin, and isolated alpha 1-acid glycoprotein. The effect of lactic acid on bupivacaine binding was also studied. Bupivacaine protein binding in serum is best characterized by a model described by two classes of binding sites and that in alpha 1-acid glycoprotein or albumin is best characterized by a model described by one class of binding sites for each protein. Albumin binding closely correlated with the data obtained for the low-affinity, high-capacity binding site in serum, while alpha 1-acid glycoprotein data closely correlated with the data obtained for the high-affinity, low-capacity site in serum. A reduction in pH resulted in a significant reduction in the affinity for the high-affinity, low-capacity site in serum. No other binding parameters were affected. These data were in excellent agreement with results of the isolated protein studies. Our data demonstrate that acidosis results in significant increases in free bupivacaine concentrations only at relatively low total bupivacaine concentrations (less than 7 micrograms/ml) and that distribution characteristics for bupivacaine are essentially unchanged over a wide concentration range.

High-performance liquid chromatography as a technique to measure the competitive adsorption of plasma proteins onto latices

Abstract: Isotherms of human serum albumin (HSA), human immunoglobulin G (HIgG), and human fibrinogen (HFb) onto a polystyrene (PS)-latex were determined by depletion of protein in the solution, which was either followed by radioactivity measurements or by UV spectroscopy. Different adsorption isotherms for the same protein were obtained when either radioactivity measurements or UV spectroscopy was used as a detection technique. In order to obtain reliable results from competitive protein adsorption experiments, a method based on the use of high-performance liquid chromatography was developed. A strong preferential adsorption of HFb was observed when adsorption studies were carried out with mixtures of HSA, HFb, and HIgG. When adsorption studies were carried out with solutions containing HSA monomer and dimer, a strong preferential adsorption of HSA dimer was also observed.

Occupational asthma from nickel sensitivity: I. Human serum albumin in the antigenic determinant.

Abstract: Occupational asthma from nickel sensitivity was confirmed in a male worker (SB) by allergy skin tests and inhalational challenge. In an ammonium sulphate coprecipitation test 63Ni was selectively precipitated from SB plasma indicating antibody with nickel related specificity. Preincubation of 63Ni with human serum albumin (HSA) increased the specificity of the coprecipitation test. Blocking experiments with nickel and copper(II) salts effectively inhibited the binding of 63Ni to antibody of SB plasma but did not affect control tests or that for antibodies to an unrelated antigen, ampicillin. Co2+ slightly inhibited the binding of 63Ni, while Zn2+ and Mn2+ failed to inhibit. This deactivation pattern corresponds to the known sequence of binding of these metals to the primary copper binding site of HSA. It is concluded that the antigenic determinant depends on the combination of Ni2+ with HSA at this specific copper/nickel plasma transport site.

Equilibrium adsorption of human serum albumin and human fibrinogen on hydrophobic and hydrophilic surfaces

Abstract: The adsorption of human serum albumin and human fibrinogen on flat surfaces was quantitatively determined by measuring the decrease in UV absorption in the adsorption solution. The applicability of the method is discussed for hydrophilic and hydrophobic materials. The values of equilibrium adsorption are presented--albumin on polyethylene, and fibrinogen on polyethylene, carbon, poly(2-hydroxyethyl methacrylate), and cellophane.

Plasma clearance, tissue distribution and catabolism of cationized albumins with increasing isoelectric points in the rat.

Abstract: The infusion of cationic substances produces acute renal failure and proteinuria, and an experimental disease very similar to disseminated coagulopathy. The purpose of this work was to investigate further, in the rat, the plasma disappearance rate, the tissue distribution and the catabolism of albumins with modified isoelectric points. Human serum albumin was cationized with hexanediamine and labelled with 125I or 131I. During 10-180 min after their intravenous injection into the rat, these modified 125I-labelled albumins were cleared from the plasma at a rate which increased with their isoelectric point. At 1 and 3 h after the injection of highly cationic proteins (isoelectric point higher than 9.5) the tissue protein bound 125I concentration was greatest (approximately 3.5% of the injected activity/g) in the spleen and liver. A significant amount of the basic proteins was found in the kidney and in the lung (0.75-1%/g). Their concentration was much lower in other tissues. The whole body radioactivity was significantly lower 24 h after the injection of 131I-labelled cationized albumins than after similarly labelled native albumin. However, expressed as a percentage of the retention at 24 h, the body radioactivity at later times was higher for cationic than for native albumin. We conclude that cationized albumins are cleared from the plasma, mainly by the reticuloendothelial system, at a rate directly related to their isoelectric point. The cationized albumins are catabolized very rapidly initially, but a fraction of the injected protein remains in the body for a longer time than native albumin.

Binding of hematoporphyrin derivative to human serum albumin.

Abstract: Dialysis of hematoporphyrin derivative fraction A (HpD-A) off human serum albumin at 38°C followed the Hill equation for cooperative binding with saturation at 5 to 8. 600 dalton porphyrin units. Approximately 15% of the HpD-A was free for concentrations typical of human serum in photoradiation therapy. Possible structures of the tumor-localizing and -photosensitizing component in HpD-A are considered. Of these, a folded-over, covalent dimer appears to be more consistent with the photophvsical properties.

IgE against ethylene oxide-altered human serum albumin patients who have had acute dialysis reactions

Abstract: Serum samples obtained from seven hemodialysis patients with immediate-type allergic reactions, from six hemodialysis patients without reactions, and from three nonatopic control subjects were analyzed for IgE against human serum albumin exposed to ethylene oxide (ETO-HSA). ETO is used to sterilize medical equipment like dialyzers that cannot withstand heat sterilization. IgE to ETO-HSA, measured by a polystyrene tube technique was found in six of seven dialysis reactor patients but in only one of six nonreactor patients (p

Human serum albumin is a major component on the surface of microfilariae of Wuchereria bancrofti.

Abstract: Wuchereria bancrofi is the major lymphatic filarial parasite of man, and endemic foci are found throughout the tropical zone; as many as 200 million people are currently infected with this nematode (Sasa 1976). Typical of most helminth infections, Bancroftian filariasis is a chronic disease encompassing a wide spectrum of conditions (Nelson 1979, Ottesen 1980) from the otherwise asymptomatic presence of blood microfilariae to the severe state of elephantiasis. Microfilaraemic individuals commonly bear over 1000 nematode larvae per ml of peripheral blood, sustaining no adverse effect from the presence'of this stage of the parasite, which may persist for over 10 years (Carme & Laigret 1979). The immunological basis for the persistence of filarial parasites is poorly understood (Ogilvie & Mackenzie 1981, Piessens 1981, Partono 1982), and studies have been hampered by the strict host-specificity of W . bancrofi (Cross et al. 1981), experimental infection being possible only in Presbytis cristatus monkeys (Palmieri et al. 1982) and other Presbytis species. An initial investigation of the antigenic composition of these parasites was therefore undertaken on the most readily available stage, microfilariae from human blood, using radiolabelling techniques recently reported to define surface components from limited quantities of material (Philipp, Parkhouse & Ogilvie 1980, Parkhouse, Philipp & Ogilvie 1981). We report here that, as in the case of the cotton-rat filarial nematode Litomosoides carinii (Philipp et al. 1984), the most prominent molecule on the surface of these human microfilarial parasites is host serum albumin. Two populations of Wuchereria bancrofti were obtained from separate endemic areas of Bihar State, India, and Jakarta, Indonesia. Both races of parasite display a characteristic nocturnal periodicity (Hawking 1975) and blood samples (20-100 ml) from patients were therefore taken at night. Microfilariae from five individuals in Bihar and one in Jakarta were purified by nucleopore filtration of haemolysed blood (Dennis & Kean 1971) by which parasites are recovered from a 3 pm pore membrane and checked * Present address: Department of Pure and Applied Biology, Imperial College, London SW7 2BR. t Present address: New England Bio-Labs, 32 Tozer Road, Beverly, Massachusetts 01915, USA.

Endocytosis of a mannose-terminated glycoprotein and formaladehyde-treated human serum albumin in liver and kidney cells from fish (Salmoalpinus L.)

Abstract: The uptake and degradation of a mannose-terminated glycoprotein, yeast invertase, in char (Salmo alpinus L.) tissue was studied after intravenously injection of the 125I-labelled protein. 125I-labelled formaldehyde-treated human serum albumin (fHSA) and native HSA was also injected for comparison. Labelled invertase was rapidly cleared from blood and at about the same rate as labelled fHSA (at 8 degrees C). Approximately 50% of the initial concentration remained in blood 15 min after the injection of the ligands. Acid soluble degradation products appeared in the circulation about 60 min after the injection of the proteins. 125I-labelled invertase was recovered in the liver, pronephros and kidney. The clearance of labelled invertase from blood and the uptake in the organs were inhibited by co-injection of excess unlabelled invertase. fHSA was taken up in the pronephros and kidney tissue, while HSA was not taken up in any organs. In vitro degradation of the labelled ligands was studied in isolated pronephros cells, which had taken up the proteins in vivo. The degradation of invertase in isolated cells was partly inhibited by ammonium chloride. Ammonium chloride and chloroquine inhibited degradation of fHSA, but not leupeptin. These results together suggest that invertase and fHSA were taken up in the organs described by the receptor-mediated endocytosis. The degradation was partly or wholly lysosomal.

Effect of late pregnancy on salicylate, diazepam, warfarin, and propranolol binding: use of fluorescent probes.

Abstract: Serum protein binding was measured in six women 38 wk pregnant and in five control subjects. Three distinct binding sites for drugs on human serum albumin have been identified. To determine whether changes in binding during pregnancy occur for common drugs or only for drugs that bind to a specific binding site, serum protein binding of three drugs—diazepam (site I), warfarin (site III), and salicylate—and four fluorescent probes—dansylsarcosine (site I), l-anilino-8-naphthalenesulfonate (site I), 7-anilinocoumarin-4-acetic acid (site II), and 5-dimethylaminonaphthalene-1-sulfonamide (DNSA) (site III)—were determined in control and pregnant sera. Unbound fractions of diazepam and salicylate in pregnant women increased but the unbound fraction of warfarin did not change. Dissociation constants (Kd1) of all fluorescent probes but DNSA were almost the same in control and pregnant sera, while the Kd1 of DNSA in pregnant serum was approximately 50% of control. Binding capacities of all probes decreased, which was attributed to decreased serum albumin concentration. We concluded that serum protein binding of drugs that bind to site I or site II on albumin decreased largely because of the reduced serum albumin concentration during pregnancy and that the binding of drugs that bind to site III changed little because of compensating effects of the decrease in serum albumin concentration and the increase in binding affinity to serum albumin. Serum concentration of α1-acid glycoprotein and serum binding of propranolol did not change in pregnant women. Clinical Pharmacology and Therapeutics (1984) 36, 201–208; doi:10.1038/clpt.1984.163