Showing papers on "Human serum albumin published in 1969"

Binding of digitoxin and some related cardenolides to human plasma proteins

Abstract: Tritium-labeled digitoxin, digitoxigenin, digoxin, and digoxigenin of established purity and chemcal authenticity were used to study the binding of these compounds to human plasma proteins. 97% of digitoxin in plasma was nondialyzable. Continuous flow paper electrophoresis of plasma containing digitoxin and dialysis experiments in which human serum albumin competed for the glycoside with plasma or plasma protein fractions demonstrated that digitoxin was almost exclusively bound by albumin. Equilibrium dialyses revealed that the interaction was characterized by a single binding site on the albumin molecule and an association constant of 9.62 x 10(4) liter/mole at 37 degrees C. At 1 degrees C the association constant was 4.64 x 10(4) liter/mole. The interaction therefore was endothermic; the gain in enthalpy of 3.5 kcal/mole and the free energy change of - 7.06 kcal/mole was derived from a large change in entropy of 33.8 cal/mole per degrees K. The direction of these thermodynamic changes suggested the formation of a hydrophobic bond between digitoxin and albumin. Quenching of the fluorescence of albumin by digitoxin indicated that the conformation of albumin was altered by the binding process.Digitoxigenin, its mono- and didigitoxosides, digoxin, and digoxigenin competed with digitoxn for its binding site on albumin. The affinity of the mono- and didigitoxosides for the site was equal to that of digitoxin, but that of digitoxigenin was only one-third as great. The ability of the digitoxose residues of the glycosides to enhance binding to albumin was also observed with digoxin, which was more extensively bound by the protein than digoxigenin. At concentrations of 2 mug/ml or less in plasma, only 23% of digoxin was bound. Albumin, which interacted with digoxin with an apparent association constant of 9 x 10(2) liter/mole at 37 degrees C, was entirely responsible for the binding. Lowering the temperature from 37 degrees to 1 degrees C decreased the fraction of digoxin bound to albumin by two-thirds. The marked difference in avidity of digitoxin and digoxin for serum albumin is reflected by the higher plasma concentrations, lower rate of urinary excretion, and longer half-time of digitoxin as compared to those of digoxin when these compounds are administered to man.

Structural changes in human serum albumin induced by ingestion of acetylsalicylic acid

Abstract: Acetylsalicylic acid (aspirin) acetylates human serum albumin under physiologic conditions in vitro. These investigations were done to determine whether this phenomenon occurs in vivo and to delineate the site(s) of acetylation on the albumin molecule. Albumin was reacted in vitro with aspirin labeled with (14)carbon at the acetyl-1 or the carboxyl carbon. The altered albumin was hydrolyzed with trypsin and peptide mapping performed. Albumin so treated contains a unique peptide, designated "A," and shows diminution of two normal peptides, designated "B" and "C." Peptide "A" is never seen in normal albumin. Amino acid analyses indicate that peptide "A" equals the sum of peptides "B" and "C." Furthermore all three peptides contain lysine but lack arginine. Thus peptide "A" is formed by the acetylation of a lysine residue which is normally susceptible to trypsin and yields peptides "B" and "C." Radioautography of the peptide maps show most of the acetyl-1-(14)C activity in peptide "A." This indicates that one of the lysine residues in this peptide is the preferential site for the transacetylation reaction. Peptide "A," used as a marker for acetylation, is found in albumin from patients who take aspirin but is not demonstrable in albumin from one of these patients while she was taking sodium salicylate. A transacetylation reaction between aspirin and human albumin occurs in vivo and is similar to that observed in vitro.

Optical Studies of Drug-Protein Complexes: II. Interaction of Phenylbutazone and Its Analogues with Human Serum Albumin

Abstract: The binding of phenylbutazone to human serum albumin generated a positive ellipticity band at 287 mµ in the circular dichroic spectrum of time protein. This extrinsic Cotton effect resulted from perturbation of the carbonyl chromophore of phenylbutazone by an asymmetrical locus at the albumin binding site. Hydrophobic interactions appeared to be important for the maintenance of a rigid drug-protein complex, since the introduction of hydrophilic groups into phenylbutazone caused considerable reduction in the magnitude of induced optical activity. Phenylbutazone competitively displaced 1-dimethylamino-naphthalene-5-sulfonyl-N-glycine (a fluorescent probe for the hydrophobic regions of proteins) from human serum albumin. This suggested that there was a hydrophobic area at or near one of the phenylbutazone-binding sites. The red shift in the ultraviolet absorption maximum of phenylbutazone on binding to albumin provided some evidence that the phenyl groups of the drug were located in a region of the protein where the dielectric constant was less than that of water.

Optical Studies of Drug-Protein Complexes

Abstract: The extrinsic Cotton effects generated by the binding of flufenamic acid [ N -(α,α,α-trifluoro- m -tolyl)anthranilic acid], meclofenamic acid [ N -(2,6-dichloro- m -tolyl)anthranilic acid], and mefenamic acid [ N -(2,3-xylyl)anthranilic acid] to human serum albumin suggested that although these anti-inflammatory drugs are bound to the same binding site, each one takes up a unique spatial orientation to the protein. Extrinsic Cotton effects generated by the binding of flufenamic acid to different albumins indicated that time drug-binding sites on human, porcine, equine, and bovine serum albumins were similar, while those of canine, ovine, and rabbit serum albumins had somewhat different asymmetries. Spectral changes which accompanied the binding of flufenamic acid to human serum albumin strongly suggested that the aromatic portion of the drug was inserted into a hydrophobic crevice in the protein, while the carboxylate group of the drug interacted with a cationic site on the protein surface. ACKNOWLEDGMENT It is a pleasure to acknowledge the skilled technical assistance of Mrs. D. K. Starkweather.

Determination of binding constants of serum albumin for penicillin.

Abstract: Free, active penicillins (benzylpenicillin, oxacillin, methicillin, nafcillin) and cephaloridine were measured in the presence of human and bovine serum albumin by means of a photometric assay of growth inhibition of Staphylococcus aureus. The decrease in antibiotic activity in the presence of albumin was assumed to reflect the antibiotic binding to the protein which other workers have demonstrated by direct physicochemical means and by the diminution of the concentration of free, active antibiotic. The percent of penicillin bound or inactivated was calcuated from the decreased antimicrobial activity in the presence of albumin. In the concentration range near therapeutic dosages, this percent antibiotic bound was independent of the antibiotic concentration and resembled a logarithmic function of the albumin concentration. Binding constants for bovine serum albumin were calculated from a simple equation for chemical equilibrium giving these values for the primary binding site: nafcillin, 3.1 X 104 M-1; oxacillin, 4.7 x 103 M-1; methicillin, 1.1x 103 M-1; benzylpenicilhin, 12 X l04 M-1 and cephaloridine, 0. These values for human serum albumin were: nafcillin, 12 X 104 M-1; methicillin, 9 x 103 M-1; benzylpenicillin, 1.1 x 103 M-1 and cephaloridine, 0. The similarity of these values with those obtained by others in the absence of bacteria supports the assumption that bound antibiotic is unavailable for bacteriostasis.

Immune reactions in polysaccharide media. The effect of hyaluronate, chondroitin sulphate and chondroitin sulphate-protein complex on the precipitin reaction.

Abstract: The influence of the connective-tissue polysaccharides hyaluronate, chondroitin 4-sulphate and a chondroitin 4-sulphate–protein complex (PP-L) from cartilage on the precipitin reaction was investigated. In a system consisting of 125I-labelled human serum albumin and the immunoglobulin G fraction from rabbit anti-albumin sera, the precipitation is greatly increased in the region of antigen excess. This effect depends on the concentration, molecular weight and configuration of the polysaccharide. The increase parallels a decrease in the amount of soluble immune complexes in the supernatant. It is suggested that the effect is due to steric exclusion of the complexes from the domains of the polysaccharides. The possibility that such a mechanism might enhance precipitation of antigen–antibody complexes in certain pathological conditions is discussed.

Specific reduction of carboxyl groups in peptides and proteins by diborane

Abstract: 1 The reaction of several peptides and proteins with diborane was studied under different conditions to determine those most suitable for the specific reduction of carboxyl groups 2 In the reaction of model peptides and the cyclic peptides bacitracin and tyrocidin, reduction at 0° was entirely specific for the carboxyl groups without affecting the peptide bonds Acid amide residues were not reduced Some tripeptides showed anomalous results in that the C-terminal residue was quite resistant to reduction 3 Specific reduction of carboxyl groups was achieved in each of the following proteins: human serum albumin, egg albumin, adult human haemoglobin, sperm-whale apomyoglobin, horse heart cytochrome c and egg-white lysozyme The C-terminal amino acid was usually reduced 4 Conditions for specific reduction of all available carboxyl groups are not easily found and may vary from one substance to another Specific reduction of a limited number of available carboxyl groups may be generally accomplished by reactions at −10° 5 It is suggested that this chemical modification, which has the advantage of permanence, may be useful in studying the role of carboxyl groups in the conformation of proteins and in the biological properties of peptides and proteins

Immunogenicity of a Fragment of Human Serum Albumin

Abstract: Summary A fragment (F1) of human serum albumin of molecular weight 6600 and carrying one of the determinant groups of HSA has been injected into rabbits and demonstrated to induce the formation of antibodies. The rabbit immune sera were shown to agglutinate red blood cells sensitized with F1 as well as with HSA. Anti-F1 antibodies were isolated by adsorption on a column of insoluble F1-benzylcellulose conjugate. They were shown to consist solely of IgG. They precipitated slightly with F1 but not with HSA. They agglutinated red blood cells sensitized with F1 or HSA. Both F1 and HSA inhibited these agglutination reactions.

Immune reactions in polysaccharide media. Experiments with specific antibodies of different affinities for serum albumin.

Abstract: Rabbit antibody fractions of different affinities for human serum albumin were prepared by an immunosorbent technique. The fractions were used in studies on the enhancement of the precipitin reaction by polymers. Dextran increased the immune precipitation to about the same extent regardless of whether antibodies with high and low affinities were used. The effect should considerably facilitate the detection of antibodies with low precipitating ability in immunological assays. The results are discussed in terms of a steric-exclusion mechanism.

The effect of antimicrobial agents on the binding of bilirubin by albumin

Abstract: Summary The effect of antibiotics, on the albumin binding capacity was studied by adding various concentrations of each drug in a standard solution of crystalline human serum albumin 4 g/ 100 ml, according to method of Porter & Waters (8). We found significant depression (above 25 %) of binding capacity of the albumin after addition of the following drugs in concentrations reaching therapeutical blood levels: Novobiocin and Sulfonamides (Elkosin, Sulfexin, Gantrisin). Moderate depression (11-25 %) was observed with the following drugs: Oxacillin, Ce-phalothin and Rifamycin. Little or no decrease of the albumin binding capacity was found after the addition of the following drugs: Penicillin, Penetracin, Methi-cillin (Staphcillin, Celbenin) Ampicillin, (Pen-trexyl, Penbritin), Cloxacillin, Lincomycin, Cephaloridin, Streptomycin, Colimycin, Chloramphenicol succinate, Erythromycin, Tetra-cyclin hydrochloride, Pyrolidynomethyl-tetra-cyclin and Oxytetracyclin.

Genetics of albumin Gainesville, a new variant of human serum albumin.

Abstract: A “NEW” slow-moving variant of serum albumin has been found in members of a single family of Irish descent from the vicinity of Gainesville, Florida1. We have demonstrated that the electrophoretic mobility of albumin Gainesville is different from the mobilities of previously reported slow-moving albumins2–5, and have provided evidence to support the hypothesis of linkage between the albumin and Gc loci6,7.

Enhancement of lysozyme activity by anodal tear protein.

Abstract: SummaryAnodal tear protein is a constituent of human and rabbit conjunctival fluid. This protein has not been identified in other body fluids. Rabbit anodal tear protein enhances the lysis of M. lysodeikticus by human and egg white lysozyme. Neither human serum albumin nor an acidic derivative of serum albumin enhanced the activity of lysozyme. The enhancing action of the acidic tear protein was noted when the assay was performed in 0.01 M phosphate buffer, pH 7.0, but was not noted when 0.1 M phosphate buffer was used.

Mechanism of the Albumin Agglutination Phenomenon

Abstract: Summary. The albumin agglutination phenomenon is due to antibodies which cause agglutination of all human red cells when these cells are suspended in an albumin medium. Two sera with this property were studied. We suggest that the reaction is due to non-specific adsorption of antigen-antibody complex onto red cells. The antibody is a gamma globulin directed at albumin which has been altered by the addition of acetyl tryptophanate or caprylate. These chemicals are added as stabilizers in the manufacture of albumin to prevent denaturation when the albumin is heated. Since unaltered (native) albumin does not react with these antibodies, blood or plasma transfusion to patients with this serologic abnormality should present no unusual hazard. A non-reactive albumin should be used in crossmatching blood for such patients. Therapeutic human serum albumin and plasma products containing stabilized albumin are probably contraindicated in these patients.

The Protein Binding of Acetylsalicylic Acid and Salicylic Acid

Abstract: Quantitative experiments to study the binding of acetylsalicylic acid (ASA) and salicylic acid (SA) by human serum albumin (HSA) were carried out using 14C-labeled ASA or SA and gel filtration to separate the free from the bound forms. Two binding procedures were employed: the ASA or SA was incubated with the HSA, or the mixture was placed in an equilibrium dialysis cell. By withdrawing samples at intervals, the extent of binding or the attainment of equilibrium could be assessed. Evidence that filtration by the gel caused unbinding of the bound SA was obtained, with resultant lower percentage binding of SA by HSA than that obtained by equilibrium dialysis without gel filtration. In either case, binding equilibrium was reached in 4-8 hr. The binding of ASA by HSA was markedly different from that of SA. The experiments both with or without gel filtration demonstrated a progressive increase in binding of ASA in the 20- to 53-hr periods studied. In addition, ASA apparently displaces SA from its binding sites on albumin, an observation that may have therapeutic implications.

The intracellular localization of two antigens after uptake in vivo by peritoneal macrophages from normal mice.

Abstract: Normal mouse macrophages, which had ingested ferritin labelled with fluorescein isothiocyanate and human serum albumin labelled with tetramethylrhodamine isothiocyanate in vivo, were fixed in formalin and embedded for electron microscopy. The examination of sections 1–2 μ thick and adjacent ultrathin sections showed that the yellow-green fluorescent droplets (due to ferritin-FITC) seen by fluorescence microscopy were in the same position as the ferritin-containing phagolysosomes as seen by electron microscopy. Normal mouse macrophages, which had ingested 125I-labelled human serum albumin ([125I]HSA) and unlabelled ferritin, were investigated by electron microscopic autoradiography. Both antigens were found to be situated within the same lysosomes.

Albumin cayemite: A Negro plasma albumin variant

Abstract: An inherited variant of human serum albumin has been found in a Negro family from Haiti. The variant is electrophoretically distinguishable from ten different previously reported types of albumin variants.

Human Serum Albumin as a Stabilizer for 99mTc-Sulfur Suspension

Abstract: Summary A nontoxic stabilizer for the 99mTc-sulfur suspension has been found. Addition of human serum albumin stabilizes the suspension for 6 hr or longer. Reaction-free liver-scanning material is now available for better-quality scans. The ability to make the preparation on a one-time daily basis is a major advantage to any productive isotope clinic.

Evaluation of the Immunosuppressive Properties of Ribonuclease Complexes

Abstract: Complexes were prepared by bisdiazobenzidine coupling of bovine pancreatic ribonuclease (RNase) and human serum albumin (HSA). These preparations prolonged skin allograft survival in mice and inhibited phytohemagglutinin stimulation of human lymphocytes in vitro . The HSA-RNase complexes that were most active in inhibiting lymphocyte stimulation failed to interfere with immune specific macrophage immobilization. The immunosuppressive potency of HSA-RNase complexes did not correlate with their measurable RNase enzyme activities. Furthermore, HSA-RNase complexes of widely differing immunosuppressive potency could not be distinguished by differences in molecular size or charge.