Showing papers on "Human serum albumin published in 1983"

Structural requirements for drug binding to site II on human serum albumin.

Abstract: Structure-activity relationships for binding at site II on human serum albumin have been investigated using drugs, fatty acids, and aliphatic amines with chain lengths C-3 to C-18. A negative charge is not required for binding provided a strongly electronegative center is present. For example, diazepam, a basic drug that exists mainly in the un-ionized form at neutral pH, also binds with high affinity to site II. However, aliphatic amines (pKa values 10-11) with chain lengths C-3 to C-12 did not displace markers from either site I or II, showing that the presence of a positive charge precludes binding at these sites. Short-chain fatty acids, C-3 to C-5, did not displace marker drugs or fluorescent probes from either site I or II when added at equimolar ratios with albumin. Displacement of site II (but not site I) markers occurred with medium-chain fatty acids, C-7 to C-11, and was maximal at C-10. Fatty acids with chain lengths C-10 to C-18 caused an enhancement of fluorescence of dansylamide bound to site I, the maximal effect occurring with C-12. Both arylpropionic acid nonsteroidal anti-inflammatory drugs and medium-chain fatty acids binding at site II had molecular lengths within the range 11-16 A. The effect of hydrophobicity (and/or molecular length) on binding affinity was much more marked with the arylpropionic acids than with the fatty acids, suggesting that bulkier aromatic molecules form more effective interactions at the binding site. The results suggest that site II is a hydrophobic cleft about 16 A deep and about 8 A wide in the albumin molecule with a cationic group located near the surface.

"Unbound analog" radioimmunoassays for free thyroxin measure the albumin-bound hormone fraction.

Abstract: We have assessed the influence of albumin-bound thyroxin (T4) on apparent free T4 values obtained by two "unbound analog" free T4 methods (AmerlexR Free T4 and Clinical Assays one-step Free T4). We evaluated sera showing three different albumin anomalies: total hereditary analbuminemia, partially corrected analbuminemia, and familial dysalbuminemic hyperthyroxinemia, where abnormal albumin-binding of analog tracer is associated with high apparent free T4 values by these methods. In hereditary analbuminemia, free T4 was almost undetectable by both assays; in contrast, free T4 by equilibrium dialysis was normal. After addition of T4-free human serum albumin, the apparent free T4 concentration in total hereditary analbuminemia became normal by the analog methods. Immunoprecipitation of [125I]T4 and the unidentified labeled kit analogs by antiserum to human albumin was negligible in untreated total hereditary analbuminemia and approximately twice normal in familial dysalbuminemic hyperthyroxinemia. Therefore, alterations in tracer binding to albumin correlate with the apparent free T4 concentrations obtained by the analog methods. The interactions of the unidentified analog tracers and T4 with albumin are such that these techniques principally reflect the albumin-bound T4 moiety.

Resonance energy transfer between cysteine-34, tryptophan-214, and tyrosine-411 of human serum albumin.

Abstract: Reaction of p-nitrophenyl anthranilate with human serum albumin at pH 8.0 results in esterification of a single anthraniloyl moiety with the hydroxyl group of tyrosine-411. The absorption spectrum of the anthraniloyl group overlaps the fluorescence emission of the single tryptophan residue at position 214. This study complements that of the preceding paper [Suzukida, M., Le, H. P., Shahid, F., McPherson, R. A., Birnbaum, E.R., & Darnall, D. W. (1983) Biochemistry (preceding paper in this issue)] where an azomercurial group was introduced at cysteine-34. Anthraniloyl fluorescence was also quenched by the azomercurial absorption at Cys-34. Thus measurement of resonance energy transfer between these three sites allowed distances to be measured between Cys-34 in domain I, Trp-214 in domain II, and Tyr-411 in domain III of human serum albumin. At pH 7.4 in 0.1 M phosphate the Trp-214 leads to Tyr-411, Tyr-411 leads to Cys-34, and Trp-214 leads to Cys-34 distances were found to be 25.2 +/- 0.6, 25.2 +/- 2.1, and 31.8 +/- 0.8 A, respectively.

Relations between high-affinity binding sites for l-tryptophan, diazepam, salicylate and Phenol Red on human serum albumin

Abstract: Binding of L-tryptophan, diazepam, salicylate and Phenol Red to defatted human serum albumin was studied by ultrafiltration at pH 7.0. All ligands bind to one high-affinity binding site with association constants of the order of 10(4)-10(5)M-1. The number of secondary binding sites was found to vary from zero to five, with association constants about 10(3)M-1. Competitive binding studies with different pairs of the ligands were performed. Binding of both ligands was determined simultaneously. L-Tryptophan and diazepam were found to compete for a common high-affinity binding site on albumin. The following combinations of ligands do not bind competitively to albumin: L-tryptophan-Phenol Red, L-tryptophan-salicylate and Phenol Red-salicylate. On the other hand, high-affinity bindings of the three ligands do not take place independently but in such a way that binding of one of the ligands results in a decrease in binding of the other ligands. The decreases in binding are reciprocal and can be accounted for by introducing a coupling constant. The magnitude of the constant is dependent on the ligands being bound. In the present study, the mutual decrease in binding was more pronounced with L-tryptophan-salicylate and Phenol Red-salicylate than with L-tryptophan-Phenol Red.

Resonance energy transfer between cysteine-34 and tryptophan-214 in human serum albumin. Distance measurements as a function of pH.

Abstract: The single cysteine residue (Cys-34) of human serum albumin was modified with the organic mercurial [4-[p-(dimethylamino)phenyl]azo]phenyl]mercuric acetate. Introduction of this chromophore into the protein results in the quenching of the protein tryptophan fluorescence spectrum due to energy transfer from the tryptophan residue to the mercurial. Since human albumin contains only a single tryptophan, it was then possible to calculate distances between the mercurial bound at Cys-34 and Trp-214 under various conditions. This distance contracted during the course of the N leads to F transition, being 34-35 A in the N conformation (pH 6-7.5) and 29.9 A in the F conformation (pH 3.6). The distance increased substantially during the course of the F leads to E transition occurring between pH 3.6 and pH 1.9 and was found to be nearly 37 A at pH 1.9. The distance between Cys-34 and Trp-214 was found to undergo a slight contraction during the N leads to B transition occurring between pH 7.0 and pH 9.0. At pH 8.5-9 where the protein is predominately in the B form, the distance was found to be slightly more than 31 A.

Photosensitized lysis of liposomes by hematoporphyrin derivative

Abstract: The spectral properties and efficiency for photosensitizing the lysis of phosphatidylcholine liposomes have been measured for the components of hematoporphyrin derivative (Hpd) after alkaline hydrolysis and fractionation by polyacrylamidc gel chromatography. Two major and two minor Hpd fractions have been identified whose spectral properties correlate with the anoxic sensitizing efficiency and the oxygen enhancement ratio (OER). The fastest moving fraction, which is the putative biologically active component, comprised one-third of the starting material and had OER = 2.7. Liposome lysis by this fraction was inhibited in the presence of human serum albumin at concentration ratios comparable to those employed for photoradiation therapy. The present results show that Hpd can act as an oxic and anoxic photosensitizer of a model biomembrane and suggest that separation from serum proteins is required for in vivo photosensitization.

Binding of anthracene to cellular macromolecules in the presence of light.

Abstract: Ultraviolet radiation (δ > 295 nm) induced covalent binding of anthracene to DNA which increased with time and was not affected by oxygen. Irradiation in the presence of anthracene induced nicking of Col E, circular DNA and decreased the thermal denaturation temperature of calf thymus DNA. These effects were oxygen dependent, and were decreased by GMP. Irradiation of anthracene and human serum albumin resulted in covalent binding of the hydrocarbon to the protein accompanied by crosslinking of the protein. Protein crosslinking decreased under anaerobic conditions. Irradiation of anthracene bound to liposomes induced lipid peroxidation which was not affected by superoxide dismutase or catalase.

Determinants of the plasma protein binding of theophylline in health.

Abstract: 1 The plasma protein binding of theophylline was determined after addition of [14C]-theophylline (15 micrograms/ml) to plasma from 24 healthy drug-free volunteers and equilibrium dialysis for 2 h at 37 degrees C. 2 The percentage of drug unbound was 60.0% +/- 2.2% (s.d.) with very little variation between individuals. The binding ratio of theophylline was not significantly related to the plasma albumin or alpha 1-acid glycoprotein (AAG) concentrations but was significantly, although weakly, negatively related to the logarithm of the non-esterified fatty acid concentration (NEFA) (r = 0.443, P less than 0.05). 3 Intravenous administration of heparin (1000 units) caused a significant rise in plasma NEFA concentration and in the percentage of drug unbound in plasma after equilibrium dialysis. 4 In human serum albumin solutions, the binding ratio of theophylline was significantly related to the albumin concentration and at the albumin concentration seen in the 24 normal subjects, the percentage of drug unbound was almost identical. Addition of AAG in physiological concentrations did not enhance theophylline binding but oleic acid, and to a lesser extent palmitic acid, reduced binding significantly. 5 The percentage of theophylline unbound in plasma varied markedly with pH so that at pH7 the percentage unbound was 52% greater than at pH 8. There was no evidence of concentration dependence of binding up to 140 micrograms/ml theophylline. 6 Theophylline appears to bind almost exclusively to albumin and its plasma protein binding varies little in healthy subjects, showing no concentration-dependence over the therapeutic range of concentrations. The binding is affected by pH and by NEFA concentration, however, and these factors may be of greater importance in disease states. Caution should be employed in the use of heparin in studies of plasma protein binding of theophylline.

Photosensitization of liposomal membranes by hematoporphyrin derivative.

Abstract: The putative tumor-localizing and -photosensitizing fraction of hematoporphyrin derivative, the fastest migrating fraction of hematoporphyrin derivative separated by polyacrylamide gel filtration (HPD-A), photosensitized lipid peroxidation and membrane lysis in egg phosphatidylcholine liposomes. The rate of membrane damage was approximately 4-fold faster in oxygen compared to anoxia, with evidence for the involvement of singlet oxygen. The diffusion of HPD-A into small liposomes led to a shift of the Soret band from 363 nm in buffer to 398 nm accompanied by 4-fold enhancement of the fluorescence. The presence of human serum albumin retarded the diffusion of HPD-A into small liposomes, which is attributed to partial complexing of the HPD-A. A different effect of serum albumin was the protection of large liposomes from photosensitized lysis by incorporated HPD-A. This protection is attributed to scavenging of singlet oxygen, as evidenced by oxidation of tryptophan in the protein.

The binding of ibuprofen to plasma proteins.

Abstract: The binding of ibuprofen to human serum albumin, normal plasma and plasma obtained from rheumatoid arthritic patients was studied using the method of ultracentrifugation. It was found that ibuprofen is more strongly bound to normal plasma than to human serum albumin although this result is probably explained by fatty acid contamination of the human serum albumin. The fraction of ibuprofen not bound to normal plasma rose significantly from a value of 0.0128 at an ibuprofen concentration of 2 mg X l-1 to 0.0155 at a concentration of 50 mg X l-1. Ibuprofen was less strongly bound to rheumatoid plasma than to normal plasma but this difference can be accounted for by the difference in albumin concentration between the two plasmas. It was found that salicylic acid can displace ibuprofen from protein binding sites, in vitro, and that this is the probable cause of the pharmacokinetic interaction between the two drugs.

Spectral study of the photochemistry of dipyrrole models for bilirubin bound to human serum albumin.

Abstract: —Dipyrromethenones, which include xanthobilirubinic acid (I), its methyl ester (II) and the endo-vinyl analog of neoxanthobilirubinic acid methyl ester (“half bilirubin” methyl ester) (III) bind to human serum albumin (HSA) and thereby exhibit greatly increased fluorescence as well as induced circular dichroism in their long wavelength transitions. As determined from fluorescence studies, the “tightness” of the binding to HSA is, inter alia, inversely related to the inherent solubility of the dipyrromethenone in aqueous buffer. Fast (ps) configurational isomerization (ZE) is the major pathway for decay of the first excited (singlet) states of the HSA-bound dipyrromethenones. A quantum yield of − 0.4 is associated with the ZE isomerization. Binding to HSA enhances the lifetimes of the less thermodynamically stable E-isomers. The results of these studies of “half bilirubins” allow a clear evaluation of the effects of protein binding upon the photophysics and photochemistry of bilirubin.

Determination of a Small Amount of Biological Constituent by Use of Chemiluminescence. I. The Flow-injection Analysis of Protein

Abstract: A new method in which luminol-hydrogen peroxide luminescent system is used has been proposed for the determination of the presence of protein. Since the catalytic activity of copper(II) for the chemiluminescent reaction between luminol and hydrogen peroxide decreased when copper(II) interacted with polypeptide linkage, this phenomenon was applied to the determination of protein. Determination of protein was carried out by a flow-injection method. The effects of reagent concentration, flow-rate, and reaction time on the analytical value were examined and the conditions for the determination of protein were established. Similar calibration curves were obtained for human serum albumin, bovine serum albumin, bovine serum α-globulin, and bovine serum γ-globulin. According to the present flow-injection method using chemiluminescent reaction, a small amount of protein could be conveniently and economically determined over a wide range of concentration, 7×10−4–7×10−2 g dm−3, with the detection limit of 0.2 μg and...

Physicochemical Properties and Blood Clearance of Human Serum Albumin Conjugated to Different Extents with Dinitrophenyl Groups

Abstract: Human serum albumin (HSA) conjugated to various extents with dinitrophenyl (DNP) groups and labelled with 125I was studied with regard to physicochemical properties and blood clearance after intravenous injection in mice. Increasing the degree of DNP conjugation increased the tendency to hydrophobic interaction, and increased the net negative charge. Heavily DNP-substituted HSA molecules tended to aggregate. The higher the degree of DNP substitution the faster was the conjugate eliminated from the circulation. Blood clearance was independent of serum complement, and was not affected by galactose, N-acetylglucosamine, mannose, alpha-methyl-D-mannoside, or fucose. It is proposed that the differences in blood clearance between the different DNP-HSA conjugates mainly depend upon differences in the tendency to hydrophobic interaction.

Hematoporphyrin photosensitization of serum albumin and subtilisin BPN

Abstract: —The photosensitized inactivation of subtilisin BPN' by free hematoporphyrin (HP) followed exponential kinetics with positive mechanistic tests for the involvement of singlet oxygen (1O2) as the principal intermediate. The photoinactivation quantum yield was 0.029 at 390 nm in oxygen-saturated, D2O buffer at pH 7.0. The effects of HP binding were investigated for tryptophan oxidation in bovine serum albumin (BSA) and human serum albumin (HSA) at high protein:HP concentration ratios where the HP was > 97% complexed. The reaction kinetics were non-exponential and mimick a second-order process in the initial stages. The rate of HP photobleaching was 30-fold faster for complexed HP compared with free HP, which was shown to account for the observed kinetics. Mechanistic tests showed that 1O2 was the dominant photooxidizing intermediate of tryptophan residues and that it was not involved in the accompanying photobleaching of HP. The quantum yield for tryptophan oxidation in BSA was 0.11 at 390 nm in oxygen-saturated, D2O buffer at pH 8.0. The reactivity of HSA was approximately 2-fold lower than BSA for equivalent conditions. Estimates of the reaction cross sections led to 3 A2 for the inactivation of subtilisin BPN' by 1O2 and 20 A2 for the oxidation of tryptophan in BSA.

Detection of microbial proteolytic activity by a cultivation plate assay in which different proteins adsorbed to a hydrophobic surface are used as substrates.

Abstract: A screening technique for microbial proteases, the thin-layer enzyme assay cultivation technique, was developed. The inner surface of a polystyrene petri dish was coated with protein and then covered with a culture agar medium. The enzymes, produced during growth of the microorganisms, reach the protein-coated surface by diffusion in the agar. Degradation of the protein was visualized by condensation of water vapor on the surface after removal of the agar medium. The wettability of the enzyme-affected protein-coated polystyrene surface was decreased compared with the unaffected protein surface. Enzyme substrates used were fibrinogen, immunoglobulin G, egg albumin, human serum albumin, bovine serum albumin, hemoglobin, mucin, and gelatin. It was possible to use a variety of culture agar media, nonselective as well as selective, in the assay. The technique provides a sensitive, convenient, and inexpensive method for screening various microbial proteases. In addition, the technique can be used for screening proteolytic enzyme activity of specific microbial species in a mixed microbial sample as well as for studies of factors that influence the cultivation conditions for protease production and activity.

Albumin-Bilirubin Binding Mechanism KINETIC AND SPECTROSCOPIC STUDIES OF BINDING OF BILIRUBIN AND XANTHOBILIRUBIC ACID TO HUMAN SERUM ALBUMIN*

Abstract: has been studied by re- cording the changes of light absorption. Similar proc- esses have been demonstrated after binding of fatty acid anion to the bilirubin-albumin complex as well as after a pH-jump from 6 to 9. Solvent perturbation spectra obtained on the addition of 20% sucrose have failed to demonstrate exposure of the bilirubin chro- mophores in the complex to the surrounding medium. Xanthobilirubinate which has a single dipyrrolic chro- mophore compared to the two of bilirubin is bound to albumin in competition with bilirubin, as concluded from co-binding studies with monoacetyldiaminodi- phenylsulfone and diazepam, probing two different binding functions of the albumin molecule. Late con- formational changes were absent after binding of xan- thobilirubinate. Binding of fatty acid to the complex and a pH-jump did not affect the spectrum of xantho- bilirubinate-human serum albumin. The findings can be explained by a model, previously proposed, in which the late spectral changes are affected by rotation of one half-domain of albumin, binding one bilirubin chromophore, relative to another half-domain to which the second bilirubin chromophore is bound, whereby a change of exiton splitting occurs. Such changes are not seen with the complex of xanthobilirubinate and albu- min, since only a single chromophore is present.