Binding Sites Of Serum Albumin Research Articles

Interactions of myristic acid with bovine serum albumin: a 13C NMR study.

Interactions of myristic acid with bovine serum albumin were studied by 13C NMR spectroscopy at 50.3 MHz using 90% isotopically substituted [1-13C]-, [3-13C]-, and [14-13C]myristic acids, either individually or in a combination of all three with albumin. At pH 7.4, two or more resonances of different intensities were observed for each 13C-enriched myristic acid. Carboxyl and methylene C-3 resonances corresponding to the major myristic acid environment(s) exhibited pH-dependent chemical shift changes indicative of protonation below pH 6.7; in contrast, carboxyl groups in minor environments were resistant to protonation. 13C NMR spectra obtained as a function of the molar ratio of [3-13C]- and [14-13C]myristic acid to bovine serum albumin (from 0.7 to 5.6) revealed at least two narrow resonances for each carbon at all molar ratios. Thus, bovine serum albumin binding sites for myristic acid are heterogeneous with respect to titration behavior and with respect to the local magnetic environment at both the polar and the nonpolar ends of the fatty acid. The narrow resonances observed for the methylene and methyl carbons are inconsistent with complete immobilization of the protein-bound acid molecules. Together with spin- lattice relaxation times and nuclear Overhauser enhancements, the linewidth results indicate that bound myristic acid has internal motions that are rapid compared with overall protein tumbling and that the C-3 methylene carbon is more restricted than the terminal methyl carbon.

Reactivity of Cu 2(lonazolac) 4, a lipophilic copper acetate derivative

An acetate-like copper complex of lonazolac (3-(p-chlorophenyl)-1-phenyl-pyrazole-4-acetate) was prepared and characterized. The copper of Cu 2-(lonazolac) 4 was spin-coupled and remained EPR-silent. Water and organic solvents did not affect this magnetic interaction. Superoxide dismutase activity of the Cu complex in micromolar concentrations was detectable in the presence of up to 900 μg per ml of serum albumin or whole serum protein. At 700 μM albumin concentration, a ternary complex between Cu 2(lonazolac) 4 and the protein was formed. The original acetate-copper coordination changed to a biuret-type copper bonding as seen from EPR and electron absorption spectrometry. Lonazolac did not induce a detectable conformational change of the protein near or at the copper binding site. Equilibrium dialysis and optical titration experiments revealed that essentially all copper of the Cu 2-(lonazolac) 4 complex was bound in the specific binding site of serum albumin. The copper complex proved to be an effective inhibitor of lipid peroxidation.

Sucrose monoesters of fatty acids: Their properties and interaction with proteins.

Critical micelle concentrations, micellar sizes and ellipticities at 220 nm were determined for sucrose monoesters of caprylic, capric and lauric acids. The interaction of these esters with proteins were also studied. The sucrose esters of caprylic and capric acids were found to bind only at discrete binding sites of native bovine serum albumin and this binding did not induce any detectable change in the protein conformation, while virtually no binding of the ligands to ovalbumin was observed, which lacks strong affinity sites in its native state. Together with the results of thermal denaturation experiments of the proteins in the presence of detergents, the present results indicate that the mode of interaction of sucrose esters with the proteins is quite similar to that of Triton X-100, a widely used nonionic detergent. Furthermore, it is suggested that the sucrose esters of caprylic and capric acids might be promising detergents for use in membrane protein research.

N-bromosuccinimide-oxidized human serum albumin as a tool for the determination of drug binding sites of human serum albumin.

In order to investigate the role of the tryptophan residue of human serum albumin (HSA) in drug binding, HSA oxidized with 8 molar equivalents of N-bromosuccinimide (NBS) was prepared at pH 4.1. The NBS-oxidized HSA had lost the lone tryptophan residue without change in tyrosine content, and the modified protein retained the gross polypeptide conformation of native HSA as far as could be judged from the circular dichroism (CD) spectrum. The binding ability of NBS-oxidized HSA for chlordiazepoxide was decreased by about 60% from that of HSA. However, the binding of phenylbutazone and warfarin at the most reactive site of the modified HSA was not very different from that to HSA. These results suggest that the lone tryptophan residue in HSA lies in or near the indole and benzodiazepine binding site and not in the primary binding site of warfarin and phenylbutazone.

Kinetic studies of proton transfer in the microenvironment of a binding site.

Excitation of 8-hydroxypyrene 1,3,6-trisulfonate to its first electronic singlet state converts the compound from weak base (pK degrees = 7.7) into a strong acid (pK* = 0.5). The dissociation of the proton in water or dilute salt solution is a very fast reaction, K12 = 1 X 10(10) S-1. In concentrated salt solutions the dissociation is slowed as an exponential function of the chemical activity of the water in the solution. This kinetic parameter has been used to gauge the properties of the microenvironment of the binding sites of bovine serum albumin at which this compound is bound. Time-resolved fluorometry reveals two distinct steps: a rapid dissociation of the proton with tau = 300 +/- 40 ps which lasts approximately 0.5 ns, followed by a slower reaction with tau = 3.3 ns. The first rapid phase represents proton dissociation taking place in the binding site. From the rate constant K = 3.3 X 10(9) s-1 we estimate that the ability of the water molecules in the site to hydrate the ejected proton is equivalent to a salt solution with water activity of 0.85. The slow phase represents the escape of the proton from the binding site. The rate of the escape, 1.4 X 10(8) s-1, is significantly slower than diffusion-controlled dissociation. It is concluded that the shape of the site or its lowered proton conductivity do not allow a rapid escape of the proton to the bulk. Still it should be remembered that the escape of the proton is 10(5)-10(6)-times faster than a typical turnover of an enzyme.U

Optical Studies on Interaction of Biliary Contrast Agents with Native and Modified Human Serum Albumin

The interaction of two homologous series of biliary contrast agents with native human and bovine serum albumin and with modified human serum albumin was investigated using circular dichroism and equilibrium dialysis. For most derivatives, extrinsic Cotton effects were observed for the interaction with both albumins. In some cases, these effects were strongly affected by only small changes in the chemical structure of the drugs. These large differences in extrinsic Cotton effects can be explained by definite effects of the chemical structures on the binding site selectivity of some drugs. For example, iopodate preferentially binds to the warfarin binding site of human Scrum albumin, while an ethyl group into the propionic acid side chain reduces the affinity for the warfarin site but strongly increases the affinity for the diazepam binding site of human serum albumin.

Binding of streptococcal lipoteichoic acid to the fatty acid binding sites on serum albumin.

The ability of the fatty acid binding sites of serum albumin to bind lipoteichoic acid of Streptococcus pyogenes was investigated. Initial studies indicated that lipoteichoic acid, but not its deacylated deprivative, protected albumin from being denatured by heat (80C for 1 h) and changed its mobility in an electrical field. Albumin covalently linked to agarose beads bound radiolabeled lipoteichoic acid, and the bound [3H]lipoteichoic acid could be specifically eluted with unlabeled lipoteichoic acid or albumin but not with other proteins tested. After binding to albumins, the lipoteichoic acid also could be quantitatively eluted with 50% ethanol and various detergents but not with up to 1.0 M sodium chloride. Binding of lipoteichoic acid to albumin followed first order kinetics, reaching saturation at 12 h. Analysis of the binding data by a Scatchard plot indicated heterogeneity of the binding sites on the albumin molecule similar to that previously reported for fatty acids. The affinity of binding of lipoteichoic acid to albumin was found to be intermediate between that previously reported for octanoic and palmitic acids, respectively. Based on these findings, we prepared affinity columns of immobilized albumin and were able to separate biologically active lipoteichoic acid from heterogeneous extracts of S. pyogenes.

Fatty acid binding sites of serum albumin as membrane receptor analogs for streptococcal lipoteichoic acid.

The ability of bovine serum albumin to inhibit the binding of group A streptococcal lipoteichoic acid (LTA) to human cells was investigated. Albumin blocked the ability of LTA to sensitize erythrocytes to agglutinate in the presence of anti-LTA in a dose-dependent manner. The inhibition of LTA binding to erythrocytes was demonstrated directly with radiolabeled LTA. At an albumin/LTA molar ratio of 1.5:1, albumin binding of the radiolabeled LTA at erythrocytes was inhibited by 45%. Analysis of the binding of radiolabeled LTA to erythrocytes in the presence of albumin indicated that albumin competitively inhibited the binding of LTA to putative cell membrane receptors, indicating that albumin and erythrocytes both bind to the same moiety on the LTA molecule.

Azapropazone binding to human serum albumin.

Azapropazone, a new non-steroidal antiinflammatory drug, is strongly bound to human serum albumin. As revealed by Scatchard analysis, one high-affinity binding site with an association constant of about 1.2 x 10(6)M-1 and two low-affinity binding sites with association constants of about 0.05 x 10(6)M-1 were found. While the high-affinity binding site of azapropazone is clearly not identical with the diazepam or digitoxin binding sites of human serum albumin, contradictory evidence was found by optical measurements and displacement studies for the similarity of the azapropazone and the warfarin binding site of human serum albumin. At present, it is suggested that both drugs bind to different areas of the same binding site. Therefore, the pronounced effects of various disease states on the plasma protein binding of azapropazone can not be explained by a binding to an unusual binding site, but seem to be due to an extreme sensitivity of the azapropazone binding area to the putative endogenous binding inhibitors, present in the blood during those disease states.

Characterization of a small apolar anion binding site of human serum albumin

Small unbranehed fatty acid anions inhibit the fast reaction between p-nitrophenylacetate and human serum albumin. Plots of reactivity versus fatty acid anion-albumin ratios resemble simple binding isotherms from which corresponding dissociation constants have been calculated. For the homologous fatty acid anions, butyrate through decanoate, dissociation constants decrease from 3.2 × 10 −4 to 1 × 10 −7 m, respectively, by uniform increments per methylene group according to the relationship −Δ G °(kcal) = 0.804 n + 2.30, where n is the number of constituent methylene groups. Small fatty acid anions thus appear to interact primarily with a single, relatively uniform apolar binding site with a capacity sufficient for nine methylene groups. Fatty acid anions larger than decanoate interact significantly with other sites and do not obey the same relationship. The reactivity of diluted human serum with p-nitrophenylacetate was found to be one-third to one-half of that expected for its content of serum albumin, but as in vitro, it could be completely inhibited by small amounts of decanoate.

Internal motion at the chloride binding sites of human serum albumin by NMR relaxation studies

FEBS LettersVolume 86, Issue 1 p. 25-28 Full-length articleFree Access Internal motion at the chloride binding sites of human serum albumin by NMR relaxation studies Thomas E. Bull, Thomas E. Bull Physical Chemistry 2, Chemical Center, POB 740, S-220 07 Lund 7, SwedenSearch for more papers by this authorBertil Halle, Bertil Halle Physical Chemistry 2, Chemical Center, POB 740, S-220 07 Lund 7, SwedenSearch for more papers by this authorBjörn Lindman, Björn Lindman Physical Chemistry 2, Chemical Center, POB 740, S-220 07 Lund 7, SwedenSearch for more papers by this author Thomas E. Bull, Thomas E. Bull Physical Chemistry 2, Chemical Center, POB 740, S-220 07 Lund 7, SwedenSearch for more papers by this authorBertil Halle, Bertil Halle Physical Chemistry 2, Chemical Center, POB 740, S-220 07 Lund 7, SwedenSearch for more papers by this authorBjörn Lindman, Björn Lindman Physical Chemistry 2, Chemical Center, POB 740, S-220 07 Lund 7, SwedenSearch for more papers by this author First published: February 01, 1978 https://doi.org/10.1016/0014-5793(78)80090-8Citations: 10AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 G. Weber, Adv. Protein Chem., 29, (1975), 1– 83. 2 I.D. Campbell, C.M. Dobson, R.J.P. Williams, Proc. Roy. Soc. London Ser. B, 189, (1975), 503– 509. 3 A. Cavé, C.M. Dobson, J. Parello, R.J.P. Williams, FEBS Lett., 65, (1976), 190– 194. 4 B.R. Gelin, M. Karplus, Proc. Natl. Acad. Sci. USA, 72, (1975), 2002– 2006. 5 I.D. Campbell, C.M. Dobson, G.R. Moore, S.J. Perkins, R.J.P. Williams, FEBS Lett., 70, (1976), 96– 100. 6 J.A. McCammon, B.R. Gelin, M. Karplus, Nature, 267, (1977), 585– 590. 7 T.E. Bull, J. Magn. Res., 8, (1972), 344– 353. 8 S.H. Koenig, Biopolymers, 14, (1975), 2421– 2423. 9 J.T. Yang, Adv. Protein Chem., 16, (1961), 323– 400. 10 P. Moser, P.G. Squire, C.T. O'Konski, J. Phys. Chem., 70, (1966), 744– 756. 11 T.E. Bull, B. Lindman, P. Reimarsson, Arch. Biochem. Biophys., 176, (1976), 389– 391. 12 Bull, T.E., to be published. 13 M.H. Cohen, F. Reif, Solid State Phys., 5, (1957), 321– 436. 14 H. Wennerström, G. Lindblom, B. Lindman, Chem. Scr., 6, (1974), 97– 103. Citing Literature Volume86, Issue1February 01, 1978Pages 25-28 ReferencesRelatedInformation

Effect of salicylate on the binding of sulfonamides to bovine serum albumin.

Displacement of eight sulfonamides by sodium salicylate from the binding sites of bovine serum albumin (BSA) was studied by the equilibrium dialysis method at pH 7.4 and 37°. Amount of sulfonamides displaced by the addition of salicylate (1 or 3 mM) were linearly correlated with their amount bound to the binding sites of BSA in the absence of salicylate. Double-reciprocal plots of the binding of sulfamethoxazole to BSA with and without salicylate, following Langmuir's isotherms, resulted in linear curves with a common ordinate intercept. This result suggests that the inhibitory effects of salicylate on the binding of sulfamethoxazole to albumin were competitively performed at the same binding site on the protein.

Asymmetric reduction of aryl trifluoromethyl ketones with an achiral NADH model compound in a chiral hydrophobic binding site of sodiumcholate micelle, β-cyclodextrin and bovine serum albumin

Asymmetric reduction of aryl trifluoromethyl ketones with an achiral NADH model compound in a chiral hydrophobic binding site of sodiumcholate micelle, β-cyclodextrin and bovine serum albumin

Studies of the thermodynamics and nature of interaction between serum albumin and penicillins

The possibility of a calorimetric determination of the number of homogeneous independent binding sites on the protein molecule surface is presented, together with the possibility of the determination of interaction heat and association constants for the binding of small molecules to one such site. Thermodynamic characteristics of interaction of 10 penicillins and methyl orange with primary and secondary binding sites of bovine serum albumin have been obtained using this technique. The data show that hydrogen bonds between the component parts of the complexes studied are absent. The main contribution to free energy change at forming these complexes is made by hydrophobic interactions. Electrostatic forces are also of some importance, their contribution into the complex formation is more significant in the case of quinacillin and carbenicillin which have an additional charged carboxyl group.

Spin assay as a general method for studying plasma protein binding. Bilirubin — Albumin binding

The binding of a spin label to pre-existing binding sites of a protein can be monitored by electron spin resonance spectroscopy and can be interpreted in terms of a spin assay for any ligand which shares these sites. As an example of this procedure, a method is presented which evaluates the reserve capacity of the bilirubin high affinity binding site of human serum albumin for additional bilirubin binding. Hyperbilirubinemia in pre-mature or low-birth-weight term infants can result in severe neurological damage. The maturation of this technique will provide a needed clinical assay in the management of jaundiced newborn infants.

Conversions of prostaglandin endoperoxides by glutathione- S-transferases and serum albumins

Serum albumins of certain animal species (cow, sheep, pig) accelerate the decomposition of prostaglandin endoperoxides, with formation of large amounts of prostaglandin D. The reaction is inhibited by arachidonic acid, which suggests an interaction of the endoperoxide with the fatty acid binding sites of serum albumin. Glutathione- S-transferases, in the presence of glutathione, convert the endoperoxide into a mixture of prostaglandin F 2α, E 2 and D 2. The prostaglandin D/E-ratio depends on the transferase used. The known rat liver transferases give mainly prostaglandin F 2α and E 2, but a new transferase in sheep lung was discovered which gives rise to large quantities of prostaglandin F 2α and D 2. The sheep lung transferase was purified to homogeneity. Two iso-enzymes with identical enzymic activity were obtained. The major component (transferase SL 2, an iso-enzyme of glutathione- S-transferase, EC 2.5.1.18) has a molecular weight of 45 000 and consists of two subunits. Its isoelectric point is 9.8–9.9. These properties, as well as the amino acid composition and the substrate specificity for typical transferase substrates, indicate a close resemblance to transferase B (ligandin), a major binding protein of rat liver. Although purified glutathione peroxidase from erythrocytes is very active in catalysing the reduction of the 15-hydroperoxy group of prostaglandins, it does not have any effect on the decomposition of the endoperoxide group.

Affinity labeling of the primary bilirubin binding site of human serum albumin.

A label for the bilirubin binding sites of human serum albumin was synthesized by reacting 2 mol of Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate) with 1 mol of bilirubin. This yielded a water-soluble derivative in which both carboxyl groups of bilirubin were converted to reactive enol esters. Covalent labeling was achieved by reacting the label with human serum albumin under nitrogen at pH 9.4 and 20 degrees. Under the same conditions, no covalent binding to the monomers of several proteins could be demonstrated. The number of binding sites for bilirubin and the label were found to be the same, and competition experiments with bilirubin showed inhibition of covalent labeling. The absorption, fluorescence and CD spectra of the label in a complex with human serum albumin were similar to those of the bilirubin human serum albumin complex. However, following covalent attachment to the spectral properties were changed, indicating loss of conformational freedom of the chromophore. Labeling ratios were selected to result in the incorporation of less than 1 mol of label/mol of human serum albumin. Under these conditions, labeling is thought to occur primarily at the high affinity binding site.

Bovine serum albumin. Study of the fatty acid and steroid binding sites using spin-labeled lipids.

Three spin-labeled derivatives of stearic acid and two derivatives of palmitic acid have been used to study the structure of the strong fatty acid binding site of bovine serum albumin. The steroid and indole binding sites have been studied using spin-labeled derivatives of androstol and indole, respectively. Paramagnetic resonance and fluorescence quenching data suggest that the fatty acid, steroid, and indole binding sites may be identical. The mobility of the nitroxyl group at C-8 of palmitic acid bound to albumin at a 1:1 molar ratio is unaffected when the carboxyl group is esterified. When the nitroxyl group is located at C-5 on this acid its motion is detectably increased by esterification of the carboxyl group but the magnitude of this change is small. This result suggests that the carboxyl group may play a minor role in the binding of fatty acids to the strongest fatty acid binding site of albumin. When stearic acid derivatives bearing the nitroxide at C-5, C-12, and C-16 are bound to albumin at a ligand to albumin ratio of 1, the order of mobility at 0-30 degrees is C-16 greater than C-12 congruent to C-5. Although motion at the methyl terminus is always greater than at the COOH terminus in the range 0-60 degrees, a simple monotonic increase in chain motion between the two termini is not observed. Arrhenius plots of the motion parameters for these bound fatty acids show two abrupt changes in slope. The temperature ranges for these changes are 15-23 degrees and 38-45 degrees. These results suggest that when one mole of spin-labeled fatty acid is bound to albumin, the protein undergoes a conformational change in each of these temperature ranges.

Binding of some phenoxyalkanoic acids to bovine serum albumin in vitro.

The phenoxyalkanoix acids are bound to bovine serum albumin at a single site of high affinity, where the interaction is most importantly influenced by strong hydrophobic bonding, with ionic bonding being of lesser importance, and also at several sites of lower affinity. The hydrophobic binding site has almost equal affinity for the phenoxy groups of 2,4-dichlorophenoxyacetic acid, 4-(2,4-dichlorophenoxy)butyric acid and 2-(2,4,5-trichlorophenoxy)propionic acid and greater affinity for the phenoxy group of 2,4,5-trichlorophenoxyacetic acid. The properties of the high affinity binding site bear some resemblance to the properties of the amino acid sequence adjoining the tryptophan residue of human serum albumin, and evidence is presented for the presence of tryptophan at the high affinity binding site of bovine serum albumin.

Coupling of dyes to biopolymers by sensitized photooxidation. Affinity labeling of a binding site in bovine serum albumin.

Coupling of dyes to biopolymers by sensitized photooxidation. Affinity labeling of a binding site in bovine serum albumin.