A Search for Specific Amino Acid Residues which are Involved in Bilirubin Binding of Human Serum Albumin
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The bilirubin binding capacity (BBC) of human serum albumin has long been studied by many investigators, but few of them reported on the nature of binding site (s), if any, of the protein. This is mainly because of lack of the knowledge on its primary structure. Recently Swaney 1) et al. have determined the adjoining structure of the lone Trp in albumin and suggested that its apolar characteristi may play a significant role in steroids binding of albumin.<BR>In this study, albumin was purified by DEAE-cellulose column chromatography, and characterized by cellulose acetate membrane electrophoresis and gel immunoelectrophoresis as a single component (Fig. 1). Disc electrophoresis, however, resolved it into 3 components, probably monomer, dimer and so on, according to other workers' published data2). The fraction of albumin was submitted to amino acid analysis. Its composition (TABLE I) roughly resembled that of Saifer's but differed therefrom in more Glu content and less Tyr content.<BR>From the structure of bilirubin and the fact that bilirubin can not be bound with ovoalbumin, it is postulated that the binding site (s) would be of hydrophobic nature and have positively charged groups and Tyr.<BR> Acetylation with acetic anhydride in 10% pyridine-water on an ice-bath yielded ε-N, acetyl (Lys)-albumin. After passage through Sephadex G-25 column and dialysis, their BBC were determined by means of absorbancy at 280nm and 460nm of proteinbilirubin complexes which had been obtained from the protein solutions in the presence of a large excess of bilirubin by gel-filtration on Sephadex G-25.<BR>The modified proteins generally had less BBC and higher acetylation degrees which were determined on the basis of ninhydrin value and dinitrophenylation value. With 5% acetylated-albumin, BBC remained 116% of that of the native one (TABLEIII). As an albumin molecule has 65 Lys residues, it might be concluded that any Lys which were preferentially acetylated is not involved in binding.<BR>The particular amino acid residue, Trp, as mentioned above, was chosen as a next target for modification. Koshland reagent5), 2-hydroxy, 5-nitrobenzyl bromide (HNB-Br), was made to react with albumin under the following conditions: albumin 0.1μmole, HNB-Br 1μmole, urea 8 M in 3 ml of 5% MeOH-water. The pH of the reaction mixture was kept at 3.0 by using pH-statt for 2 hrs. The modified protein was obtained by gel-filtration on Sephadex G-25 column equilibrated with 8M urea. It was then subjected to gel-filtration on Sephadex G-100 equilibrated with 0.01N NaH<SUB>2</SUB>PO<SUB>4</SUB>. The protein emerged as a single peak was named HNB-Alb.<BR>In the third attempt to elucidate the bilirubin binding site (s) qualitatively, Vallee's reagent6), N-acetylimidazole (N-AI), was used for Trp modification. The conditions: albumin 0.1μmole: N-AI 100μmole: 0.1M phosphate, pH 7.0 at room temperature. The reaction proceeded under continuous stirring. Each 1.5ml of the reaction mixture was pipetted out at 1 hr and 2 hrs intervals and the aliquot was gel-filtrated on Sephadex G-10 to give the modified proteins-I and-II, respectively.<BR>Judging from the absorption spectrum of HNB-Alb, Trp in the protein was modified by approximately 100% (TABLE II), and BBC of the modified protein was decreased but similar in behavior with respect to bilirubin concentration to that of native albumin (Fig. 2). O-AcAlbs whose amounts of unmodified Tyr were determined on the basis of absorbance at 278nm showed a marked decrease in their BBC. The specimen II which had more modified Tyr were much more decreased in BBC than I (Fig. 2). The exact number and location of the modified Tyr in the primary structure is under investigation.
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