Chemical Modification of the High‐Affinity Bilirubin‐Binding Site of Human‐Serum Albumin

Abstract

A number of derivatives of human serum albumin were prepared by chemical modification of native albumin with acetylsalicylic acid, succinic anhydride, maleic anhydride, methyl acetimidiate hydrochloride, N‐acetylimidazole, acetic anhydride, tetranitromethane, diethylpyrocarbonate, glyoxal, ethyl diazoacetate and o‐nitrophenylsulfenyl chloride. The conformational changes associated with the modification reactions were detected by Sephadex G‐200 gel chromotography. The affinity of bilirubin for the modified albumins was estimated by the peroxidase method at a molar ratio of 0.5 bilirubin per albumin. With increased unfolding of albumin, due to amino group modification, the affinity decreased. Acetylation of 22 or amidination of 52 amino groups or sulfenylation of the single tryptophan did not change the binding affinity significantly. Blocking of 10 arginine, 2 histidine or 8 tyrosine residues resulted in decreased affinity for bilirubin. The results indicate that the binding of bilirubin to the first binding site on albumin involves both hydrophobic and electrostatic forces. The results also suggest that arginine, tyrosine and histidine residues are close to or located in the strong binding site. Copyright © 1972, Wiley Blackwell. All rights reserved