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

Abstract

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.