Structural features of bromocresol purple and its binding sites on human serum albumin for a proton-exchange reaction

Abstract

Human serum albumin (HSA) levels of identical samples are different when determined using the bromocresol green (BCG) and bromocresol purple (BCP) methods. The aim of this study was to determine this reason for this difference. The pH-dependent color change of the Sulton pH indicators (BCG, BCP, bromophenol blue, bromothymol blue, thymol blue, and phenol red [PR]) complexed with HSA (or poly- L -lysine) in acidic (pH 2.47.8) and basic (pH 7.611.2) solutions was determined with and without inhibition of complex formation by warfarin and ibuprofen. The structures surrounding the drug-binding sites were analyzed by determining the required characteristics of target residues predicted from the experimental data based on the crystallographic data of the drugHSA complexes (2BXD and 2BXG). BCP specifically bound to the warfarin- and ibuprofen-binding sites in acidic pH, but BCG did not. In basic pH, all of the indicators, except for PR, bound to their sites. All of the residues for color change, proton exchange, and binding via electrostatic interaction were present in the binding sites. Given that BCP binds to the warfarin- and ibuprofen-binding sites, the influence of coexisting substances can be experimentally evaluated to facilitate precise measurement using the BCP method.