Cobinding of bilirubin and sulfonamide and of two bilirubin molecules to human serum albumin: A site model

Abstract

Differential light absorption spectra of the bilirubin-albumin 1:1 complex, obtained on addition of 20 different sulfonamides, differ with respect to shape and amplitude. This finding seems to indicate that the sulfonamide molecule is bound in direct touch with the bilirubin. The light absorption spectrum of bilirubin-albumin 1:1 undergoes changes on cobinding of a fatty acid anion, laurate, and on variation of pH, previously explained by a change of dihedral angle between the two chromophores of the bilirubin molecule. In bilirubin-albumin 2:1, binding of laurate and variation of pH cause little change of the spectrum. This is best explained by binding of the two bilirubin molecules in close proximity, preventing conformational changes in the complex. From measurements of fluorescence of the lone tryptophan group in albumin and quenching on binding of bilirubin, we calculated the distance of 22 Å from tryptophan to the first bound bilirubin molecule, and of 18 Å to the second. Mutual quenching of the bilirubin fluorescence from two bound bilirubin molecules seemed to indicate that the two are bound closely together. A model of bilirubin-albumin with a binding site capable of accommodating one bilirubin and one sulfonamide molecule, or two molecules of bilirubin, is compatible with our findings.