Fluorescence stopped-flow study of relaxation processes in the binding of bilirubin to serum albumins

Abstract

Bilirubin binding to albumins of different species has been studied with stopped-flow measurements of protein fluorescence quenching and the appearance of bilirubin fluorescence. Binding, itself, as indicated by quenching of protein fluorescence, occurs at an immeasurably fast rate. The visible fluorescence of bilirubin, however, appears more slowly. The development of this fluorescence is a first-order process with a half-time of 50–100 msec, depending on the species of albumin, and may be related to the relaxation of bilirubin to a helical conformation. A slower secondary quenching of protein fluorescence occurs after the initial quenching associated with binding itself. The secondary process reduces the already quenched fluorescence by another 50%, is a first-order reaction, and has a half-time of 300–400 msec. From energy transfer calculations, a 4 Å migration of bound bilirubin along the albumin binding crevice toward tryptophans could account for this degree of secondary quenching, but a protein conformational change is not ruled out. Multiple relaxation processes in macromolecular binding may prove to be rather general phenomena.