Optical properties of bilirubin-serum albumin complexes in aqueous solution: I. Dependence on pH

Abstract

Circular dichroism (CD) measurements in the wavelength range 300 to 550 nm of bilirubin (2.5 · 10 −5 M)-human serum albumin (5.0 · 10 −5 to 12.5 · 10 −5 M) complexes in aqueous solution (27°C) were carried out with differently treated serum albumins and at various pH values in the pH range 3.5 to 10. In all cases, two large and proximate bands of opposite sign were recorded between 400 and 500 nm and, in some cases, one or two smaller additional bands were observed either in the range of 300 to 400 nm or near 500 nm. Large differences in the magnitude of the various bands were measured for complexes involving either charcoal-treated or untreated serum albumins. Bilirubin complexes of all human serum albumin preparations showed a reversible inversion with pH in the sign of the Cotton effects of the two main bands occurring around pH 5, with some concomitant shifts in the position of band extrema. In contrast to CD, the light-absorption spectra did not differ largely under various conditions. The main Cotton effects observed in the visible region are interpreted as coupling between electric transition dipole moments of bound bilirubin leading to exciton splitting. This explanation is substantiated by computer analysis into Gaussian curves of observed light-absorption and CD spectra. From these data, and on the basis of some assumptions, both distances and relative positions in space of the transition dipoles are estimated for various cases. Other possible contributions to the optical activity are also considered. Apart from their theoretical significance, the large and diverse effects of optical activity observed under different conditions are also considered to serve as very sensitive probes for changes in the protein environment at the binding site. Moreover, the CD spectra of the complex bilirubin-serum albumin may be of analytical value for the differentiation between preparations of serum albumin and also between serum albumins from different species.