Competitive interaction of biliverdin and bilirubin only at the primary bilirubin binding site on human albumin

The binding of biliverdin-IXα by human albumin and serum was quantitated, using three different binding techniques, to study the effects of biliverdin on bilirubin-albumin binding. The apparent equilibrium association constants (K ± SD) and binding capacities (n) of defatted albumin, pooled adult sera, and pooled umbilical cord sera for biliverdin are: K = 1.3 ± 2 × 10^{6 −1}, n = 1.00; K = 13.0 ± 3 × 10^{6 −1}, n = 0.90; and K = 6.8 ± 0.1 × 10^{6 −1}, n = 0.85, respectively. Although bilirubin binds at more than one albumin site, competitive studies showed that biliverdin binds only at the primary (highest affinity) bilirubin site. Sulfisoxazole, previously thought to compete with bilirubin for the primary binding site, was found to displace bilirubin from both primary and secondary bilirubin binding sites. Biliverdin, because of its specific binding and spectral characteristics, could be a useful probe for determining the capacity of the primary bilirubin-albumin binding site.