A dynamic model for bilirubin binding to human serum albumin

Site-directed mutagenesis of human serum albumin was used to study the role of various amino acid residues in bilirubin binding. A comparison of therm odynamic, proteolytic, and x-ray crystallographic data from previous studie s allowed a small number of amino acid residues in subdomain 2A to be selec ted as targets for substitution. The following recombinant human serum albu min species were synthesized in the yeast species Pichia pastoris: K195M, K 199M, F211V, W214L, R218M, R222M, H242V, R257M, and wild type human serum a lbumin. The affinity of bilirubin was measured by two independent methods a nd found to be similar for all human serum albumin species. Examination of the absorption and circular dichroism spectra of bilirubin bound to its hig h affinity site revealed dramatic differences between the conformations of bilirubin bound to the above human serum albumin species. The absorption an d circular dichroism spectra of bilirubin bound to the above human serum al bumin species in aqueous solutions saturated with chloroform were also exam ined. The effect of certain amino acid substitutions on the conformation of bound bilirubin was altered by the addition of chloroform. In total, the p resent study suggests a dynamic, unusually flexible high affinity binding s ite for bilirubin on human serum albumin.