STUDIES OF THE AFFINITY OF HUMAN SERUM ALBUMIN FOR BINDING OF BILIRUBIN AT DIFFERENT TEMPERATURES AND IONIC STRENGTH

Abstract

The association constants for the binding of bilirubin to human serum albumin (HSA) have been determined at four different temperatures by measurements of the rate of the peroxidase catalyzed oxidation of unbound bilirubin. The change of enthalpy is determined from a van't Hoff plot (ln K_ass versus 1/T) to about —13.5 kcal/mol. ΔGd̀ is calculated from the binding constants, and ΔSd̀ is obtained from: ΔGd̀=ΔHd̀— TΔSd̀. The results show that the large negative ΔGd̀ (— 11 kcal/mol) for binding of bilirubin to HSA is a consequence of the negative ΔHd̀. The entropy was found to be about — 8.5 cal/mol/degree and tends to diminish the numerical value of ΔGd̀. The binding constant has also been determined at varying ionic strength. The results show a decrease in binding for increasing salt concentration. The data from the two sets of experiments suggest that hydrogen bonds and salt linkages rather than hydrophobic interactions are the main factor in the binding of bilirubin to its primary site on HSA.