Studies of the affinity of human serum albumin for binding of bilirubin at different temperatures and ionic strength.

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 Kass versus 1/T) to about -13.5 kcal/mol. deltaG degrees is calculated from the binding constants, and deltaS degrees is obtained from: deltaG degrees = deltaH degrees--TdeltaS degrees. The results show that the large negative deltaG degrees (--11 kcal/mol) for binding of bilirubin to HSA is a consequence of the negative deltaH degrees. The entropy was found to be about--8.5 cal/mol/degree and tends to diminish the numerical value of deltaG degrees. 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.