Dichroic Probe of the Equilibrium Constant of the Distribution of Bilirubin to Human and Bovine Serum Albumins

Abstract

We describe here a method to evaluate the equilibrium constant of the distribution of a ligand, bilirubin, to two different albumins (human and bovine serum albumins, HSA and BSA) and hence to determine the association constant of the ligand to an albumin (in this case HSA) with the knowledge of the association constant of the ligand to the other albumin (in this case BSA). The circular dichroic (CD) spectra of bilirubin (BR) induced by HSA and BSA are characteristically different. If in a pre-formed BSA-BR complex HSA is added, the negative bisignate CD spectrum of BSA-BR progressively changes sign characteristic to that of HSA-BR (positively bisignate). This change in dichroism has been used to calculate the equilibrium constant K of the process: BSA-BR + HSA = HSA-BR + BSA, the value of K comes to be 1.25. The individual association constant of BSA-BR has been determined fluorimetrically to be 2.7 × 10⁷ M-¹. Since, K of the above process must be the ratio of the individual association constants of HSA-BR and BSA-BR. the association constant of HSA-BR comes to be 3.37 × 107_M⁻¹