42 Biochimica et Biophysica .4cta, 703 (1982) 42-48 Elsevier Biomedical Press BBA 31136 THE KINETICS OF THE REACTION BETWEEN BOVINE SERUM ALBUMIN AND BILIRUBIN A SECOND LOOK R. KOREN a E. NISSANI b and B. PERLMUTTER-HAYMAN b Department of Pharmacology, Hadassi~h Medical School and l, Department of Physica/ Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904 (Israel) (Received August 8th, ! 981) Key words: Albumin; Bilirubin; Complex formation," N---, B transition The kinetics of the reaction between bilirnbin and bovine serum albumin have been re-investigated at 20°C. The results of previous authors concerning both human and bovine serum albumin were largely confirmed, namely the existence of two first-order configurational changes after a primary complex is formed in a fast, bimoleeular step. From the kinetic behaviour it is concluded that this primary complex is present in non-negiigible concentration after equilibrium is reached, and that it exchanges bilirnbin with the surround- ings with a rate constant of at least 23 s-1. This also means that the secondary complex, and, possibly, also the final product, are in dynamic equilibrium with each other and with the primary complex, and, therefore, only the sum of their formation and dissociation rate constants can be measured. The dependence of the two observable relaxation times on pH does not parallel the N-, B transition. On the other hand, a pH jump between 7.4 and 9.0 is assumed to monitor the N-~ B transition, both the free albumin and the complex in its various forms undergoing this transition at identical rates. This transition, although influencing the absorptiv- ity of the complex, was found not to influence the strength of the binding site. Introduction The binding of organic molecules to serum pro- teins may be a controlling factor in the distribu- tion of drugs, essential ligands and sometimes toxic substances into various tissues and body compartments [1]. This process has been studied extensively under equilibrium conditions in a number of systems and by several different tech- niques [2]. However, the distribution and elimina- tion of absorbed compounds involve kinetics and may be affected not only by the extent of binding to serum proteins, but also by the protein-ligand association-dissociation rate-constants. Owing to both experimental and theoretical difficulties, the information about the kinetics of protein-ligand interaction is much less complete than that about 0167-4838/83/0000-0000/$02.75 © 1982 Elsevier Biomedical Press equilibrium binding, both with respect to quantita- tive analysis and to mechanistic interpretation. One of the best-studied of these systems is the interaction between bilirubin and human or bovine serum albumin. This reaction is of special physio- logical importance, because it reduces the con- centration of a highly toxic substance to a very low value in the bloodstream. The subject has recently been reviewed extensively by Brodersen [3]. The albumin was found to contain one very strong binding site and possibly one or even two much weaker ones [3-8]. All the kinetic experi- ments were carried out under conditions where the strong complex is the dominant species [6,9-12]. Although the results are somewhat contradictory, it seems clear that the primary reaction between the two reactants is very fast, and is followed by