Positive cooperativity in binding by albumin: the system bovine serum albumin and alizarin yellow G cobinding by salicylic acid.

The binding of alizarin yellow G--an azo derivative of salicylic acid--by bovine serum albumin has been investigated using the method of equilibrium dialysis. Six strong and a number of additional, weak binding sites have been found to be present. The system is characterized by strong positive cooperativity between the first and second sites. Six binding constants have been determined on the basis of a simplified mathematical model. The results are approximately 2 X 10(4) M-1 for the first binding site, 6 X 10(5) M-1 for the second, and between 4 X 10(4) and 10(5) M-1 for the rest. The phenomenon is discussed in terms of the existence of various conformers or of the conformational adaptability of albumin. Cobinding by salicylic acid does not displace alizarin yellow G but induces a conformational change in the protein which affects the absorption spectrum of the bound dye. As expected for this kind of heterotropic interaction, the spectrum of the system albumin-salicylic acid is similarly affected by the cobinding of alizarin yellow G.

The kinetics of the reaction between bovine serum albumin and bilirubin. A second look.

The kinetics of the reaction between bilirubin and bovine serum albumin have been re-investigated at 20 degrees 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, bimolecular step. From the kinetic behaviour it is concluded that this primary complex is present in non-negligible concentration after equilibrium is reached, and that it exchanges bilirubin with the surroundings with a rate constant of at least 23 s-1. This also means that the secondary complex, and, possible, 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 leads to B transition. On the other hand, a pH jump between 7.4 and 9.0 is assumed to monitor the N leads to B transition, both the free albumin and the complex in its various forms undergoing this transition at identical rates. This transition, although influencing the absorptivity of the complex, was found not to influence the strength of the binding site.