Kinetic studies of the interaction of bromphenol blue with bovine serum albumin by pressure-jump method.

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The interaction of Bromphenol Blue (BPB) with bovine serum albumin (BSA) was studied statically, by spectrophotometry, and kinetically, by the pressure-jump method. The absorbance changes of BPB were monitored at 20 °C, pH 7.00 in 0.2 M phosphate buffer. The static measurements showed that there were two binding classes. The number of binding sites and the binding constants for each class are n1=1, K1=1.4×107 M−1 and n2=3, K2=9.5×104 M−1. Kinetically, the binding of BPB to the primary binding site of BSA proceeds via at least 4 steps. Two models are offered for the possible binding mechanism. In these models, a fast, probably diffusion controlled, second order step is followed by three first order steps. A correlation between the number of binding steps and the magnitude of the binding constant is discussed. The positive activation entropies associated with the backward reactions of the second and third steps show that these reactions proceed through disordered activation states. From the comparison of the present results to those for other ligands, it was found that the ligand is intimately involved in which activated configuration is adopted.
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