Preparative binding of Coomassie brilliant blue to bovine serum albumine at alkaline pH

Laboratory scale preparation of bovine serum albumin (BSA) stained with Coo massie brilliant blue (CBB) at alkaline pH is first described. Physical-che mical analyses of CBB-BSA showed that the unprotonated (anion) CBB dye bind s tightly to BSA in buffered media of pH 8.2. Characteristic differences in spectra lambda(max) and molar absorptivities were found for the free anion CBB dye versus the CBB-BSA complex. Binding studies with low versus high d ye/protein concentration ratios at alkaline pH gave values for n, binding s ite numbers, and K, intrinsic binding coefficient, consistent with those re ported in analytical studies under acidic pH, but higher than values for ne utral pH. Comparative analyses of Beer's law plots for the alkaline CBB-BSA complex under different experimental conditions showed its high stability toward various interferences, such as pH, strong detergents, temperature, l ight, prolonged storage, as well as high affinity for tannins. The hydropho bic nature of the CBB-BSA association at alkaline pH was tested.