RESUMO
Laboratory scale preparation of bovine serum albumin (BSA) stained with Coomassie brilliant blue (CBB) at alkaline pH is first described. Physical-chemical analyses of CBB-BSA showed that the unprotonated (anion) CBB dye binds 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 dye/protein concentration ratios at alkaline pH gave values for n, binding site numbers, and K, intrinsic binding coefficient, consistent with those reported in analytical studies under acidic pH, but higher than values for neutral 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, light, prolonged storage, as well as high affinity for tannins. The hydrophobic nature of the CBB-BSA association at alkaline pH was tested.
Assuntos
Corantes de Rosanilina/química , Soroalbumina Bovina/química , Animais , Sítios de Ligação , Bovinos , Detergentes , Concentração de Íons de Hidrogênio , Indicadores e Reagentes/química , Cinética , Modelos Químicos , Ligação Proteica , Corantes de Rosanilina/metabolismo , Sensibilidade e Especificidade , Soroalbumina Bovina/metabolismo , Espectrofotometria , Taninos/química , Taninos/metabolismoRESUMO
A quantitative model is derived for tannin-protein binding and protein precipitation in solution. This model is based on the assumption that precipitation occurs when the number of tannin molecules associated with one protein molecule attains a critical value. Precipitation occurs at this point because tannin crosslinking causes formation of large protein aggregates. Analytical expressions were derived for the dependence of protein precipitate yields upon the concentrations of the protein and tannin in solution. This expression fits reasonably well the experimentally observed bell-shaped dependencies of bovine serum albumin or gelatin precipitation upon total protein and tannin concentrations.