The interaction of Bromophenol Blue with proteins in acidic solution

The interactions of Bromophenol Blue (BPB) with bovine serum albumin and γ-globulin in acidic solutions were investigated by a spectrophotometric method. It was considered that the electrostatic force is the main binding force, and that the color change during the combination is due to the transformation of dye species of free acidic form into bound basic form as well as to the bathochromic and hyperchromic effects of conjugation. The formation of an isosbestic point in the absorption spectra was explained based on a new consideration about the solution equilibria. Two conditional constants, apparent binding constant and maximum binding number, were defined to express the binding ability of a dye to a certain protein under a given set of conditions, and two linear regression equations were derived to determine these two parameters and the molar absorptivity of bound dye. The Scatchard model is not appropriate in the treatment of data obtained here. The factors which influence the sensitivity of a dye binding protein assay were discussed, and the Sandell index was used to express the sensitivity of protein detection. It was found that sodium chloride concentration and acidity of the solutions have significant effect on the sensitivity of BPB protein assay.