Abstract: Under simulated physiological conditions of animals and at various temperatures, the binding reaction of Bromophenol blue (BPB) to bovine serum albumin (BSA) was studied by fluorescence spectrum and ultra-violet spectrum spectroscopy. The fluorescence quenching data were analyzed according to Stem-Volmer equation and Lineweaver-Burk double-reciprocal equation. The study show that BSA reacted with BPB and formed a certain new compound, which belonged to static fluorescence quenching. The formation constants of the compound K_LB, the thermodynamic parameters and the number of binding sites were obtained. And the binding power between them is mainly the electrostatic acting force. Site marker competitive experiments showed that the binding of BPB to BSA primarily took place in site Ⅰ(sub-domain ⅡA)of BSA. The effect of BPB on the conformation of BSA was analyzed by synchronous fluorescence spectra and three-dimensional fluorescence spectra, which indicates that the polarity microenvironment around Trp residues decreased, while hydrophobic forces increased. These provide important information for enucleating the dyeing mechanisms, the toxicity effects and biological effects of BPB.

Key words: bromophenol blue, bovine serum albumin, fluorescence quenching spectrum, three dimensional fluorescence spectrum, thermodynamic parameters