Abstract: The binding-site number was calculated by using fluorescence spectroscopic method with bovine serum albumin(BSA) and Indo-1 as protein and ligand models, respectively. The method for calculating binding-site number in BSA for Indo-1 was developed based on the relationships between the changes of Indo-1 fluorescence intensity and the analytical concentration of BSA. And the interaction of BSA with Indo-1 was investigated comprehensively by using fluorescence techniques as well as fluorescence resonance energy transfer, and the thermodynamic parameters were calculated according to the changes of enthalpy on temperature. Three binding sites in BSA for Indo-1 were revealed, and the distances from Trp212 in BSA to the three binding sites were 2.93, 2.57 and 2.40 nm, respectively. It was also proved that Indo-1 embedded into the three hydrophobic cavities of BSA by hydrophobic association. This paper provided a use for reference on calculating the binding-site number in protein for ligand and studying their interactions by fluorescence spectroscopic methods. In fluorescent quenching experiments, fluorescence changes were automatically recorded in real time by combining Microlab 500 Series Dispenser and PTI fluorescence apparatus.

Key words: Bovine serum albumin, Fluore scence probe Indo-1, Binding-site number, Interaction, Fluorescence spectroscopic method