Abstract: The effects of plasticizer dibutyl phosphate (DBP) on the spectral properties,structure and catalytic activity of trypsin were investigated using fluorescence,UV-vis absorption and circular dichroism (CD) spectroscopy along with atomic force microscopy (AFM) and molecular simulation under simulative physiological conditions (pH 7.4).The result of fluorescence quenching indicated that a ground state complex was formed between DBP and trypsin through hydrogen bonds and Van Der Waals forces,resulting in the intrinsic fluorescence quenching of trypsin.There was a single class of binding sites on trypsin for DBP.Analysis of synchronous fluorescence,UV-vis absorption and CD spectra demonstrated that the addition of DBP led to the conformational alteration of trypsin,decreases in the α-helix,β-sheet and β-turn contents and an increase in the random coil content.The AFM topography image showed that the binding of DBP with trypsin caused the surface morphology change of trypsin and the protein aggregation.The molecular modeling results exhibited that the binding site of DBP on trypsin was adjacent to the S1 binding pocket,and three hydrogen bonds formed between the amino acid residues His 57,Ser 195 and Gly 193 of trypsin and the oxygen atom of DBP.The enzymatic activity assay indicated that the binding interaction led to the inhibition of the trypsin activity.

Key words: dibutyl phosphate, trypsin, binding characteristic, molecular modeling