Studies on the Binding Mechanism of VB₁ and VB₉ with Trypsin

Abstract

The binding characteristics of vitamin B 1 (VB 1 ) and vitamin B 9 (VB 9 ) with trypsin were investigated by fluorescence spectrometry and UV/vis spectrophotometry under simulated physiological conditions. With the addition of VB 1 or VB 9 , the intrinsic fluorescence emission intensity of trypsin was quenched by the nonradiative energy transfer mechanism. The fluorescence quenching process of trypsin may be mainly governed by a static quenching mechanism. The binding parameters such as the binding constants and the number of binding sites can be evaluated by fluorescence quenching experiments. The numbers of the apparent binding constant K b of VB 1 -trypsin at different temperatures were 0.4948 and 4.8340 × 10 4 L/mol and the numbers of binding sites n were 0.9359 and 1.1820. Similarly, the numbers of the apparent binding constant K b of VB 9 -trypsin at different temperatures were 5.9310 and 13.040 × 10 4 L/mol and the numbers of binding sites n were 0.9908 and 1.0750. The thermodynamic parameters, with a negative value of Δ G , revealed that the bindings are spontaneous processes and the positive values for both enthalpy change (Δ H ) and entropy change (Δ S ) indicate that the binding powers of VB 1 and VB 9 with trypsin are mainly hydrophobic interactions. And synchronous spectrums were used to study the conformational change of trypsin. In addition, the binding distances of VB 1 -trypsin and VB 9 -trypsin were estimated to be 0.55 nm and 0.87 nm according to the F ö rster’s resonance energy transfer theory.