Studies on the Binding Mechanism of VB₁ and VB₉ with Trypsin

Yan Gao, Congying Shao, Wanru Ji, Min Xiao, Fan Yi, Tong Zhou, Yanqin Zi

Department of Chemistry and Materials Science, Huaibei Normal University, Huaibei, China.

DOI: 10.4236/ajac.2013.412094                

Abstract

The binding characteristics of vitamin B₁ (VB₁) and vitamin B₉ (VB₉) with trypsin were investigated by fluorescence spectrometry and UV/vis spectrophotometry under simulated physiological conditions. With the addition of VB₁ or VB₉, 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 VB₁-trypsin at different temperatures were 0.4948 and 4.8340 × 10⁴ 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 VB₉-trypsin at different temperatures were 5.9310 and 13.040 × 10⁴ L/mol and the numbers of binding sites n were 0.9908 and 1.0750. The thermodynamic parameters, with a negative value of ΔG, revealed that the bindings are spontaneous processes and the positive values for both enthalpy change (ΔH) and entropy change (ΔS) indicate that the binding powers of VB₁ and VB₉ with trypsin are mainly hydrophobic interactions. And synchronous spectrums were used to study the conformational change of trypsin. In addition, the binding distances of VB₁-trypsin and VB₉-trypsin were estimated to be 0.55 nm and 0.87 nm according to the Förster’s resonance energy transfer theory.

Keywords

Trypsin; VB₁ and VB₉; Fluorescence Spectrometry; Nonradiative Energy Transfer Mechanism

Conflicts of Interest

The authors declare no conflicts of interest.

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