TITLE:
Studies on the Binding Mechanism of VB1 and VB9 with Trypsin
AUTHORS:
Yan Gao, Congying Shao, Wanru Ji, Min Xiao, Fan Yi, Tong Zhou, Yanqin Zi
KEYWORDS:
Trypsin; VB1 and VB9; Fluorescence Spectrometry; Nonradiative Energy Transfer Mechanism
JOURNAL NAME:
American Journal of Analytical Chemistry,
Vol.4 No.12,
December
27,
2013
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 Kb 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 Kb 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.