Studies on the Binding Mechanism of VB 1 and VB 9 with Trypsin

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 × 10 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 × 10 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.


Introduction
There are main functions of VB 1 in sugar metabolism, energy metabolism and digestive system normal work [1].VB 9 is the floorboard of the compounds which contains pteroylglutamic acid and is well studied and separated from spinach leaf [2].Proteins are important and widespread in kinds of biological macromolecules in living organisms and take part in almost all life processes.Trypsin is a serine proteinase, which hydrolyzes proteins and peptides at the carboxyl sides of arginine and lysine residues.Many investigations between proteins and vitamins have been published including bovine serum albumin [3][4][5][6] and human serum albumin [7][8][9], but the studies on the interaction between vitamin B and trypsin have not been reported.In this paper, the interactions between vitamin B and trypsin have been studied at different temperatures under physiological conditions using UV/vis spectrophotometry and fluorescence spectrometry.The effects of VB 1 and VB 9 on the trypsin have been evaluated and compared, such as quenching mechanism, binding constants, binding sites, binding mode and so on.

Apparatus and Reagents
An FP-8300 fluorescence spectrometer (Jasco, Japan) was used to record the fluorescence spectra in 1.00 cm quartz cell, a TU1901 UV/vis Spectrophotometer (PGeneral, Beijing, China) was employed to record the absorption spectra and a PHS-3C meter (Shanghai Precision Scientific Instrument Co., Ltd China) was used to measure the pH values of B-R buffer solutions.
VB 1 solutions (1.00 × 10 −4 mol/L) were prepared by diluting 0.0094 g (337.27Da, Sinopharm Chemical Rengent Co., Ltd, Shanghai, China) in 250.00 mL of deionized water.VB 9 solutions (1.00 × 10 −4 mol/L) were dissolved by diluting 0.0110 g (441.41Da, Tianjin recovery fine chemical industry research institute, Tianjing, China) in 250.00 mL of deionized water.Trypsin solutions (1.00 × 10 −4 mol/L) were prepared by diluting 0.6005 g of trypsin (24000 Da, Sinopharm Chemical Rengent Co., Ltd, Shanghai, China) in 250.00 mL of water.Britton-Robinson (B-R) buffer solutions (pH = 7.90) were prepared by combining a mixed acid (composed of 0.04 mol/L of H 3 PO 4 , HAc, and H 3 BO 3 ) with 0.20 mol/L of NaOH in equal proportions.NaCl (0.20 mol/L) were dissolved to adjust the ionic strength of the VB 1 -trypsin and VB 9trypsin solutions so as to study the effects of electrolytes on binding.All solutions were prepared using double-distilled, deionized water and the reagents were of analytical reagent grade.In the experiments, a known volume standard of VB 1 or VB 9 solutions were added in 10.00 mL calibrated tubes with deionized water and mixed well.

General Procedure
In 10.00 mL calibrated tubes, 1.00 mL B-R buffer solutions (pH = 7.90), 1.00 mL of 1.00 × 10 −4 mol/L trypsin solutions and a known volume of the standard VB 1 or VB 9 solutions were added.Then the mixture were diluted to 10.00 mL with NaCl (0.20 mol/L) and mixed thoroughly by shaking.After reaction for 30 min, the solutions were taken into the optical cell.The system's fluorescence spectra wavelengths were recorded from 290 nm to 450 nm and the bandwidths were 5 nm.

Fluorescence Quenching Spectra and
Quenching Mechanism of VB 1 and VB 9 with Trypsin Figure 1 shows that fluorescence emission spectra of trypsin with the increasing concentrations of VB 1 and VB 9 following an excitation wavelength at 281 nm.Trypsin shows a fluorescence emission with a peak at 340 nm.The fluorescence intensity of trypsin decreased gradually with the increasing concentrations of vitamin B, and higher concentrations led to more efficient quenching of the tryptic fluorescence.By comparison, it was known that VB 9 led to more apparently efficient quenching of the protein fluorescence than VB 1 .Such a quenching clearly indicated the binding of VB 1 and VB 9 with trypsin.Meanwhile, there are not a shift of maximum emission peaks, indicating that vitamin B didn't influence the microenvironment around trypsin.The fluorescence quenching mechanisms usually contain dynamic quenching and static quenching, which are caused by diffusion and ground-state complex formation spectively [10,11].In order to further clarify the fluorescence quenching mechanism induced by vitamin B, the Stern-Volmer equation get used to evaluate the data.
where F 0 and F represent the steady-state fluorescence intensities in the absence and presence of the quencher, respectively;   Q is the concentration of quencher; sv K is the Stern-Volmer quenching constant; q K is the bimolecular quenching rate constant and q K is equal to 0 sv K  ; 0  is the average lifetime of the molecule without any quencher and this 0  = 10 −8 s [12].The Stern-Volmer curves at two temperatures were shown in Figure 2. The values of sv K and q K derived from Equation (1) are listed in Table 1.The minimum value of q K as shown in Table 1 is 8.621 × 10 11 L/(mol•s), which is greater than the maximum diffusion collision quenching rate constant of 2.0 × 10 10 L/(mol•s) [13].So it indicated that the fluorescence quenching process of trypsin with VB 1 and VB 9 may be mainly governed by a static quenching mechanism.

Binding Constant and Number of Binding Site
In static quenching process, when small molecules are bound independently to a set of equivalent sites on a macromolecule, the equilibrium between free and bound molecules is given by Equation (2) [14]: Open Access AJAC Y. GAO ET AL.

Thermodynamic Parameters and Nature of
The es contributing to drug-biomolecule that there are a strong interaction and a comp formation between trypsin with VB 1 and VB 9 .Furthermore, it can be inferred from the values of n that there is an independent class of binding sites on trypsin with VB 1 and VB 9 .But it appears that the binding constants and the number of binding sites also increase with higher temperature [15,16].So this may be because the capacity of VB 1 and VB 9 binding to trypsin is enhanced with increasing temperature. ) for a binding interaction at different temperatures c be determined by the Equation ( 4).
where b K is the binding constant, R is the gas constant and T is the absolute temperature.The fluorescence quenching of trypsin after binding VB 1 and VB 9 indiactes that the tranfer of energy has occurred.According to Förster's resonance energy transfer theory [17], the distance between two interacting molecules and the efficiency of energy transfer can be discribed by the following equation: where E is the energy transfer efficiency, F is the fluor- where K 2 is the spatial orientation factor of the dipole, N is the refractive index of the medium, Φ is the fluorescence quantum yield of the donor, J is the overlap integral of the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor.J is calculated using the equation: where F(λ) is the fluorescence intensity of the fluorescence donor when the wavelength is λ and ε(λ) is the molar absorbance coefficient of the acceptor when the wavelength is λ.It has been reported that K 2 = 2/3, N = 1.336, and Φ = 0.118.Figure 5 shows that the spectral overlap between the fluorescence emission spectrum of trypsin and UV/vis absorption spectrum of VB 1 and VB 9 .
From the above relationships, J = 3.79× 10 −19 cm 3 •l•mol −1 , R 0 = 0.45 nm, E = 0.79 and R = 0.55 nm for trypsin and VB 1 .Similarly, J = 7.96 × 10 −17 cm 3 •l•mol −1 , R 0 = 0.87 nm, E = 0.23 and R = 0.87 nm for trypsin and VB 9 .The distance R < 8 nm between donor and acceptor indicates that the energy transfer from trypsin to VB 1 and VB 9 occurred with high possibility.This obeyed the conditions of Förster energy transfer theory.nteractions between two kinds of vita-

Conclusion
In this paper, the i min B and trypsin have been investigated under simulated physiological conditions using spectrometries.The fluorescence of trypsin was quenched by two kinds of vitamin B mainly through static quenching.The enthalpy change ( H  ) and entropy change ( S  ) for the systems were calculated respectively.The positive H  and S  values indicated that hydrophobic interactions played main roles in the binding between trypsin and vitamin B. A binding distance R of 0.55 nm and 0.87 nm between donor and acceptor was obtained.According to the data, the two B vitamins have similar interactions with trypsin.The results obtained are of important biological significance in pharmacology and clinical medicine.

3. 4 . 3 . 5 .
Synchronous Fluorescence Spectroscopy sed Synchronous fluorescence spectroscopy is usually u to investigate the microenvironment around the fluorophore functional groups.At   = 60 nm, the synchronous fluorescence spectra ar ttributed to tryptophan, while e a   = 15 nm, the spectra are attributed to tyrosine.Synchr us fluorescence spectra of trypsin with addition of VB 1 are shown in Figure 4(a) and these with addition of VB 9 are shown in Figure 4(b).From Figure 4, the emission maxima have no shifts with regards to VB 1 and VB 9 , which indicates that there was no change of the microenvironment of the tryptophan and tyrosine.ono Energy Transfer and Binding Distance with