Studies on the Binding Mechanism of VB<sub>1</sub> and VB<sub>9</sub> with Trypsin

doi:10.4236/ajac.2013.412094

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American Journal of Analytical Chemistry, 2013, 4, 771-775

Published Online December 2013 (http://www.scirp.org/journal/ajac)

http://dx.doi.org/10.4236/ajac.2013.412094

Open Access AJAC

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

Email: [email protected]

Received November 16, 2013; revised December 18, 2013; accepted December 25, 2013

permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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) in-

dicate 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

1. Introduction

There are main functions of VB1 in sugar metabolism,

energy metabolism and digestive system normal work [1].

VB9 is the floorboard of the compounds which contains

pteroylglutamic acid and is well studied and separated

from spinach leaf [2]. Proteins are important and wide-

spread 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 vi-

tamins have been published including bovine serum al-

bumin [3-6] and human serum albumin [7-9], 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 dif-

ferent temperatures under physiological conditions using

UV/vis spectrophotometry and fluorescence spectrome-

try. The effects of VB1 and VB9 on the trypsin have been

evaluated and compared, such as quenching mechanism,

binding constants, binding sites, binding mode and so on.

2. Experimental

2.1. 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, Bei-

jing, China) was employed to record the absorption spec-

tra and a PHS-3C meter (Shanghai Precision Scientific

Instrument Co., Ltd China) was used to measure the pH

values of B-R buffer solutions.

VB1 solutions (1.00 × 10−4 mol/L) were prepared by

diluting 0.0094 g (337.27 Da, Sinopharm Chemical Ren-

gent Co., Ltd, Shanghai, China) in 250.00 mL of deion-

ized water. VB9 solutions (1.00 × 10−4 mol/L) were dis-

solved by diluting 0.0110 g (441.41 Da, 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.,

Y. GAO ET AL.

772

Ltd, Shanghai, China) in 250.00 mL of water. Britton-

Robinson (B-R) buffer solutions (pH = 7.90) were pre-

pared by combining a mixed acid (composed of 0.04 mol/L

of H3PO4, HAc, and H3BO 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 VB1-trypsin and VB9-

trypsin solutions so as to study the effects of electrolytes

on binding. All solutions were prepared using double-dis-

tilled, deionized water and the reagents were of analytical

reagent grade. In the experiments, a known volume stan-

dard of VB1 or VB9 solutions were added in 10.00 mL

calibrated tubes with deionized water and mixed well.

2.2. General Procedure

In 10.00 mL calibrated tubes, 1.00 mL B-R buffer solu-

tions (pH = 7.90), 1.00 mL of 1.00 × 10−4 mol/L trypsin

solutions and a known volume of the standard VB1 or

VB9 solutions were added. Then the mixture were diluted

to 10.00 mL with NaCl (0.20 mol/L) and mixed thor-

oughly by shaking. After reaction for 30 min, the solu-

tions were taken into the optical cell. The system’s fluo-

rescence spectra wavelengths were recorded from 290

nm to 450 nm and the bandwidths were 5 nm.

3. Results and Discussion

3.1. Fluorescence Quenching Spectra and

Quenching Mechanism of VB1 and VB9 with

Trypsin

Figure 1 shows that fluorescence emission spectra of

trypsin with the increasing concentrations of VB1 and

VB9 following an excitation wavelength at 281 nm. Tryp-

sin 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 VB9 led to more apparently efficient quenching of

the protein fluorescence than VB1. Such a quenching

clearly indicated the binding of VB1 and VB9 with tryp-

sin. Meanwhile, there are not a shift of maximum emis-

sion peaks, indicating that vitamin B didn’t influence the

microenvironment around trypsin. The fluorescence quen-

ching 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 me-

chanism induced by vitamin B, the Stern-Volmer equa-

tion get used to evaluate the data.

 

sv q

QKQ



  (1)

where F0 and F represent the steady-state fluorescence

intensities in the absence and presence of the quencher,

300 350 400 450

200

400

600

Fluorescence intensity

Wavelength(nm)

VB1

(a)

320 360 400440

200

400

600

Fluorescence intensity

Wavelength(nm)

VB9

(b)

Figure 1. (a) Evolution of fluorescence spectra of trypsin in

presence of VB1 with different concentrations. (b) Evolution

of fluorescence spectra of trypsin in presence of VB9 with dif-

ferent concentrations. [Trypsin] = 1.0 × 10−5 mol/L; [VB1] ×

10−5 mol/L, 1-10: 0.00, 0.50, 1.00, 1.50, 2.00, 2.50, 3.00, 3.50,

4.00, 4.50, [VB9] × 10−5 mol/L, 1-10: 0.00, 0.10, 0.30, 0.50,

0.70, 0.90, 1.10, 1.30, 1.50, 1.70, λex = 281 nm, λem = 340 nm;

pH = 7.90; T = 300 K.

respectively;





Q is the concentration of quencher;

is the Stern-Volmer quenching constant; q

is the bi-

molecular quenching rate constant and q

is equal to

0sv



; 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

and q

derived from

Equation (1) are listed in Table 1. The minimum value of

as shown in Table 1 is 8.621 × 1011 L/(mol·s),

which is greater than the maximum diffusion collision

quenching rate constant of 2.0 × 1010 L/(mol·s) [13]. So it

indicated that the fluorescence quenching process of

trypsin with VB1 and VB9 may be mainly governed by a

static quenching mechanism.

3.2. 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]:



loglog log

 (2)

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Y. GAO ET AL. 773

1.2 2.4 3.6 4.8

1.1

1.2

1.3

1.4

FO/F

[Q]( 10-5

300K

VB1

309K

(a)

0.0 0.5 1.0 1.5

1.2

1.6

2.0

2.4

FO/F

[Q]( 10-5

300K

309K

VB9

(b)

Figure. 2. The Stern-Volmer plots of the trypsin-VB1 and

trypsin-VB9 systems at different temperatures.

Table 1. Stern-Volmer quenching constants for the VB1-

trypsin and VB9-trypsin systems at pH = 7.9.

Drug T

KSV

104 L/mol R Kq

1012L/(mol·s)

VB1 300 ± 1

309 ± 1

0.9159

0.8621

0.9977

0.9159

0.8621

VB9 300 ± 1

309 ± 1

7.0920

6.0720

0.9973

0.9963

7.0920

6.0720

where b

is the binding constant and n is the number of

binding sites. The values for b

and n at different tem-

peratures can be derived in Figure 3 from the intercept

and slope of plots of

log



versus





log Q based

on Equation (2), which are listedTabl e in 2. b

shows

3.3. Thermodynamic Parameters and Nature of

The es contributing to drug-biomolecule

that there are a strong interaction and a comp forma-

tion between trypsin with VB1 and VB9. Furthermore, it

can be inferred from the values of n that there is an inde-

pendent class of binding sites on trypsin with VB1 and

VB9. But it appears that the binding constants and the

number of binding sites also increase with higher tem-

perature [15,16]. So this may be because the capacity of

VB1 and VB9 binding to trypsin is enhanced with in-

creasing temperature.

lex

Binding Mode

intermolecular forc

interactions with drugs may include hydrogen bonds,

-5.0 -4.8 -4.6 -4.4

-1.2

-0.9

-0.6

-0.3

lg[(FO-F)/F]

lg[Q]

300K

309K

VB1

(a)

-6.0 -5.5 -5.0

-1.5

-1.0

-0.5

0.0

lg[(FO-F)/F]

lg[Q ]

300K

309K

VB9

(b)

Figure 3. lg(F0-F)/F against lg[Q] at two temperatures of

Table 2. Binding constants an thermodynamic parameters

Drug 105 oln R

Kl K l

ΔS

J/(

two systems.

of VB1 and VB9 with trypsin at two temperatures.

T K ΔG ΔH

KL/m J/mo J/mo mol·K)

VB1

0. 0.300

309

0.4948

4.8340

9359

1.1820

9982

0.9981

−21.22

−21.71

195.2

721.3

VB9

300

309

5.9310

13.040

0.9908

1.0750

0.9971

0.9986

−27.41

−30.26

67.46

316.2

Van der Waals interactions, electrostatic interactions and

hydrophobic force, etc. The thermodynamic parameters

were analyzed by temperature in order to provide strong

evidence for the presence of binding forces. The value of

enthalpy change (



) and entropy change (S



) can be

determined by the n’t Hoff Equation (3), if the en-

thalpy change (



) does not vary significantly with

temperature. Thee of free energy change (G

valu



) for a

binding interaction at different temperatures cbe de-

termined by the Equation (4).

ln b

KRT R







(3)

ln b

GRTK



 (4)

where b

is the binding constant, R is the gas constant

and T is the absolute temperature. The values of



and G



are listed in Table 2. A negative valf ue o



reveals that the binding process is spontaneous.

ophobic interactions play main roles in the binding

between trypsin with VB1 and VB9 because the values of

Hydr



and S



are positive.

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3.4. Synchronous Fluorescence Spectroscopy

sed Synchronous fluorescence spectroscopy is usually u

to investigate the microenvironment around the fluoro-

phore functional groups. At





= 60 nm, the synchro-

nous fluorescence spectra arttributed to tryptophan,

while

e a



 = 15 nm, the spectra are attributed to tyrosine.

Synchrus fluorescence spectra of trypsin with addi-

tion of VB1 are shown in Figure 4(a) and these with ad-

dition of VB9 are shown in Figure 4(b). From Figure 4,

the emission maxima have no shifts with regards to VB1

and VB9, which indicates that there was no change of the

microenvironment of the tryptophan and tyrosine.

ono

3.5. Energy Transfer and Binding Distance

with The fluorescence quenching of trypsin after binding

VB1 and VB9 indiactes that the tranfer of energy has oc-

curred. According to Förster’s resonance energy transfer

theory [17], the distance between two interacting mole-

cules and the efficiency of energy transfer can be dis-

cribed by the following equation:

EFRR

  (5)

where E is the energy transfer efficiency, F is the fluor-

scence intensity of the donor in the presence of equal

amounts of accepter, F0 is the fluorscence intensity of the

donor in the absence of equal amounts of accepter, R0 is

280 320 360

150

300

450

nm

Flourenscence Indencity

Wavelength(nm)

nm

VB1

(a)

300 350 400 450

150

300

450

nm

nm

Flourenscen c e Indencity

Wavelength(nm)

VB9

(b)

Figure 4. (a) Evolution of syronous fluorescence spectra

absence and presence of VB9, T = 300 K.

nch

of trypsin in the absence and presence of VB1; (b) Evolu-

tion of synchronous fluorescence spectra of trypsin in the

the critical distance, and R is the distance between ac-

ceptor and donor. The quantity 6

R is

0calculated by the

following equation:

6254

2

8.8 10KN



 (6)

where K2 is the spatial orientation factor of the dipole, N

is the refractive index of the medium, Φ is the fluores-

cence quantum yield of the donor, J is the overlap inte-

gral of the fluorescence emission spectrum of the donor

and the absorption spectrum of the acceptor. J is calcu-

lated using the equation:





















 (7)

where F(λ) is the fluorescence intensity of the fluores-

cence 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 K2 = 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 VB1 and VB9.

From the above relationships, J = 3.79× 10−19 cm3·l·mol−1,

R0 = 0.45 nm, E = 0.79 and R = 0.55 nm for trypsin and

VB1. Similarly, J = 7.96 × 10−17 cm3·l·mol−1, R0 = 0.87

nm, E = 0.23 and R = 0.87 nm for trypsin and VB9. The

distance R < 8 nm between donor and acceptor indicates

that the energy transfer from trypsin to VB1 and VB9

occurred with high possibility. This obeyed the condi-

tions of Förster energy transfer theory.

300 330 360 390

0.00

0.01

0.02

Wavelength( nm)

Abosorbance

VB1

100

200

300

400

Fluroscence Inensity

(a)

300 330 360 390

0.0

0.3

0.6

0.9

Wavelength( nm)

Abosorbance

VB2

100

200

300

400

Fluroscence Inensity

(b)

Figure 5. Spectral overlap een fluorescence spectrum

of trypsin (B) and absorbanpectrum of VB (A) and

betw

ce s1 1

VB9 (A2) at 300 K; [trypsin] = [VB1] = 5.0 × 10−5 mol/L,

[trypsin] = [VB9] = 5.0 × 10−5 mol/L.

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nteractions between two kinds of vita-

4. Conclusion

In this paper, the i

min B and trypsin have been investigated under simu-

lated physiological conditions using spectrometries. The

fluorescence of trypsin was quenched by two kinds of

vitamin B mainly through static quenching. The enthalpy

change (

) and entropy change (S) for the systems

were calculated respectively. The positive



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 signifi-

cance in pharmacology and clinical medicine.

5. Acknowledgements

This work was supported by the Natural Science Founda-

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