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
Received November 16, 2013; revised December 18, 2013; accepted December 25, 2013
Copyright © 2013 Yan Gao et al. This is an open access article distributed under the Creative Commons Attribution License, which
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.
 
0
0
11
sv q
F
K
QKQ
F
   (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)
1
10
VB1
(a)
320 360 400440
200
400
600
Fluorescence intensity
Wavelength(nm)
1
10
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;
s
v
K
is the Stern-Volmer quenching constant; q
K
is the bi-
molecular quenching rate constant and q
K
is equal to
0sv
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
s
v
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 × 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]:

0
loglog log
b
FF
K
nQ
F
 (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 M)
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 M)
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
K
KSV
104 L/mol R Kq 1012L/(mol·s)
VB1 300 ± 1
309 ± 1
0.9159
0.8621
0.9977
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
K
is the binding constant and n is the number of
binding sites. The values for b
K
and n at different tem-
peratures can be derived in Figure 3 from the intercept
and slope of plots of
0
log
F
F
F

versus


log Q based
on Equation (2), which are listedTabl e in 2. b
K
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.
d
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
195.2
721.3
721.3
VB9
300
309
5.9310
13.040
0.9908
1.0750
0.9971
0.9986
−27.41
−30.26
67.46
67.46
316.2
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 (
H

) and entropy change (S

) can be
determined by the n’t Hoff Equation (3), if the en-
thalpy change (
va
H

) 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).
an
ln b
H
S
KRT R



(3)
ln b GRTK

 (4)
where b
K
is the binding constant, R is the gas constant
and T is the absolute temperature. The values of
H

,
S

and G

are listed in Table 2. A negative valf ue o
G

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
H

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:
6
0
6
00
1R
6
EFRR   (5)
where E is the energy transfer efficiency, F is the fluor-
F
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
0
150
300
450
nm
Flourenscence Indencity
Wavelength(nm)
1
10
1
10
nm
VB1
(a)
300 350 400 450
0
150
300
450
nm
nm
1
10
10
Flourenscen c e Indencity
Wavelength(nm)
1
VB9
(b)
Figure 4. (a) Evolution of syronous fluorescence spectra
absence and presence of VB9, T = 300 K.
J
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
0
R
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:



4
F
JF

 

 

 (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
B
A1
100
200
300
400
Fluroscence Inensity
(a)
300 330 360 390
0.0
0.3
0.6
0.9
Wavelength( nm)
Abosorbance
B
A2
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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Y. GAO ET AL.
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775
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 (
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 signifi-
cance in pharmacology and clinical medicine.
5. Acknowledgements
This work was supported by the Natural Science Founda-
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