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DOI: 10.1039/C9FO00183B
Food & Function

ARTICLE

Molecular mechanism of interaction between resveratrol and

trypsin by spectroscopy and molecular docking

Food & Function Accepted Manuscript

Received 00th January 20xx, a a a a a a
Guoyan Ren *, He Sun , Jinying Guo , Jinling Fan , Gen Li , Saiwen Xu

Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Published on 07 May 2019. Downloaded on 5/8/2019 2:47:27 AM.
Abstract Molecular mechanism of interaction between resveratrol and trypsin was studied using fluorescence
spectroscopy (intrinsic fluorescence, synchronous fluorescence, Three-dimensional fluorescence), ultraviolet-visible (UV-
vis) spectroscopy, fourier transform infrared (FT-IR) spectroscopy and molecular docking method, as well as enzyme
activity and antioxidant assay. The fluorescence experiments (The Stern−Volmer quenching constants (K sv )) indicated
that resveratrol quenched the intrinsic fluorescence of trypsin through the static quenching mechanism. The number of

www.rsc.org/ binding site was about one. The thermodynamic functionsΔG<0 andΔH>0，ΔS>0 of the binding process, which
indicated that combination of resveratrol and trypsin process was spontaneous exothermic reaction and hydrophobic
effect was the main force between them. Uv-vis spectra, synchronous fluorescence spectra and three-dimensional
fluorescence spectra analysis showed that combination of resveratrol and trypsin induced changes in the
microenvironment around fluorophores of trypsin, resulting in alteration in the spatial structure of trypsin. FT-IR
spectroscopy indicated that the contents of α-helix and β-turn in trypsin were decreased and the contents of β-sheet,
random coil and antiparalled β-sheet were increased. All these experimental results were verified and reasonably
explained by molecular docking result. Upon resveratrol and trypsin binding, the enzyme catalytic activity of trypsin and
antioxidant of resveratrol were decreased. Results from this study should be useful to elucidate molecular mechanisms of
interaction between resveratrol and trypsin and contribute to make full use of resveratrol in the food industry.

Keywords Spectroscopy ； Molecular docking ； Resveratrol ； Trypsin ； Interaction

Introduction digestion and absorption of organisms, affecting the activities

Resveratrol (RES) was a non-flavonoid polyphenol
compound. It was widely found in many plants such as the
grape family and the lily family. With a variety of biological
activities and pharmacological effects, it was a natural active
substance having significant impacts on human health [1]. RES
molecule (C14H12O3, 3,5,4'-trihydroxydistyrene) had cis- and
trans- structure (Fig. 1), cis-RES structure could be converted
into trans-RES structure under heating or ultraviolet radiation.
Therefore, trans-resveratrol was more stable and functional
than cis-resveratrol [2]. Studies had shown that RES had many
biological functions, such as antioxidant [3], cancer prevention
[4-6], anti-inflammatory and cell protection [7], cardiovascular
disease prevention [8], inhibition of apoptosis and autophagy
in cerebral ischemia/reperfusion cells [9], anti-aging and
immunity improvement [10]. The functional activity of RES was
mainly achieved through its digestion, absorption and
metabolism in the digestive tract. However, a large number of
studies had shown that polyphenols interacted with digestive
enzymes (such as pepsin, trypsin, etc.) in the process of
of polyphenols and digestive enzymes [11-12]. Therefore, it
was necessary to study the interaction mechanism between
polyphenols and digestive enzymes.
Trypsin (EC3.4.21.4), as a typical serine protease, was a
soluble globulin and digestive enzyme in the pancreas. It
consisted of 223 amino acids with a molecular weight of
23.3kDa. Four tryptophan residues (Trp), ten tyrosine residues
(Tyr) and six phenylalanine residues (Phe) were its intrinsic
fluorescence groups [13]. In the three-dimensional structure of
bovine trypsin, it was composed of two domains of similar size
which connecting by six disulfide bonds. Each structural
domain consists of six antiparallel β-sheet [14]. Like other
serine proteases, the catalytic activity of trypsin was affected
by two sites: one was the catalytic site consisting of His57, Asp
102 and Ser 195, and the other was the primary substrate
binding site (S1 binding pocket) containing 189-195, 214-220
and 225-228 residues [15]. In addition to digestion and
deconstruction of food proteins, trypsin also played an
important role in other physiological processes such as
apoptosis, hemostasis, signal transduction, reproduction and
immune response [16]. Phenolic compounds (including RES)
extracted from plants could inhibit the activity of trypsin [17].
However, the mechanism of alone RES inhibiting trypsin
activity had not been reported. So it was important to

This journal is © The Royal Society of Chemistry 20xx J. Name., 2019, 00, 1-12 | 1
 Food & Function
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understand the mechanism of interaction between RES and
trypsin. It could provide some important theoretical
information for the metabolism, distribution and biological
activity changes of RES in vertebrates (including humans).
In this paper, fluorescence spectrometry, ultraviolet
spectroscopy, FT-IR spectroscopy and molecular docking
method, as well as enzyme activity and antioxidant assay
techniques were used to detect interaction between RES and
trypsin, which provided important information for elucidating
the mechanism of interaction between RES and trypsin.
Fig.1 Structures of resveratrol
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Experiment
Chemicals
Bovine trypsin (T105531) purchased from Aladdin
Reagent Co.Ltd (Shanghai, China) was used directly without
further purification. Resveratrol (A506244) was purchased
from Sangon Biotech Co.Ltd (Shanghai, China). All other
reagents were of analytical pure grade, the deionized water
used in this work was passed through a Milli-Q purification
system. Tris-HCl buffer solution with concentration of 0.05
-1
mol· L and pH value of 7.4 was prepared. 0.600 g trypsin was
-1
dissolved in 100 mL of 0.05 mol·L Tris-HCl buffer (pH 7.4) to
-4
form trypsin stock solution with the concentration of 2.5×10
-1
mol·L . All above stock solutions were stored at 4 °C.
UV–visible absorption spectra
Absorption spectra were recorded in the range of 185-
700 nm using UV-2600 UV-visible spectrophotometer （
Shimadzu Corporation，Japan） with a 1.0 cm quartz cell.
Spectra analysis was carried out with the software in the
-4 -1
instrument. Pipette 375 μL of 2.5×10 mol·L trypsin stock
solution fixed volume to 5 mL using Tris-HCl buffer solution
(pH 7.4) to obtain a trypsin test solution with a molar
-5 -1
concentration of 1.875×10 mol·L . 93 μL resveratrol stock
-3 -1
solution (1.0×10 mol·L ) was taken and adjusted to 5 mL with
Tris-HCl buffer solution (pH 7.4) to obtain resveratrol sample
-5
solution having a molar concentration of 1.875×10 mol·L .
The RES+trypsin mixture sample solution was obtained by
-4 -1
adding 375μL 2.5 ×10 mol·L trypsin stock solution and
-3 -1
1.0×10 mol·L resveratrol stock solution of 93 μL into a 5mL
volumetric flask and using Tris-HCl buffer solution (pH7.4) for a
constant volume (the molar concentration ratio of RES and
trypsin in the system was 1:1). After sufficiently mixed, the
RES+trypsin complex solution were allowed to stand for 30min
at room temperature before detection. Using the Tris-HCl
solution as a reference, the absorption spectra of trypsin
2 | J. Name., 2019, 00, 1-12
Page 2 of 15
Journal Name
solution, RES+trypsin complex solution and RES solution
View ArticleinOnline
the
range of 185-700 nm were scanned. DOI: 10.1039/C9FO00183B
Fluorescence spectroscopic measurements
-4 -1
Trypsin stock solution (2.5×10 mol·L ) was diluted to
-5 -1
2.5×10 mol·L by Tris-HCl buffer solution for fluorescence
spectroscopic measurements. Fluorescence spectra of all
trypsin solutions under simulated physiological conditions (pH
7.4) in the absence and presence of RES were recorded using a
Cary Eclipse fluorescence spectrometer (Agilent, USA)
Food & Function Accepted Manuscript
equipped with a 1.0 cm quartz cell at three different
temperatures (298 K, 304 K, and 310 K). The excitation
wavelength was set to 280 nm. The slit width of excitation and
emission were both set to 5 nm. The steady-state fluorescence
spectra were recorded in the wavelength range of 300-500
nm. In all fluorescence detection experiments, the trypsin
-5 -1
concentration was maintained at 2.5×10 mol·L , and
different doses of RES were added to the trypsin solution to
form a concentration gradient of 0, 0.625, 1.250, 1.875, 2.500,
-5 -1
3.125, 3.750, 4.325, 5.000, 5.625×10 mol·L .
All fluorescence intensities were corrected in this study
based on the following relationship [18]:
( )
(1)
Fcorr and Fobs were the fluorescence intensity corrected
and observed respectively, ODex and ODem were the
fluorescence absorption intensity of the system at excitation
and emission wavelengths, respectively.
Synchronous fluorescence measurements
The synchronous fluorescence spectra of trypsin solution
in the absence and presence of RES were recorded using a
Cary Eclipse fluorescence spectrometer (Agilent, USA)
equipped with a 1.0 cm quartz cell at the region of 200-500 nm
(at 298K). Simultaneously, the excitation and emission spectra
of constant wavelength differences ∆λ=15 nm (for tyrosine)
and ∆λ=60 nm (for tryptophan) were scanned with 5/5 nm slit
width. All the solution concentration and fluorescence
intensities calculation method were the same as the
fluorescence spectra experiment.
Three-Dimensional fluorescence measurements
Three-dimensional fluorescence spectra of trypsin
−5 −1
(2.5×10 mol·L ) and RES+trypsin complex (1:1 molar ratio)
were collected using a Cary Eclipse fluorescence spectrometer
-1
(Agilent, USA) equipped with a 1.0 cm quartz cell at room
temperature (298K). The excitation and emission wavelength
range was set to 200-400 nm and 200-500 nm, respectively.
The slit width of excitation and emission were both fixed at 5
nm.
FT-IR measurements
FT-IR spectra of trypsin in the presence and absence of
RES were recorded using a VERTEX 70 FT-IR spectrometer
This journal is © The Royal Society of Chemistry 20xx
 Page 3 of 15 Food & Function
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(Bruker, Germany) with calcium fluoride (GaF2) film. RES
-5 -1
solution (5×10 mol·L ) was prepared in Tris-HCl buffer at pH
7.4. Complex solution of RES and trypsin were prepared at 1:1
molar ratio and incubated at room temperature for 15 min.
Under the same conditions as the sample determination, the 4
-1
cm resolution was scanned for 1000 times to collect the
water vapor spectra. OMNIC8.2 data processing software was
used to automatically perform water vapor correction, and the
background was collected in the open state at room
temperature. Then, the samples were uniformly spread on the
GaF2 film, and the spectra of the samples were scanned in the
-1
spectral range of 2000-1100 cm for 600 times under the
-1
resolution of 4 cm .
-1
A smooth straight line between 1800 and 2200 cm was
taken as the criterion for the spectra of the samples solution
minued the buffer solution without samples. The subtracted
spectra were corrected for two baselines within the spectral
-1
band range 1600-1700 cm (amide band Ⅰ). After smoothing
with 9-point, the second derivative and Fourier deconvolution
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were performed to estimate the peak position and half-peak
width of each sub-peak. The peak position and peak width of
each sub-peak were selected manually. The spectrum was
fitted with Gausse function, and the different spectral bands
overlapping the minimum residuals were completely
distinguished by multiple fitting. When the corresponding
relationship between each sub-peak and different secondary
structures was determined. The relative percentage content of
various secondary structures was calculated according to their
integral area.
Molecular docking studies
The crystal structure of bovine trypsin was obtained from
PDB Entry (PDB Entry: 2ZQ1). After removing all the crystal
water, the structure was directly used as the main body in the
molecular docking process. The ligand molecular structure
(PDB ID: STL) was obtained directly from the protein database.
The simulation of molecular docking was performed by using
the Autodock 4.2 program. The Gird was selected as
120×126×126 Å, and the grid spacing was 0.375 Å, ensuring
sufficient RES accessible space. RES was set as flexible
molecule docking with rigid molecule trypsin. The results were
optimized by Lamarckian Genetic Algorithm (LGA), and several
important parameters in the Algorithm were set as follows:
the running times was 1000, energy evaluation was 2500000,
maximum number of generations was 27 000, the population
size 150 by default. Finally, the RES-trypsin complex with the
minimum binding energy was obtained by cluster analysis of
the molecular docking results with a root mean square 2.0 Å
tolerance. The software PyMol (version 1.5.0.3) was used to
visualize the docking conformation and calculate the distance
between possible hydrogen bond pairs.
Trypsin activity measurement
The catalytic activity of trypsin was determined by a
modified Lowry assay [19]. The specific methods of operation
This journal is © The Royal Society of Chemistry 20xx
ARTICLE
were as follows: Different volumes of RES stockView solution (0,
Article Online
125, 250, 375, 500, 625, 750, 875, 1000 DOI:
μL), 10.1039/C9FO00183B
casein solution (1
-1
mL 10.0 g.L ) and 500 μL trypsin stock solution were added to
a 5 mL volumetric flask and dilute to volume with Tris-HCl
buffer (pH 7.4) to obtain different samples. The above samples
were incubated at 40 ° C for 10 minutes. The hydrolysis
-1
reaction was terminated by adding 2 mL of 0.4 mol·L
trichloroacetic acid solution to each sample. The mixture was
kept at 40 ° C for 20 minutes, and the precipitate was removed
-1
by filtration. 1 mL of the supernatant, 5 mL of 0.4 mol·L
Na2CO3 and 1 mL of Folin-phenol reagent were mixed, colored
Food & Function Accepted Manuscript
at 40 ° C for 20 minutes, and after cooling to room
temperature, the absorbance of the mixture was measured at
a wavelength of 680 nm. Each experiment was carried out in
triplicates. The specific enzyme activity could be calculated by
the following formula:
( ) ( ) ( )
Where A and a were the enzyme activities in the absence
and presence of RES, respectively.
Antioxidant assay
In this experiment, Superoxide anion radicals scavenging
rate and DPPH (1,1-diphenyl-2-picrylhydrazyl) radical
scavenging rate were used as two indicators of antioxidant
activity.
The scavenging activity of superoxide anion radicals was
determined according to the method of B.A.M. Rocha et al.
-3 -1
[20]. Briefly, 100, 200, 300, 400, 500 μL of 1.0×10 mol·L RES
methanol solution was taken to prepare a sample of RES
-4 -1
scavenging superoxide anion radical. 50 μL of 2.5×10 mol·L
trypsin solution was added to the above sample to prepare a
sample of RES+trypsin scavenging superoxide anion radical.
Pipette 1.0 mL of the above sample solution, adding 0.4 mL of
−1
25 mmol·L pyrogallol solution to the screw seal tube, and
−1
adding 4.5 mL Tris-HCl buffer solution (pH 8.2, 50 mmol·L )
-1
and mixing uniform. 1.0 mL of hydrochloric acid (8 mmol·L )
was added to stop the reaction. The absorbance of the
reaction mixture to the blank reagent was measured at 299
nm. Each experiment was carried out in triplicates. Superoxide
anion radical scavenging rate (SARSA) was calculated by the
following formula(3):
( ) ( ) (3)
Where A0, AS, and AS0 were the absorbance values
determined at 299 nm of the blank, the sample and the
reagent blank, respectively.
DPPH was determined according to the method described
by V. Rahul et al. and slightly modified [21]. The specific
-4 -1
experimental methods were as follows: 2.0 mL 1×10 mol·L
-3 -1
DPPH methanol solution was mixed with 1.0×10 mol·L RES
methanol solution of different volume (100, 200, 300, 400, 500
μL). After 30 minutes of avoiding light at room temperature,
the absorbance was measured at 515 nm, and the scavenging
effect of different concentrations of RES on DPPH free radicals
-4 -1
was detected. 50 μL of 2.5×10 mol·L trypsin solution was
added to the above solution, and the scavenging effects of
different concentrations of RES+trypsin on DPPH free radicals
J. Name., 2019, 00, 1-3 | 3
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ARTICLE Journal Name

were detected according to the same detection method. Each
experiment was carried out in triplicates, and scavenging
effect of DPPH was calculated by the following formula(4):
( ) ( )
(4)
Where AS was the sample absorbance (DPPH solution with
RES or RES+trypsin). A0 was blank absorbance (solution
without DPPH and sample). A was the control absorbance
(DPPH solution without sample).
quenching would increase with the increasing of temperature.
View Article Online
The combination of dynamic quenchingDOI:and10.1039/C9FO00183B
static quenching
caused by simultaneous collision and complex formation in the
same quenching medium. For both dynamic and static
quenching, the dependence of F0/F on [Q] was linear.
However, for the combination of dynamic and static
quenching, F0/F dependence on [Q] was characterized by

Results and discussion

Food & Function Accepted Manuscript

Fluorescence quenching of trypsin by RES
Fluorescence spectroscopy was an effective method for
studying the interaction of small molecules and proteins, and
could provide a wealth of information about protein
conformation [22]. In fact, the intrinsic fluorescence of trypsin
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was almost composed of 4 Trp and 10 Tyr residues. These

residues could act as intrinsic fluorophore to provide
information on ligand-trypsin interactions and ligand-induced
conformational changes. Fig. 2A depicted the fluorescence
emission spectra of trypsin in the absence and presence of RES
at different concentrations at 298K. It could be seen that the
fluorescence intensity of trypsin gradually decreased with the
increasing of RES concentration, and the maximum emission
peak (342nm) showed significant red-shift. This indicated that
the specific biochemical reaction between RES and trypsin was
formed after the addition of RES. RES was bound to the
substrate binding site of trypsin or formed directly non-
covalent interaction with aromatic Trp and Try residues, which
brought about quenching the fluorescence of trypsin [23].
The fluorescence quenching mode were classified as static
quenching, dynamic quenching and combination of dynamic
quenching and static quenching based on different quenching
mechanisms[24]. These quenching mechanisms could be
distinguished by the dependence of their different constants
on temperature and viscosity, or more precisely by the
variation of their fluorescence lifetime. The static quenching
caused by a formation ground state complex of protein with
quenchers. In the case of static quenching, the stability of the
complex decreased with the temperature increasing.
Therefore, the value constant (K SV) of the static quenching
decreased with the increasing of temperature. The dynamic
quenching caused by the collision of protein and quenchers. In
the process of dynamic quenching, the diffusion coefficient
increased and electron transfer promoted with the increasing
of temperature. The value constant (K SV) of the dynamic

Table 1 Quenching rate constants and correlation coefficients of RES-trypsin.

Ksv Kq
T/K R2
(104 L·mol-1) (1012 L·mol-1·S-1)
298 3.3683±0.0944 3.3683±0.0944 0.9904

304 3.0466±0.0689 3.0466±0.0689 0.9901

310 2.9280±0.1070 2.9280±0.1070 0.9960

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 Page 5 of 15 Food & Function

Journal Name ARTICLE

bending upward and concave to the Y-axis [25]. The analysis of increasing temperature indicated that weak bound
View Article Online
the quenching mechanism could deeply understand the nature complexation were formed between DOI: RES10.1039/C9FO00183B
and trypsin. In
12 -1 -1
of the interaction between RES and trypsin, as well as the addition, the value of Kq (>10 L·mol ·s ) was much larger
parameters of quenching constant (K sv) and binding affinity than the maximum diffusion collision quenching temperature
10 −1 −1
(Kq) during the interaction process. Therefore, in order to constant (2.0 × 10 m ·s ) [27]. These results indicated that
examine the quenching mechanism involved in RES+trypsin the quenching of RES to intrinsic fluorescence of trypsin was
complex, temperature-dependent fluorescence experiments static quenching mechanism.
were carried out at 298K, 303K and 308K. The classical stern-
volmer relation (Eq 5) was used to analyse the emission data
obtained. The results obtained from the linear Stern-Volmer Binding constants and the number of binding
plot (Fig 2B) were listed in table 1 [26]: sites

Food & Function Accepted Manuscript

(5)
Binding constants and binding sites could be calculated
It could be seen from Fig. 2B that the dependence of F0/F
from the slope and intercept data of the plot between lg[(F 0-
on [Q] was linear in the range of RES concentration in the
F)/F] and lg[Q] by using double logarithmic equation(6)(Fig.2C)
experiment, indicating that the quenching mechanism of RES
and the results obtained were listed in Table 2 [28-29]:
on trypsin might be dynamic quenching or static quenching.
( ) (6)
The results in Table 1 showed that Ksv values of RES+trypsin
Where K was the binding constant, n was the number of
interaction were 3.37, 3.04 and 2.92 at three different
binding sites, and [Q] was the quencher concentration.
temperatures, respectively. The Ksv value decrease with
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 Food & Function Page 6 of 15

ARTICLE Journal Name

4 -1
It could be seen that the K value was about 10 M , which der Waals forces, hydrophobic interactions and hydrogen
View Article Online
suggested binding affinity of the interaction between RES and DOI: be
bonds [32]. The intermolecular force could 10.1039/C9FO00183B
determined by
6
trypsin was moderate compared to strong binding affinity (10 - thermodynamic parameters such as entropy change (ΔS),
8 -1
10 M ) of other protein-ligand [30]. At the studied enthalpy change (ΔH), and Gibbs free energy change (ΔG).
temperature, the value of n was about 1, which might indicate When the temperature did not change much, we could use the
the existence of one binding site of RES in trypsin. RES was van't Hoff equation （Eq7-8）to calculate ΔH and ΔS as shown
likely to bind near the Trp and/or Tyr residues (intrinsic below [33].
fluorophore of trypsin) and caused the fluorescence quenching (7)
of trypsin [31]. (8)
Where T was the experimental temperature, R was the
gas constant, and K was the association constant at the
Thermodynamic parameters and binding mode

Food & Function Accepted Manuscript

corresponding T. ΔH and ΔS were obtained from the slope and
The interaction forces between small molecule substrates intercept of the curve, which plots lnK versus 1/T according to
and biomacromolecules include electrostatic interactions, van Equation (7).
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Table 2 Binding constants, number of binding sites and thermodynamic parameters for RES binding into trypsin.

T/K K N R2 ΔG ΔH ΔS
(105 L·mol-1) (kJ·mol-1) (kJ·mol-1) (J·mol-1·K-1)
298 0.9077±0.0284 1.1110±0.0759 0.9920 -(28.2842±1.8455)
304 1.8444±0.0167 1.1916±0.0229 0.9967 -(30.6456±0.5974) +(80.1519±3.6904) +(364.0723±1.2778)
310 3.1725±0.0335 1.2483±0.0700 0.9987 -(32.6483±1.8621)

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Fig. 4 The three-dimensional fluorescence contour spectra of trypsin (A), RES+trypsin systems (B), the projection spectra of trypsin (C),
RES+trypsin systems (D). C(trypsin)= 2.5×10-5mol·L-1, C(RES) =2.5×10-5mol·L-1. T=298K
The resultant thermodynamic parameters were listed in
Table 2. ΔG < 0 indicated that RES and trypsin binding process
was a spontaneous process. The negative ΔH suggested that
RES binding to trypsin forming complexation was an
exothermic process. According to the theory of Ross and
Olsson, when ΔH<0 and ΔS<0, hydrogen bond and van der
Waals force were dominant, while when ΔH> 0 and ΔS>0,
hydrophobic interaction played a major role in the binding
reaction. In addition, the electrostatic interaction was the main
driving force when ΔH<0 and ΔS>0 [34+. The values of ΔH and
-1
ΔS during the binding were +80.1519 kJ·mol and +364.0723
-1 -1
J·mol ·K , respectively, indicating that the interaction
between RES and trypsin was mainly attributed to hydrophobic
forces.
Synchronous fluorescence spectra
Synchronous fluorescence spectra provided information
about the molecular microenvironment near the functional
fluorophore. When the wavelength interval (Δλ) excitation and
emission wavelength was 15 nm and 60 nm respectively, the
spectra provided characteristic information of the tyrosine
(Tyr) residue and tryptophan (Trp) residues information. The
shift in the position of the maximum fluorescence emission
This journal is © The Royal Society of Chemistry 20xx
Food & Function
ARTICLE
View Article Online
DOI: 10.1039/C9FO00183B
Food & Function Accepted Manuscript
peak corresponded to alteration in polarity around the
fluorophore [35]. After adding different concentrations of RES,
the synchronous fluorescence spectra of trypsin was showed
in Fig. 3. As the concentration of RES increasing, the
fluorescence intensity of trypsin decreased gradually and the
position of the maximum emission wavelength was
significantly red-shifted from 294 nm to 296 nm at Δλ= 15 and
from 278 nm to 287 nm at Δλ = 60 nm. The decrease of
intensity of Tyr and Trp residues and the red-shift of peaks
suggested that RES might bind trypsin. Intervention of
exogenous substance RES leaded to the destruction of the
natural tertiary structure of trypsin. Tyr and Trp residues
originally surrounded by non-polar amino acids were exposed,
and their environment changed from weak polarity to more
polarity [36].
Three-dimensional fluorescence spectroscopy
Three-dimensional (3D) fluorescence spectroscopy could
provide more scientific and reliable details of protein structure
and microenvironment changes. Fig.4 showed the 3D
fluorescence spectra of native trypsin and RES+trypsin
complexes, respectively. The native trypsin displayed four
characteristic peaks: peak a (λex = λem) and peak b (2λex =
J. Name., 2019, 00, 1-3 | 7
 Food & Function
ARTICLE
λem) represented first and second order Rayleigh scattering
peaks respectively. Peak 1 (λex/λem = 270 / 352 nm) was
attributed to the π-π* transition of aromatic amino acids in
trypsin, being the intrinsic fluorescence spectral behaviour of
Trp and Tyr residues. Peak 2 (λex/λem: 235/340 nm) resulted
from the excitation of higher excited electronic states of the
aromatic (Trp and Tyr) residues of protein. Therefore, when
excited at 280 nm, peak 2 only comed from the local
environment of Trp and Tyr aromatic residues, and its
variation should be similar to that of peak 1 [37].
Fig. 4 A, B, C and D clearly described the change of 3D
fluorescence behavior of native trypsin upon binding with RES.
The intensity of peak 1 (λex/λem =270/352 nm) decreased
significantly from 219.8 (Fig. 4A) to 145.7 (Fig. 4B) with a
significant red-shift from 340 nm to 352 nm. The intensity of
peak 2 (λex/λem=240/336 nm) decreased significantly from
105 (Fig. 4A) to 65.7 (Fig. 4B) with a slight red-shift from 336
nm to 338 nm. The fluorescence intensity of peak 1 and peak 2
indicating that the polarity increased near the Trp and Tyr
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microenvironment [38]. These results were consistent with the
steady-state fluorescence and synchronous fluorescence.
UV–vis absorption spectra
UV-vis absorption spectra could be used to explore the
changes in the microenvironment around the chromophore of
proteins and to study the formation of protein ligand
complexes [39]. Fig. 5 showed the absorption spectra of RES
(curve a), trypsin (curve b), RES+trypsin (curve c). The curve d
was obtained by subtracting the RES spectra curve (a) from the
RES+trypsin curve (curve c). As could be seen from Fig. 5, the
spectra of trypsin had two absorption peaks. The strong
absorption peak at 219 nm reflected the framework
conformation of the protein. The weak absorption peak at 289
nm was due to the absorption of ultraviolet by aromatic amino
acids (major of Trp, Tyr and minor of Phe), and was often used
to reflect changes in protein conformation [40]. If there was no
interaction between trypsin and RES, the spectra of trypsin
8 | J. Name., 2019, 00, 1-12
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Journal Name
and [(RES+trypsin)-RES] should be the same. By Viewcomparing
Article Online
DOI: 10.1039/C9FO00183B
curve b and curve d in Fig. 5, it could be seen that the shape of
absorption peak at approximately 219nm was changed slightly,
meanwhile the absorption peak 289 nm increased slightly and
the red-shift occurred after the interaction of RES. This
suggested that RES and trypsin formed new complexation. The
addition of RES affected the conformation of trypsin, changed
the microenvironment around the aromatic amino acid (Trp,
Tyr) residues of trypsin and exposed the chromophore of
trypsin to the molecular surface. These results were consistent
with the experimental results of steady-state fluorescence
Food & Function Accepted Manuscript
spectroscopy, simultaneous fluorescence and three-
dimensional fluorescence results.
FT-IR measurements
The effect of RES on the conformational changes of
trypsin was further studied by infrared spectroscopy. FT-IR
spectra of the protein showed many amide bands representing
different vibrations of the peptide portion [41]. Among all
-1
protein amide bands, the amide I band of 1600-1700cm and
-1
the amide II band of 1500-1600cm had important relations
with the secondary structure of proteins. The amide I raised
mainly from C=O stretch, minor contribution from the C–N
out-of-phase stretching vibration, the C–C–N deformation and
the N–H in-plane bending vibration. The amide II band
assigned to mainly out-of phase combination of the N–H in-
plane bending vibration and the C–N stretching vibration and
smaller contribution of the C=O in-plane bending vibration and
-1
the C–C and C–N stretching vibration at near 1550 cm [42].
However, the amide I band was more sensitive to the change
of the secondary structure of protein than amide II band,
which had been widely used in protein conformation studies
[43]. Therefore, in order to further clarify the effect on the
secondary structure of trypsin after binding RES, the FT-IR
spectra of trypsin amide I band in the presence or absence of
RES were monitored. The curve-fitting procedures were shown
in the Fig. 6. Generally speaking, the peak positions of amide I
-1
band of proteins in the spectral region of 1600-1700 cm were
mainly composed of secondary structure β-sheets (1610-1640
-1 -1 -1
cm ), random coils (1640-1650 cm ), α-helix (1650-1660 cm ),
-1
β-turn (1660-1680 cm ) and antiparalled β-sheet (1680-1690
-1
cm ) [44]. All peaks in Fig. 6 were processed with self-
deconvolution with second derivative resolution
enhancement, and the results were shown in Table 3. The
results showed that native trypsin contained 17.81% α-helix,
27.45% β-sheet, 26.85% β-turn, 19.58% random coil and 8.31%
antiparalled β-sheet. Upon RES interaction, a major decrease
of α-helix from 17.81% to 10.61%,β-turn from 26.85% to
14.43% with an increase in the β-sheet content from 27.45% to
Table 3 The influence of RES on the secondary structure of trypsin
System α-helix Antiparalled β-sheet β-turn Random
(%) β-sheet (%) (%) (%) coil (%)
Free trypsin 17.81 8.31 27.45 26.85 19.58
*RES+trypsin+-RES 10.61 11.48 41.13 14.43 22.35
This journal is © The Royal Society of Chemistry 20xx
 Page 9 of 15 Food & Function
Journal Name
a a
b b
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41.13%, random coil from 19.58% to 22.35% and antiparalled
β-sheet from 8.31% to 11.48% were observed. The decrease of
α-helix and β-turn content and the increase of β-sheet,
random coil and antiparalled β-sheet content in the secondary
structure of trypsin indicated that the secondary structure of
trypsin became loose and stretch, and the exposure of polar
groups resulted in the enhancement of protein polarity and
the weakening of hydrophobicity. These results validated the
results of fluorescence experiments and infrared experiments.
-1
The infrared spectra region 1500-1100 cm corresponded to
the symmetric and asymmetric bending vibrations of the CH 2
and CH3 functional groups and had been used to characterize
the presence of hydrophobic contacts in protein-ligand
complexation [45-46]. Fig.6 (B) illustrated the frequency of
vibration of native trypsin before and after interaction with
-1
RES in the region of 1500-1100 cm . The results showed that
the positions of the vibrational peak of the CH2 and CH3 groups
of trypsin changed after complexation with RES, indicating that
there was hydrophobic force in the interaction between
trypsin and RES [47].
Molecular docking
To understand more about the binding of RES to trypsin,
molecular docking studies were performed. The stereo view of
the best energy ranked result of the binding mode between
This journal is © The Royal Society of Chemistry 20xx
ARTICLE
View Article Online
DOI: 10.1039/C9FO00183B
Food & Function Accepted Manuscript
RES and trypsin was shown in Fig. 7. As shown in Fig. 7C and
Fig. 7B, RES was found in the vicinity of (Distances 4Å involved)
Gly226,Cys220,Gln192,Trp215,Gly219,Asp189,Gly216,Tyr228,S
er214,Ser190 and Val213. Fig. 7A showed that the binding site
of RES to trypsin was not located catalytic triad (His 57, Asp
102 and Ser 195) of trypsin active site, but rather at the
hydrophobic cavity-S1 binding pocket (the primary substrate-
binding pocket)of the trypsin active site. RES had hydrophobic
interaction with its surrounding amino acid residues and one
hydrogen bond formed between RES and residue Val227 of
trypsin (2.70 Å). This suggested that hydrophobic force played
a major role in the RES+trypsin interaction [48]. 4 Tyr and 2 Trp
residues were shown in the molecular docking diagram
(Supplementary Fig.3) (12Å), while only Trp215 and Tyr228
residues were shown in the molecular docking diagram (4Å)
Fig7B. This indicated that the binding site of RES and trypsin
was close to Trp215 and Tyr228, and the change of their
microenvironment was the main cause of fluorescence
quenching. This might explain the phenomenon of the
previous experimental results. The binding energy of
-1
molecular docking was -29.59 kJ·mol , which was very close to
the results calculated based on fluorescence experimental
-1
data (-28.28 kJ·mol , 298k), indicating the reliability of docking
results.
J. Name., 2019, 00, 1-3 | 9
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Effect of RES on trypsin activity
Changes in the structure of the protease by RES affected
the catalytic activity of the enzyme. Therefore, the effect of
RES on trypsin could be determined by measuring the catalytic
activity of the trypsin. The effects of different concentrations
of RES on the activity of trypsin were presented in Fig. 8. The
Fig. 8 Relative activity of trypsin in the absence and presence of RES.
10 | J. Name., 2019, 00, 1-12
Page 10 of 15
Journal Name
View Article Online
DOI: 10.1039/C9FO00183B
Food & Function Accepted Manuscript
activity of trypsin decreased with the increasing concentration
of RES. These results indicated that the addition of RES could
inhibit trypsin activity. The enzymatic mechanism of trypsin
was similar to that of other serine proteases. There were 2
fundamental characteristics of the active site region. The first
one was formed by the 3 catalytic residues His 57, Asp 102,
and Ser 195; the second one was the primary substrate-
binding pocket (S1 binding pocket, hydrophobic cavity)
consisting of residues189-195, 214-220 and 225-228, which
defining the cut position of substrate binding and the peptide
bond. 3-catalytic-residue and the primary substrate-binding
pocket (S1 binding pocket, hydrophobic cavity) residues
accomplished the catalytic activity of trypsin. Asp189 residues
were located at the bottom of S1 and had the function of
substrate recognition [49]. When RES binded to the S1 pocket
of trypsin, it had hydrophobic interaction and a hydrogen bond
with surrounding amino acid residues (including Asp189),
which might affect casein (the substrate) in the reaction
system binding to the S1 pocket, resulting in decreased trypsin
activity [50].
Antioxidant activities
This journal is © The Royal Society of Chemistry 20xx
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Free radical scavenging activities in vitro of free RES and
RES adding trypsin were analyzed by the DPPH assay and
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This journal is © The Royal Society of Chemistry 20xx
Food & Function
ARTICLE
superoxide anion radicals scavenging assay as View described
Article Online
earlier. This experiment could verify theDOI: 10.1039/C9FO00183B
effect of trypsin on
the antioxidant activity of RES. It could be seen from Fig. 9 and
Fig. 10, the DPPH scavenging rate and the superoxide anion
radicals scavenging rate of RES were showed similar tendency
to in the presence of trypsin. The results showed that the
presence of trypsin could significantly reduce (p<0.05) the
scavenging ability of RES to DPPH and superoxide anion free
radicals. The potential free radical scavenging capacity of
polyphenols were related to hydroxyl groups in molecules [51].
In this work, a hydrogen bonding formed between hydroxyl
Food & Function Accepted Manuscript
group of RES and Val227 of trypsin might be responsible for
the decrease of free radical scavenging capacity of RES.
Conclusions
In this study, the binding interaction of RES with trypsin
was studied using multiple spectroscopic and molecular
docking methods. The experimental results revealed that RES
and trypsin complexation was formed and trypsin fluorescence
could be quenched by RES with static quenching mechnism.
The process of RES binding to trypsin was spontaneous
exothermic reaction, and the hydrophobic forces played a
main role in stabilizing the RES+trypsin complexation. The
results of synchronous fluorescence and three-dimensional
fluorescence showed that the microenvironment of the
fluorophores of trypsin changed after binding with RES. The
changed of UV-vis spectroscopy demonstrated that
intervention of exogenous substances of RES led to the
destruction of the original tertiary structure of trypsin, the
spatial structure became loose, and polar amino acids were
exposed on the molecular surface, resulting in decreased
hydrophobicity and enhanced polarity of trypsin molecules. FT-
IR spectra showed the alterations in the secondary structure of
trypsin upon binding to RES. Molecular docking experiments
showed that RES binded in the S1 pocket of trypsin. The
relevant information obtained from molecular docking
confirmed the conclusion of the previous experiment, and
provided an explanation for the reduction of trypsin catalytic
activity and RES antioxidant activity after the combination of
RES and trypsin. This work was helpful for us to understand the
interact mechanism of RES on trypsin, and could provide the
basic data for RES as functional food ingredient in vivo.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
We gratefully acknowledge the financial support of the
National Natural Science Foundation of China (grant
No.31571800).
Notes
RES Resveratrol
J. Name., 2019, 00, 1-3 | 11
 Food & Function
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UV Ultraviolet absorption spectrum
Tris-HCl Trisaminomethane- hydrochloric acid
DPPH 1,1-diphenyl-2-picrylhydrazyl
FT-IR Fourier Transform Infrared
Na2CO3 Sodium Carbonate
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360-367.

Food & Function Accepted Manuscript

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Graphic Abatract: DOI: 10.1039/C9FO00183B

Food & Function Accepted Manuscript

Mechanism of interaction between resveratrol and trypsin and its effect on their biological activity
Published on 07 May 2019. Downloaded on 5/8/2019 2:47:27 AM.