Investigations of conformational structures and activities of trypsin and pepsin affected by food colourant allura red

Article abstract of DOI:10.1016/j.molliq.2020.114359

Herein, binding interactions of food colourant allura red (AR) with trypsin and pepsin were comparably investigated for deep revelations of conformational structures and activities of proteinases affected by food colourant. Various results indicated that

Full text of DOI:10.1016/j.molliq.2020.114359

Journal of Molecular Liquids 319 (2020) 114359
Contents lists available at ScienceDirect
Journal of Molecular Liquids
journal homepage: www.elsevier.com/locate/molliq
Investigations of conformational structures and activities of trypsin and
pepsin affected by food colourant allura red
⁎
Qi Xiao, Jiandan Liang, Huajian Luo, Haimei Li, Jing Yang, Shan Huang
Guangxi Key Laboratory of Natural Polymer Chemistry and Physics, College of Chemistry and Materials, Nanning Normal University, Nanning 530001, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 July 2020
Received in revised form 19 August 2020
Accepted 16 September 2020
Available online 19 September 2020
Herein, binding interactions of food colourant allura red (AR) with trypsin and pepsin were comparably investi-
gated for deep revelations of conformational structures and activities of proteinases affected by food colourant.
Various results indicated that one AR bound with one proteinase to form novel ground state complex under
the binding forces of van der Waal interactions and hydrogen bonds. Intrinsic fluorescence of proteinases was
quenched by AR via static fluorescence quenching mode. Conformational structures of proteinases were all
changed obviously after their binding interactions with AR, resulting in their structure transformation to the β-
sheet structure. AR bound with the allosteric site of proteinases to inhibit their activities via non-competitive
manner. Finally, AR protected human serum albumin from the digestion of proteinases efficiently. These results
revealed the exact binding mechanisms of food colourant AR with proteinases, which illuminated the possible
biological risk of food colourant AR on human beings.
Keywords:
Allura red
Trypsin
Pepsin
Binding interaction
Conformational structure
Enzymatic activity
© 2020 Elsevier B.V. All rights reserved.
1. Introduction
systematically evaluate the biological risks of AR on human beings. Un-
doubtedly, the biosafety evaluation of AR becomes the vital issue that
Allura red (AR), which is a kind of artificial azo dye, has been widely
used in food and beverage industries [1,2]. Since AR can interact with
target cells and cause malignant tumors, the excessive intake of AR
into the human body can damage several living organisms and cause
potential carcinogenic effects on human beings [3]. Consequently, on
its first safety assessment, the acceptable daily intake of AR is 7 mg/kg
body weight per day according to the Joint FAO/WHO Expert Committee
on Food Additives [4]. Due to the vital multiple biological functions of
serum proteins in blood plasma, the biological influences of AR on sev-
eral serum proteins have been deeply investigated [5]. Several re-
searches groups investigated the binding property between AR and
human serum albumin (HSA) at the molecular level through several
spectroscopic approaches and molecular modeling techniques [6–8].
Lelis and co-workers studied the interaction of AR with bovine serum al-
bumin and further illustrated their binding thermodynamics [9]. All
such investigations confirmed the binding interaction of AR with
serum proteins and the structural variations of these proteins affected
by AR. Consequently, the biological functions of serum proteins were
changed by AR, supporting the hypothesis of the potential biological
risk of AR to human health. However, the binding mechanism of AR
on important biofunctional proteins is exceedingly complicated and re-
mains largely unknown, although such revelations can be used to
must be clarified for their widespread application.
Proteinases play important roles in numerous catalyzing reactions
during several physiological and pathological processes [10]. The inves-
tigations of conformational structures and activities of proteinases are
very imperative to the exploration of effective and targeted drugs for
proteinases-related diseases therapy. Trypsin (EC 3.4.21.4) is a type of
serine proteinase [11,12] while pepsin (EC 3.4.23.1) is a representative
aspartic proteinase [13,14]. Trypsin and pepsin are proved to play im-
portant biological roles in the digestion and decomposition of several
physiological processes in human digestive system. Consequently,
both of them are often chosen as binding model proteinases to illumi-
nate the biological influences of small molecules those founded in
food on their conformational structures and activities. Freitas et al. stud-
ied the interaction between grape seed procyanidins and trypsin, and
their results proved that procyanidins affected the conformational
structure of trypsin slightly [15]. Wang et al. investigated the interac-
tions between five chlorophenols and trypsin, and their results showed
that the number of chlorine atoms of chlorophenols partly affected the
binding ability of them with trypsin [16]. The binding interactions of
sunset yellow, curcumin, and Ligupurpuroside A with pepsin have also
been revealed [13,14,17]. The influences of phenylpropanoid glycosides
and folic acid on the conformations and activities of trypsin and pepsin
were studied by Wu's group and Fei's group [18,19]. However, the var-
iations of conformational structures and activities of trypsin and pepsin
after their interactions with food colourant AR have not been reported
⁎
Corresponding author.
E-mail address: huangs@whu.edu.cn (S. Huang).
https://doi.org/10.1016/j.molliq.2020.114359
0167-7322/© 2020 Elsevier B.V. All rights reserved.

Q. Xiao, J. Liang, H. Luo et al.
Journal of Molecular Liquids 319 (2020) 114359
Fig. 1. (A) UV–vis absorption spectra of trypsin, AR, AR-trypsin system, and the difference absorption spectrum between AR-trypsin system and AR. (B) UV–vis absorption spectra of
pepsin, AR, AR-pepsin system, and the difference absorption spectrum between AR-pepsin system and AR. The concentrations of proteinases and AR were all 4.0 × 10−6 mol L−1
.
(C) Fluorescence decay traces of trypsin before and after the addition of AR. (D) Fluorescence decay traces of pepsin before and after the addition of AR. The concentrations of
proteinases and AR were all 4.0 × 10−6 mol L−1. τ is the fluorescent lifetime of proteinase and b is the normalized pre-exponential factor, respectively.
as far as we know. Such investigations can be used to evaluate the bio-
logical risk of AR on human beings more deeply and objectively.
Herein, the binding interactions of AR with trypsin and pepsin were
investigated by using several experimental approaches and molecular
modeling technique. Detailed binding mechanisms between protein-
ases and AR will be elucidated through this research. Such researches
can further explore the variations of conformational structures and ac-
tivities of proteinases after their binding interactions with AR. Through
such investigation, we expect to provide valuable and meaningful infor-
mation to the biological effect evaluation of food colourant AR in health-
related fields.
between AR-proteinase systems and AR were measured by using AR
as the reference. The measuring wavelength was decreased from 360
to 200 nm with 1 nm interval and the scan speed was 600 nm min−1
.
Quartz cells with 1.0 cm path-length were used for all measurements.
The concentrations of proteinases and AR were all 4.0 × 10−6 mol L−1
.
Proteinase activity experiments were proceeded by using BApNA
and hemoglobin as substrates for trypsin and pepsin according to the re-
ported literature [20,21]. For evaluation of trypsin activity, 5.0 mL of
BApNA with different concentration, 0.2 mL of trypsin (0.25 g L−1),
and 4.8 mL of Tris-HCl buffer (pH 8.2) were mixed completely and incu-
bated at 37 °C for 10 min. The absorbance of the solution at 400 nm was
measured to evaluate the trypsin activity. For evaluation of pepsin activ-
ity, 2.0 mL of hemoglobin with different concentration, 1.0 mL of pepsin
(1.0 g L−1), and 2.0 mL of citric acid buffer (pH 2.0) were mixed
completely and incubated at 37 °C for 30 min. Then, 2.0 mL of trichloro-
acetic acid (10%) was added to stop the enzymatic reaction. The mixture
was centrifuged at 12,000 rpm for 10 min, and the supernatant was
transferred into another new test tube. Finally, the absorbance of the so-
lution at 275 nm was measured to evaluate the pepsin activity. The scan
speed was 600 nm min−1 and quartz cells (1.0 cm path-length) were
used for all measurements. The concentration of trypsin and pepsin
were 2.1 × 10−7 and 5.6 × 10−6 mol L−1, respectively. The concentra-
2. Material and methods
2.1. Materials
Trypsin (from bovine pancreas), pepsin (from porcine gastric mu-
cosa), HSA, AR, hemoglobin (from bovine erythrocyte), N-alpha-Ben-
zoyl-DL-arginine-4-nitroanilide hydrochloride (BApNA, ≥98%), and p-
nitroaniline (pNA) were all purchased from Sigma-Aldrich Co., Ltd. (St.
Lousi, USA). Trypsin was dissolved in 20 mM of phosphate buffer saline
(PBS, pH 7.4). Pepsin was dissolved in 20 mM of citric acid solution
(pH 2.0). All other chemical reagents were of analytical reagent grade
and used as received without any additional processes. Ultrapure
water with a resistivity of 18.2 MΩ cm was produced by Millipore-Q Ac-
ademic purification set (Millipore, Bedford, MA, USA) and used in the
whole experiments.
tion of BApNA was increased from 5.7 × 10−5 to 9.2 × 10−4 mol L−1
.
The concentration of hemoglobin was increased from 7.8 × 10−6 to
7.8 × 10−5 mol L−1. The concentration of AR was increased from 0 to
3.6 × 10−4 mol L−1 with interval of 1.2 × 10−4 mol L−1
.
2.3. Fluorescence spectroscopy
2.2. UV–vis absorption spectroscopy
Time-resolved fluorescence spectra of proteinases and AR-
proteinase systems were recorded on Horiba Scientific QM-8075 high
sensitivity steady-state transient fluorescence spectrometer (HORIBA,
Japan) with time-correlated single-photo counting system.
UV–vis absorption spectra of proteinases, AR, and AR-proteinase sys-
tems were recorded on Cary 100 UV–vis spectrophotometer (Agilent
Technologies, Inc., Australia). The difference absorption spectra
2

Q. Xiao, J. Liang, H. Luo et al.
Journal of Molecular Liquids 319 (2020) 114359
Fig. 2. Steady-state fluorescence spectra of trypsin with increasing concentration of AR at 298 (A), 304 (B), and 310 K (C). Steady-state fluorescence spectra of pepsin with increasing
concentration of AR at 298 (D), 304 (E), and 310 K (F). The concentrations of proteinases were all 4.0 × 10−6 mol L−1. The concentrations of AR were (1–11, ×10−6 mol L−1): 0, 1, 2,
3, 4, 5, 6, 7, 8, 9, and 10, respectively.
Fluorescence decay traces were recorded at room temperature with ex-
citation/emission wavelengths of 278/350 nm/nm, respectively. The
slits of the excitation and the emission were all 8.0 nm. Fluorescence
decay traces were fitted with the biexponential function. The average
fluorescent lifetime (<τ>) was calculated by the equation of <τ> =
Στibi (τ is the fluorescent lifetime of proteinase and b is the normalized
pre-exponential factor) [22]. Each data was the average of three times
successive scanning. The concentrations of proteinases and AR were
researching the influences of co-existed metal ions on AR-proteinase
systems, the steady-state fluorescence spectra of AR-proteinase systems
were recorded at 298
K after the addition of metal ions
(4.0 × 10−6 mol L−1). Other experimental parameters were all
remained as previously mentioned.
Three-dimensional fluorescence spectra of proteinases and AR-
proteinase systems were performed on Perkin-Elmer LS 55 lumines-
cence spectrometer (Waltham, MA, USA). The excitation wavelength
was increased from 200 to 350 nm and the emission wavelength was
changed from 200 to 500 nm with 5 nm increment. The slits of the ex-
citation and the emission for trypsin and AR-trypsin system were 15
and 18 nm, respectively. The slits of the excitation and the emission
for pepsin and AR-pepsin system were 9 and 6.5 nm, respectively. The
scan speed was 600 nm min−1. The concentrations of proteinases and
all 4.0 × 10−6 mol L−1
.
Steady-state fluorescence spectra of proteinases with different con-
centrations of AR at 298, 304, and 310 K were performed on Perkin-
Elmer LS 55 luminescence spectrometer (Waltham, MA, USA) with a
thermostatic bath. Fluorescence spectra were recorded with excita-
tion/emission wavelengths of 280/350 nm/nm, respectively. The slits
of the excitation and the emission for AR-trypsin system were 15 and
18 nm, respectively. The slits of the excitation and the emission for
AR-pepsin system were 9 and 6.5 nm, respectively. The scan speed
was 600 nm min−1. Each spectrum was the average of three times suc-
cessive scanning. The concentrations of proteinases were all
4.0 × 10−6 mol L−1, and the concentration of AR was increased from 0
to 1.0 × 10−5 mol L−1 with interval of 1.0 × 10−6 mol L−1. When
AR were all 2.0 × 10−6 mol L−1
.
2.4. Fourier transform infrared spectroscopy
Fourier transform infrared (FT-IR) spectra of proteinases and AR-
proteinase systems were recorded on Perkin-Elmer Frontier spectrom-
eter (Waltham, MA, USA) via the zinc selenide attenuated total
3

Q. Xiao, J. Liang, H. Luo et al.
Journal of Molecular Liquids 319 (2020) 114359
Fig. 3. Modified Stern-Volmer plots of AR-trypsin system (A) and AR-pepsin system (B) at three different temperatures. Double-logarithmic plots of AR-trypsin system (C) and AR-pepsin
system (D) at three different temperatures.
reflection method with 128 interferograms and resolution of 4 cm−1
.
systems with different molar ratio of AR to proteinase under different
reaction temperatures, the temperature was increased from 20 to
90 °C in 5 °C steps with 300 s increments. The melting temperature
(Tm) and the molar enthalpy change (ΔHm) at that temperature were
measured by CD spectrometer equipped with the Global Analysis Soft-
ware. The percentages of different secondary structures of proteinases
were analyzed via the CD neural networks (CDNN) software [25]. The
scan speed was 500 nm min−1 and the response time was 0.5 s. Each
spectrum was the average of three times successive scanning. The con-
The difference FT-IR spectra between AR-proteinase systems and AR
were measured by using AR as the reference. The subtraction of the ref-
erence spectrum from the spectrum of the buffer solution was carried
out in accord with the criteria that straight baseline was obtained be-
tween 2000 and 1750 cm−1 [23]. The percentages of different second-
ary structures of proteinases were estimated by Omnic data
processing software combined with Fourier deconvolution and curve
fitting from 1700 to 1600 cm−1 [24]. The peak positions and the half
widths of each sub-peak were estimated with the peak width of 48.8
and the enhancement of 3.2. After determining the correspondences be-
tween each sub-peak and different secondary structures, the relative
percentages of various secondary structures were calculated according
to their integrated areas. The concentrations of proteinases and AR
centrations of trypsin and pepsin were 2.5
×
10−5 and
2.5 × 10−6 mol L−1. The concentrations of AR were one or two times
of the proteinase concentrations.
2.6. Electrochemical investigation
were all 1.0 × 10−3 mol L−1
.
Electrochemical experiments were all performed on Chenhua CHI-
660E electrochemical workstation (Shanghai, China) with conventional
three-electrode electrochemical testing system. The electrolyte is
5.0 mM of K3Fe(CN)6/K4Fe(CN)6 solution with 10 mM of KCl. Gold elec-
trode (GE), Ag/AgCl electrode, and platinum wire were served as work-
ing electrode, reference electrode, and counter electrode, respectively.
For the preparation of proteinases modified GE (Trypsin/GE and
2.5. Circular dichroism spectroscopy
Circular dichroism (CD) spectra of proteinases, AR, and AR-
proteinase systems were recorded on Chirascan CD spectrometer (Ap-
plied Photophysics, England) at 25 °C under the constant nitrogen
flush protection. When recording the CD spectra of AR-proteinase
Table 1
Binding constants Ka, binding constants Kb, binding number n, and relative thermodynamic parameters in AR-proteinase systems.
System
T (K)
Ka
R2a
Kb
n
R2a
ΔH
ΔG
ΔS
R2a
(104 L mol−1
)
(104 L mol−1
)
(kJ mol−1
)
(kJ mol−1
)
(J mol−1 K−1
)
298
304
310
298
304
310
5.12
4.27
3.32
5.91
4.72
3.77
0.54
0.25
0.35
0.49
0.17
0.21
0.999
0.999
0.999
0.999
0.999
0.999
6.11
5.13
4.12
6.79
5.10
4.31
0.08
0.34
0.41
0.25
0.12
0.07
1.03
1.06
1.04
1.04
1.04
1.04
0.999
0.999
0.999
0.999
0.998
0.999
−26.75
−26.31
−25.86
−27.30
−26.52
−25.75
0.007
0.001
0.007
0.17
0.20
0.22
AR-trypsin
AR-pepsin
−48.73
−65.72
0.16
0.98
−73.78
0.52
0.999
0.999
−128.93
3.86
a
R
2 is the correlation coefficient.
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Q. Xiao, J. Liang, H. Luo et al.
Journal of Molecular Liquids 319 (2020) 114359
50 mV s−1 and EIS was measured within the frequency range from 0.1
to 100 kHz. Each spectrum was the average of three times successive
scanning. The concentration of AR was increased from 6.0 × 10−6 to
Table 2
Binding constants Ka of AR-proteinase systems in the absence and presence of different
common metal ions.
6.0 × 10−5 mol L−1 with interval of 6.0 × 10−6 mol L−1
.
System
Ka (104 L mol−1
)
R2a
S.D.b
K/Ka
AR-trypsin alone
AR-trypsin-Mg2+
AR-trypsin-Ca2+
AR-trypsin-Cu2+
AR-trypsin-Al3+
AR-trypsin-Zn2+
AR-trypsin-Cr3+
AR-trypsin-Pb2+
AR-trypsin-Mn2+
AR-trypsin-Fe2+
AR-trypsin-Fe3+
AR-pepsin alone
AR-pepsin-Mg2+
AR-pepsin-Ca2+
AR-pepsin-Cu2+
AR-pepsin-Al3+
AR-pepsin-Zn2+
AR-pepsin-Cr3+
AR-pepsin-Pb2+
AR-pepsin-Mn2+
AR-pepsin-Fe2+
AR-pepsin-Fe3+
5.12
4.78
4.86
4.61
4.79
4.72
4.71
4.67
4.82
4.88
4.89
5.91
5.29
5.14
5.29
5.15
5.25
4.92
5.21
5.12
5.17
5.23
0.54
0.05
0.19
0.23
0.13
0.05
0.31
0.08
0.18
0.14
0.25
0.49
0.20
0.17
0.05
0.13
0.27
0.13
0.18
0.12
0.08
0.12
0.999
0.999
0.999
0.999
0.999
0.999
0.999
0.999
0.998
0.999
0.999
0.999
0.999
0.999
0.999
0.999
0.999
0.999
0.999
0.999
0.999
0.999
0.01
0.04
0.09
0.17
0.19
0.13
0.18
0.11
0.07
0.13
0.16
0.01
0.20
0.17
0.05
0.13
0.06
0.13
0.18
0.12
0.12
0.08
1
0.93
0.95
0.90
0.94
0.92
0.92
0.91
0.93
0.95
0.96
1
0.90
0.87
0.90
0.87
0.89
0.83
0.88
0.87
0.87
0.89
2.7. Molecular modeling
Crystal structures of trypsin (PDB ID: 2ZQ1) and pepsin (PDB ID:
3PEP) were taken from the RCSB Protein Data Bank [26,27]. The binding
interactions of proteinases with AR were researched by using Surflex
Dock program in Sybyl-X 2.0 software with energy termination gradient
of 0.01 kcal mol−1 [28]. The molecular structure was optimized by the
Tripos force field. The AR molecule was charged by the Gasteiger and
Hückel methods. The protomol for trypsin and pepsin were all gener-
ated by the ligand mode. The threshold was 0.50 and the bloat was
3.0. The parameters in the docking work were same with the previously
reported data [28].
2.8. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) of substrate HSA with proteinases at the present of different con-
centration of AR was performed using vertical electrophoresis system
(Bio-Rad, U.K.) [29]. The concentrations of HSA, trypsin, and pepsin
were 6.0 × 10−6, 2.5 × 10−5, and 1.1 × 10−5 mol L−1, respectively.
The concentration of AR was increased from 0 to 2.4 × 10−2 mol L−1
with interval of 8.0 × 10−3 mol L−1. AR with different concentration
and proteinase were firstly mixed completely and incubated at 37 °C
water bath for 30 min. Then, HSA was added into the solution and the
mixture was continuously heated at 37 °C water bath for 10 min.
Phenylmethanesulfonyl fluoride was added to terminate the enzymatic
reaction. The treated sample was mixed with four times of the loading
buffer (0.5 mol L−1 of Tris-HCl, pH 6.8, 5% (w/v) SDS, 20% (v/v) glyc-
erol). The mixture was boiled for 5 min and then was cooled to room
a
R
2 is the correlation coefficient.
S.D. is standard deviation.
b
Pepsin/GE), 10 μL of proteinases solution (5.0 × 10−6 mol L−1) were
dropped onto the surface of bare GE. The electrode was then incubated
at 4 °C for 1 h. The proteinases modified GE were washed by ultrapure
water for three times and then were dried in nitrogen airflow. For cyclic
voltammograms (CV) and electrochemical impedance spectrum (EIS)
measurements, different concentration of AR was added into the elec-
trolyte solution and then stirred for 5 min. The reaction system was at
rest for 3 min before testing. CV was recorded with scan rate of
Fig. 4. (A) CV of GE and Trypsin/GE with different concentration of AR. (B) CV of GE and Pepsin/GE with different concentration of AR. Linear relationships between 1/ΔIp and 1/c in AR-
trypsin system (C) and AR-pepsin system (D). The concentrations of AR were (1–11, ×10−6 mol L−1): 0, 6, 12, 18, 24, 30, 36, 42, 48, 54, and 60, respectively.
5

Q. Xiao, J. Liang, H. Luo et al.
Journal of Molecular Liquids 319 (2020) 114359
Fig. 5. (A) EIS of GE and Trypsin/GE with different concentration of AR. (B) EIS of GE and Pepsin/GE with different concentration of AR. Linear relationships between Rct(i)/Rct(0) and c in AR-
trypsin system (C) and AR-pepsin system (D). The concentrations of AR were (1–11, ×10−6 mol L−1): 0, 6, 12, 18, 24, 30, 36, 42, 48, 54, and 60, respectively.
temperature gradually. The experiments were performed in 5% (w/v)
stacking gel and 14% (w/v) separating gel. The low molecular weight
marker from 14.3 to 97.2 KDa was used to estimate the molecular
weight of the sample. After electrophoresis, the gel was stained with
Coomassie brilliant blue R-250 in staining solution (30% (v/v) methanol,
10% (v/v) acetic acid, and 60% (v/v) water) for 30 min. The gel was de-
stained in de-staining solution (30% (v/v) methanol, 10% (v/v) acetic
acid, and 60% (v/v) water) for additional 30 min. Finally, the clear pro-
tein bands on the gel indicated the presence of protease activity.
equation of <τ> = Στibi (b is the normalized pre-exponential factor)
[22]. As shown in Fig. 1C and D, the average fluorescent lifetimes of tryp-
sin and pepsin were calculated to be around (2.27 0.01) and (5.09
0.02) ns, respectively. The addition of AR did not change the average
fluorescent lifetimes of proteinases at all. The fluorescent lifetime of
fluorophore remains to its initial value if compound binds with
fluorophore to form ground state complex [31]. Thus, AR should bind
with trypsin and pepsin to form ground state complexes, which agrees
perfectly with the previous results.
3.1.2. Binding constants and binding numbers
3. Results and discussion
Binding constants among AR-proteinase systems can be obtained via
recording the fluorescence spectra of proteinase with increasing con-
centration of AR under 298, 304, and 310 K. As shown in Fig. 2, two pro-
teinases exhibited characteristic and intrinsic fluorescence peak at
350 nm under the excitation of 278 nm, but AR showed no absorption
ability during the same wavelength range. In addition, the influence of
temperature on the fluorescence intensity of pepsin was much higher
than that of trypsin (red and blue dotted lines in Fig. 2). The intrinsic
fluorescence of proteinases was all quenched by AR through
concentration-dependent manner. Comparably, 1.0 × 10−5 mol L−1 of
AR quenched the intrinsic fluorescence of trypsin to be about 43.1% at
298 K, while 1.0 × 10−5 mol L−1 of AR quenched the intrinsic fluores-
cence of pepsin to be about 37.4% at 298 K, confirming the stronger fluo-
rescence quenching ability of AR on trypsin. Due to the ground state
complexes formation between AR and proteinases, AR can quench the
intrinsic fluorescence of proteinases through static quenching mode.
Undoubtedly, binding constant (Ka) among AR-proteinase systems can
be calculated via modified Stern-Volmer equation [32]:
3.1. Binding interactions between proteinases and AR
3.1.1. Binding mechanisms
Binding mechanisms between proteinases and AR are firstly verified
by using UV–vis absorption spectroscopy. As exhibited in Fig. 1A and B,
trypsin and pepsin showed a strong absorption peak at 204 nm and a
typical absorption peak at 280 nm. These peaks are ascribed to peptide
structures and the amino acid residues [tryptophan (Trp), tyrosine
(Tyr), and phenylalanine (Phe)] in the structure of proteinases [22,30].
AR showed almost no obvious absorption from 200 to 360 nm. Mean-
while, the difference absorption spectrum between AR-trypsin system
and AR did not cover perfectly with the UV–vis absorption spectrum
of trypsin. Same situation was existed in pepsin and AR-pepsin system.
It has been reported that if the absorption spectrum of fluorophore
changes after the addition of compound, this compound should bind
with fluorophore to form novel ground state complex [31]. Conse-
quently, AR may bind with trypsin and pepsin to construct ground
state complexes.
I0
1
1
1
Such speculation can be further proved by time-resolved fluores-
cence spectroscopy. Fluorescence decay traces of proteinases without
or with AR were illustrated in Fig. 1C and D. The fluorescent lifetime
(τ) of proteinases consists of two parts: a short lifetime τ1 and a long
lifetime τ2. The average fluorescent lifetime (<τ>) is fitted with the
¼
þ
ð1Þ
I0−I f aKa ½Qꢀ f a
In Eq. (1), I0 and I are the fluorescence intensities of proteinases
without and with AR, [Q] is the concentration of AR, and fa is the mole
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Q. Xiao, J. Liang, H. Luo et al.
Journal of Molecular Liquids 319 (2020) 114359
Fig. 6. Lowest binding energy conformation of the surface binding mode of AR with trypsin (A) and pepsin (B). Surrounded amino acid residues of trypsin (C) and pepsin (D) in AR
molecule. Hydrogen bonds between amino acid residues of trypsin (E) and pepsin (F) and AR molecule.
fraction of solvent-accessible fluorophore, respectively [32]. Modified
Stern-Volmer plots of AR-proteinase systems at three different temper-
atures were shown in Fig. 3A and B. The Ka values of AR-proteinase sys-
tems at three different temperatures were calculated and listed in
Table 1. Since the Ka values of AR-proteinase systems were decreased
gradually with the increase of temperature, reconfirming the static fluo-
rescence quenching mechanism of AR-proteinase systems [23]. The Ka
values of AR-proteinase systems were decreased to some extent when
common metal ions were present (Table 2), indicating that common
metal ions affected the binding interactions of AR with trypsin and pep-
sin obviously.
double-logarithmic plots of AR-proteinase systems were shown in
Fig. 3C and D, and both the Kb values and the n values were all illustrated
in Table 1. The Kb values were all decreased gradually with the incre-
ment of temperature, but the n values were almost constant to be one.
Therefore, AR strongly bound with one proteinase to form ground
state complexes.
On the other hand, binding constant can be also calculated through
electrochemical methods, including CV and EIS [23,33]. As indicated in
Fig. 4A and B, after the successful assembling of proteinases on the sur-
face of GE, the redox peak currents of K3Fe(CN)6/K4Fe(CN)6 were de-
creased significantly due to the enhanced steric hindrance effect. The
redox peak currents of K3Fe(CN)6/K4Fe(CN)6 were decreased step by
step after the addition of increasing concentrations of AR, informing
the improved electrostatic repulsion of K3Fe(CN)6/K4Fe(CN)6 toward
the modified electrodes [23]. All these phenomena confirmed the bind-
ing interactions between AR and proteinases. The binding constant ob-
tained by CV (KCV-a) can be calculated through the plot of the reciprocal
of the current drop (ΔIp) and the reciprocal of AR concentration (c) ac-
cording to the Langmuir equation [23]:
Binding constant (Kb) is further calculated by the traditional double-
logarithmic equation [33]:
I0−I
I
log
¼ logKb þ n log½Qꢀ
ð2Þ
The binding number (n) can be also obtained through Eq. (2) if the
compound binds with fluorophore at the same site [33]. Finally, the
7

Q. Xiao, J. Liang, H. Luo et al.
Journal of Molecular Liquids 319 (2020) 114359
Fig. 7. Distances between AR molecule and the nearest fluorescent amino acids of trypsin (A) and pepsin (B). Distances between AR molecule and the catalytic amino acids of trypsin
(C) and pepsin (D).
values were 1.16 × 104 L mol−1 in AR-trypsin system and
a
1
1
1
3.11 × 104 L mol−1 in AR-pepsin system, respectively. Due to the natural
conformation variation of proteinases and the enhanced steric hin-
drance effect, the binding interaction between proteinases and AR can
be inhibited. Consequently, the binding constants calculated by electro-
chemical approaches are a little lower than those obtained through
spectroscopic methods.
¼
þ
ð3Þ
ΔIp ΔIp; max ΔIp; maxKCV−ac
The plots between 1/ΔIp and 1/c of AR-proteinase systems were
shown in Fig. 4C and D. The linear fitting equations of AR-proteinase
systems were 1/ΔIp [μA−1] = 1.401 + 41.48/c [L μmol−1] for AR-
trypsin and 1/ΔIp [μA−1] = 1.368 + 33.65/c [L μmol−1] for AR-pepsin
with correlation coefficient of 0.999. The KCV-a values were calculated
to be 3.38 × 104 L mol−1 in AR-trypsin system and 4.10 × 104 L mol−1
in AR-pepsin system, respectively.
As further shown in Fig. 5A and B, the semicircle diameter at higher
frequency of EIS curve was increased dramatically after surficial assem-
bling of proteinases on GE. This semicircle diameter often indicates the
charge-transfer resistance (Rct) of the electrode [34], so the Rct value
was increased after the modification of proteinases on GE surface. The
Rct value was continuously increased with the increasing concentration
of AR, ascribing to the enhanced charge-transfer resistance of the mod-
ified electrodes after the binding interactions of AR with proteinases.
The binding constant obtained by EIS (KEIS-a) can be calculated through
the plot of the Rct value and the AR concentration (c) according to the
following equation [34]:
3.1.3. Binding forces and binding sites
Binding forces during AR-proteinase systems can be speculated from
the variations of thermodynamic parameters including enthalpy change
(ΔH), entropy change (ΔS), and free energy change (ΔG). The relation-
ships among these thermodynamic parameters are expressed as: ln
Ka = −ΔH / RT + ΔS/R and ΔG = ΔH − TΔS (R and T are the gas con-
stant and the temperature, respectively) [34]. Through the fluorescent
data of AR-proteinase systems at three different temperatures, the lin-
ear fitting equations of AR-proteinase systems were ln Ka = 5861.38 /
T − 8.87 (J mol−1) in AR-trypsin system and ln Ka = 7903.93 /
T − 15.51 (J mol−1) in AR-pepsin system, respectively. The calculated
ΔH, ΔS, and ΔG values were all negative (Table 1). Negative ΔG values
suggested that the binding processes between AR and proteinases
were occurred spontaneously [35]. Binding forces between compound
and protein mainly include electrostatic interactions, hydrophobic
bonds, hydrogen bonds, and van der Waals interactions [36]. Both neg-
ative ΔH and ΔS values in AR-proteinase systems indicated that hydro-
gen bonds and van der Waals interactions played vital roles among
these binding interactions [35].
RctðiÞ
Rctð0Þ
¼ KESI−ac þ 1
ð4Þ
The plots between Rct(i)/Rct(0) and c were shown in Fig. 5C and D. The
linear fitting equation of AR-trypsin was Rct(i)/Rct(0) = 0.01161c
[μmol L−1] + 1 with correlation coefficient of 0.999 (Fig. 5C), while
the linear fitting equation of AR-pepsin was Rct(i)/Rct(0) = 0.03447c
[μmol L−1] + 1 with correlation coefficient of 0.999 (Fig. 5D). The KEIS-
Detail binding forces and binding sites can be elucidated by molecu-
lar modeling [25]. Fig. 6A and B shows the lowest binding energy con-
formations of AR-trypsin system and AR-pepsin system. The docking
8

Q. Xiao, J. Liang, H. Luo et al.
Journal of Molecular Liquids 319 (2020) 114359
Fig. 8. Three-dimensional fluorescence spectra of trypsin (A), AR-trypsin system (B), pepsin (C), and AR-pepsin system (D). The concentrations of proteinases and AR were all
2.0 × 10−6 mol L−1, respectively.
scores (stand for − logKd, and Kd is the dissociation constant) among
AR-proteinase systems were 4.06 for AR-trypsin system and 5.96 for
AR-pepsin system, suggesting that AR bound with proteinases from a
very different binding direction. Since the higher docking score
corresponded with the stronger binding ability between compound
and the binding site [28], AR bound with pepsin more tightly than
with trypsin. As further shown in Fig. 6C, eleven amino acid residues, in-
cluding glutamine-50 (Gln-50), Trp-51, serine-86 (Ser-86), lysine-87
(Lys-87), Lys-107, leucine-108 (Leu-108), Lys-109, Ser-110,
isoleucine-242 (Ile-242), alanine-243 (Ala-243), and asparagine-245
(Asn-245), took part in the binding interaction between AR and trypsin.
As exhibited in Fig. 6D, twenty-one amino acid residues, containing
aspartic acid-32 (Asp-32), glycine-34 (Gly-34), Ser-35, Ile-73,
threonine-74 (Thr-74), Tyr-75, Gly-76, Thr-77, Ile-128, Tyr-189, Ile-
213, Asp-215, Gly-217, Thr-218, Ser-219, Leu-220, Thr-222, glutamic
acid-287 (Glu-287), methionine-289 (Met-289), proline-292 (Pro-
292), and Ile-300, took part in the binding interaction between AR and
pepsin. Obviously, the amino acid residues participated in the binding
interaction of AR-pepsin system were more than those of AR-trypsin
system, indicating the stronger binding ability of AR with pepsin. Ser,
Tyr, Thr, and Lys amino acid resides are all polar amino acid residues,
so van der Waals interactions should be involved in these binding inter-
actions [37]. Furthermore, six hydrogen bonds were existed between
the amino acid residues Gln-50 (2.06 Å), Trp-51 (2.74 Å), Lys-107
(2.10 and 2.28 Å), Lys-109 (1.93 Å), and Asn-245 (2.20 Å) of trypsin
and AR molecule (Fig. 6E). Also, six hydrogen bonds were existed be-
tween the amino acid residues Thr-74 (1.99 Å), Gly-76 (2.28 Å), Tyr-
189 (1.92 Å), Ser-219 (1.90 Å), Leu-220 (2.11 Å), and Glu-287
(2.10 Å) of pepsin and AR molecule (Fig. 6F). Thus, hydrogen bonds
and van der Waals interactions played major roles in the binding inter-
actions between AR and proteinases.
In addition, the distance between AR molecule and the nearest fluo-
rescent amino acid Trp-51 of trypsin was calculated to be 2.63 Å
(Fig. 7A), while the distances of two hydrogen bonds of AR with the
fluorescent amino acid Tyr-75 and Tyr-189 of pepsin were 1.92 and
2.11 Å (Fig. 7B). Since the distances between AR and proteinases were
very close, AR can quench the intrinsic fluorescence of proteinases dra-
matically. Moreover, the distances of AR molecule with the catalytic
triad histidine-57 (His-57), aspartic acid-102 (Asp-102), and serine-
195 (Ser-195) of trypsin [26] were 22.68, 20.67, and 21.61 Å, respec-
tively (Fig. 7C). However, the distances of AR molecule with the catalytic
twosome Asp-32 and Asp-215 of pepsin [27] were 4.81 and 3.89 Å,
9

Q. Xiao, J. Liang, H. Luo et al.
Journal of Molecular Liquids 319 (2020) 114359
Fig. 9. FT-IR spectrum of trypsin and difference FT-IR spectrum between AR-trypsin system and AR (A), curve fitting of infrared amide I band in FT-IR spectrum of trypsin (B), and curve
fitting of infrared amide I band in difference FT-IR spectrum between AR-trypsin system and AR (C). FT-IR spectrum of pepsin and difference FT-IR spectrum between AR-pepsin system
and AR (D), curve fitting of infrared amide I band in FT-IR spectrum of pepsin (E), and curve fitting of infrared amide I band in difference FT-IR spectrum between AR-pepsin system and AR
(F). The concentrations of proteinases and AR were all 1.0 × 10−3 mol L−1
.
respectively (Fig. 7D). Thus, AR did not bind directly with the enzymatic
active-site residues of proteinases, and the enzymatic activity of pepsin
should be affected by AR more obviously.
512.9 to 440.5 for peak 1 and from 336.7 to 243.5 for peak 2, while
the intensities of scattering peaks were changed from 619.9 to 645.1
for peak a and from 166.2 to 104.6 for peak b (Fig. 8B). These variations
suggested the increment of the scattering effect and the significant
change of conformational structure after the binding interaction of AR
with trypsin. Same situations can be observed in the binding interaction
3.2. Conformational structures of proteinases affected by AR
between AR and pepsin except only one fluorescent peak 1 (λex/em
=
3.2.1. Three-dimensional fluorescence spectrometry
290/348 nm/nm) in pepsin and AR-pepsin system [13]. After the addi-
tion of AR, the intensity of fluorescent peak 1 was decreased from
829.6 to 716.3 and the intensity of scattering peak b was decreased
from 92.8 to 73.8 (Fig. 8C and D). These phenomena also proved the
binding interaction of AR with pepsin and the conformational structure
variation after such interaction.
Three-dimensional fluorescence spectrometry can be used to inves-
tigate the conformational structures of proteinases under the influence
of AR. Fig. 8 presents the three-dimensional fluorescence spectra of pro-
teinases and AR-proteinase systems. As shown in Fig. 8A, trypsin exhib-
ited two fluorescent peaks (peak 1 and peak 2) and two scattering peaks
(peak a and peak b). Fluorescent peak 1 (λex/em = 290/345 nm/nm)
represented the fluorescent properties of amino acid residues and fluo-
rescent peak 2 (λex/em = 235/342 nm/nm) meant the π → π* transition
in polypeptide backbone structure, respectively [38]. Scattering peak a
and peak b indicated the Rayleigh (λem = λex) and the second-order
(λem = 2λex) scattering peak, respectively [23]. After the addition of
AR, both intensities of fluorescent peaks were all decreased from
3.2.2. FT-IR spectrometry
The secondary structures of proteins can be studied by FT-IR spec-
trometry, especially the curve fitting of the infrared amide I band of pro-
tein [12,24]. Usually, the infrared amide I band of protein is attributed as
1692 to 1680 cm−1 for β-antiparallel, 1680 to 1660 cm−1 for β-turn,
10

Q. Xiao, J. Liang, H. Luo et al.
Journal of Molecular Liquids 319 (2020) 114359
Table 3
interacted with trypsin, resulting in the rearrangement of the secondary
structure in trypsin [24]. According to the curve fitting spectrum of in-
frared amide I band of trypsin (Fig. 9B), the peaks at 1688, 1670, 1655,
1643, and 1628 cm−1 were ascribed to the β-antiparallel, β-turn, α-
helix, random coil, and β-sheet structures of trypsin, respectively. The
relative percentages of various secondary structures were calculated
and the results were exhibited in Table 3. The relative percentages of
secondary structures of trypsin were 10.1% of α-helix, 43.3% of β-sheet
(main secondary structure), 16.8% of β-turn, 2.4% of β-antiparallel,
and 27.4% of random coil [12], respectively. At the present of AR, the
peaks at 1683, 1670, 1655, 1643, and 1629 cm−1 were ascribed to the
β-antiparallel, β-turn, α-helix, random coil, and β-sheet structures of
trypsin, respectively (Fig. 9C). The relative percentages of secondary
structures of trypsin were changed to 9.2% of α-helix, 48.5% of β-
sheet, 15.1% of β-turn, 2.9% of β-antiparallel, and 24.3% of random coil,
respectively. Therefore, the secondary structure of trypsin was changed
from the mainly β-turn and random coil structures to the β-sheet
structure.
On the other hand, the infrared amide I band and the infrared amide
II band of pepsin were at around 1640 and 1551 cm−1, respectively
(Fig. 9D). These two bands of pepsin were changed to 1635 cm−1 for in-
frared amide I band and to 1550 cm−1 for infrared amide II band after
binding with AR (black curve in Fig. 9D). As further shown in Fig. 9E,
the peaks at 1685, 1671, 1656, 1644, and 1625 cm−1 reflected the β-
antiparallel, β-turn, α-helix, random coil, and β-sheet structures of pep-
sin, respectively. As shown in Table 3, the relative percentages of sec-
ondary structures of pepsin were 7.5% of α-helix, 37.9% of β-sheet
(main secondary structure), 20.7% of β-turn, 3.4% of β-antiparallel,
and 30.5% of random coil [12], respectively. At the present of AR, the
peaks at 1685, 1671, 1656, 1644, and 1626 cm−1 meant the β-
antiparallel, β-turn, α-helix, random coil, and β-sheet structures of pep-
sin, respectively (Fig. 9F). The relative percentages of secondary
Curve fitting data of infrared amide I bands of proteinases and AR-proteinases systems.
System
Peak
FWHM
Area
Relative percentage of area
(%)
(cm−1
)
(cm−1
)
Trypsin
1628
1643
1655
1670
1688
1629
1643
1655
1670
1683
1625
1644
1656
1671
1685
1626
1644
1656
1671
1685
27.4
20.6
10.7
16.5
7.8
25.8
15.2
11.2
15.1
9.6
21.4
19.3
8.4
18.5
9.7
0.433
0.274
0.101
0.168
0.024
0.485
0.243
0.092
0.151
0.029
0.379
0.305
0.075
0.207
0.034
0.489
0.273
0.070
0.147
0.021
43.3
27.4
10.1
16.8
2.4
48.5
24.3
9.2
15.1
2.9
37.9
30.5
7.5
AR-trypsin
Pepsin
20.7
3.4
48.9
27.3
7.0
AR-pepsin
20.6
13.2
4.7
12.9
7.6
14.7
2.1
1660 to 1649 cm−1 for α-helix, 1648 to 1638 cm−1 for random coil, and
1637 to 1615 cm−1 for β-sheet structures, respectively [12,24]. As ex-
hibited in Fig. 9A, trypsin exhibited an infrared amide I band at around
1639 cm−1 (C_O stretch), reflecting the secondary structures of trypsin
[12]. Trypsin exhibited an infrared amide II band at about 1550 cm−1
(C\\N stretch and N\\H bending mode) reflecting the main peptide vi-
bration bands in trypsin [12]. These bands of trypsin were changed to
1638 cm−1 for infrared amide I band and to 1548 cm−1 for infrared
amide II band after binding with AR (black curve in Fig. 9A). So, AR
Fig. 10. (A) CD spectra of AR and trypsin in the presence of different concentrations of AR. (B) Percentages of different secondary structures of trypsin in the presence of different
concentrations of AR. (C) CD spectra of AR and pepsin in the presence of different concentrations of AR. (D) Percentages of different secondary structures of pepsin in the presence of
different concentrations of AR. The concentrations of trypsin and pepsin were 2.5 × 10−5 and 2.5 × 10−6 mol L−1, while the concentrations of AR were one or two times higher than
the concentrations of proteinases.
11

Q. Xiao, J. Liang, H. Luo et al.
Journal of Molecular Liquids 319 (2020) 114359
Fig. 11. (A to C) Temperature dependent CD spectra of trypsin and AR-trypsin system with different molar ratios. (D to F) Percentages of different secondary structures of trypsin in the
presence of different concentrations of AR. The concentration of trypsin was 2.5 × 10−5 mol L−1, while the concentration of AR was increased from 0 to 5.0 × 10−5 mol L−1 with interval of
2.5 × 10−5 mol L−1
.
structures of pepsin were 7.0% of α-helix, 48.9% of β-sheet, 14.7% of β-
turn, 2.1% of β-antiparallel, and 27.3% of random coil in AR-pepsin sys-
tem, respectively. Obviously, the secondary structure of pepsin was
changed from the mainly β-turn and the random coil structures to the
β-sheet structure. Such variations were highly consistent with those in
AR-trypsin system. All these results implied that AR induced the sec-
ondary structure variations of both trypsin and pepsin, resulting in the
enhanced hydrophobic environments and more stable conformations
of proteinases. Comparably, after the addition of AR, the variation of
the β-sheet structure in pepsin was much bigger than that in trypsin,
suggesting that the conformational structure of pepsin was affected by
AR more significantly and efficiently.
2.5 × 10−5 mol L−1 of AR was present, the contents of α-helix and β-
sheet structures were changed to 9.9% and 42.2%, respectively. When
5.0 × 10−5 mol L−1 of AR was present, the contents of α-helix and β-
sheet structures were changed to 7.2% and 46.4%, respectively. Same
changes were existed in AR-pepsin system with different concentra-
tions of AR (Fig. 10C and D). Pepsin showed a characteristic negative
peak at around 200 nm [14], and the absorbance of such peak was de-
creased gradually after the addition of increasing concentration of AR.
The percentages of secondary structures of pepsin were calculated to
be 7.8% of α-helix, 37.2% of β-sheet, 22.9% of β-turn, and 32.1% of ran-
dom coil structures, respectively (Fig. 10D). When 2.5 × 10−6 mol L−1
of AR was present, the contents of α-helix and β-sheet structures
were changed to 6.5% and 39.6%, respectively. When
5.0 × 10−6 mol L−1 of AR was present, the contents of α-helix and β-
sheet structures were changed to 6.0% and 41.8%, respectively. These
variations suggested that AR combined with the amino acid residues
of proteinases and induced their partial structure folding [25], which
agreed well with the results obtained by FT-IR spectrometry.
3.2.3. CD spectroscopy
The secondary structures of proteinases are continuously researched
by CD spectroscopy [25,39]. Fig. 10A exhibited the CD spectra of AR and
trypsin with different concentrations of AR. AR did not show any CD sig-
nal while trypsin showed a typical negative peak at around 206 nm [25],
and AR caused a significant decrease of the absorbance of this band. Ac-
cording to the CDNN software [25], the percentages of secondary struc-
tures of trypsin were 11.4% of α-helix, 38.2% of β-sheet, 18.9% of β-turn,
and 31.5% of random coil structures, respectively (Fig. 10B). When
Besides those, to investigate the thermal stability of proteinases af-
fected by AR, both Tm and ΔHm values were measured by CD spectrom-
eter equipped with the Global Analysis Software. Figs. 11 and 12
showed the temperature dependent CD spectra of AR-proteinase
12

Q. Xiao, J. Liang, H. Luo et al.
Journal of Molecular Liquids 319 (2020) 114359
Fig. 12. (A to C) Temperature dependent CD spectra of pepsin and AR-pepsin system with different molar ratios. (D to F) Percentages of different secondary structures of pepsin in the
presence of different concentrations of AR. The concentration of pepsin was 2.5 × 10−6 mol L−1, while the concentration of AR was increased from 0 to 5.0 × 10−6 mol L−1 with
interval of 2.5 × 10−6 mol L−1
.
systems with different molar ratios. Finally, the calculated Tm values of
trypsin and pepsin were (66.8 3.5) and (63.3 0.8) °C, respectively.
The Tm value of trypsin was decreased to (55.8 1.7) and (49.9 0.1)
°C separately when the concentration of AR was one or two times higher
than the concentration of trypsin. The Tm value of pepsin was also de-
creased to (57.3 0.1) and (46.1 0.1) °C separately when the concen-
tration of AR was one or two times higher than the concentration of
pepsin. Meanwhile, the ΔHm value of trypsin was decreased from
BApNA and hemoglobin were often degraded by trypsin and pepsin
separately [20,21]. BApNA can be digested by trypsin to produce pNA,
and the generation of pNA per minute (mol L−1 min−1) is used to eval-
uate the trypsin activity [20]. Meanwhile, hemoglobin can be digested
by pepsin to produce tyrosine, so the generation of tyrosine per minute
(mol L−1 min−1) is absolutely used to evaluate the pepsin activity [21].
The Michaelis-Menten curves from data of two enzymatic reactions
with different concentrations of AR were shown in Fig. 13A and B. It is
obvious that the enzyme reaction velocity (V) value was increased ac-
cordingly with the increase of the concentration of substate but this
value was decreased dramatically with the addition of increasing con-
centration of AR under same substate concentration. Lineweaver-Burk
plot was created by plotting the reciprocal of enzyme reaction velocity
(V) against the reciprocal of the substrate concentration ([s]). Michael's
constant (Km) and the maximum reaction velocity (Vmax) were ob-
tained from the double-reciprocal plot [20,21]:
(254.7
7.1) to (178.1
7.4) and (100.5
18) kJ mol−1 when the
10−5 and
concentration of AR was increased to 2.5
×
5.0 × 10−5 mol L−1, respectively. The ΔHm value of pepsin was also de-
creased from (260.2 0.92) to (210.7 6.2) and (143.5 30) kJ mol−1
when the concentration of AR was increased to 2.5 × 10−6 and
5.0 × 10−6 mol L−1, respectively. Both the reductions of Tm and ΔHm
values confirmed that AR inhibited the thermal denaturation proce-
dures of proteinases and reduced their thermal stabilities [25,39].
1
V
Km
1
1
3.3. Activities of proteinases
¼
þ
ð5Þ
Vmax ½sꢀ Vmax
3.3.1. Kinetic parameters of proteinases
Kinetic parameters of proteinases can be obtained through enzy-
matic reaction of trypsin on BApNA and pepsin on hemoglobin, since
The double-reciprocal plots of proteinases with various concentra-
tions of substrates and AR were exhibited in Fig. 13C and D. As can be
13

Q. Xiao, J. Liang, H. Luo et al.
Journal of Molecular Liquids 319 (2020) 114359
Fig. 13. (A) Michaelis-Menten curves from activity data of trypsin with different concentrations of AR. (B) Double-reciprocal plots of trypsin with different concentrations of AR.
(C) Michaelis-Menten curves from activity data of pepsin with different concentrations of AR. (D) Double-reciprocal plots of pepsin with different concentrations of AR. The
concentrations of trypsin and pepsin were 2.1 × 10−7 and 5.6 × 10−6 mol L−1. The concentration of BApNA was increased from 5.7 × 10−5 to 9.2 × 10−4 mol L−1. The concentration
of hemoglobin was increased from 7.8 × 10−6 to 7.8 × 10−5 mol L−1. The concentration of AR was increased from 0 to 3.6 × 10−4 mol L−1 with interval of 1.2 × 10−4 mol L−1
.
seen from Table 4, the Km and Vmax values of trypsin alone were (2.60
0.01) × 10−4 mol L−1 and (1.23 0.02) × 10−5 mol L−1 min−1, respec-
tively. Obviously, after the addition of AR, the Vmax values was decreased
dramatically but the Km values was kept almost constant, reflecting the
non-competitive inhibition mode of AR in the enzymatic activity of
trypsin [40,41]. Meanwhile, the Km and Vmax values of pepsin alone
were (2.81 0.10) × 10−5 mol L−1 and (1.17 0.02) × 10−5 mol L−1-
min−1, respectively. When AR was present, the Km values remained al-
most constant and the Vmax values was decreased gradually, also
suggesting the non-competitive inhibition mode of AR in the enzymatic
activity of pepsin [40]. Thus, AR could reduce the activities of trypsin
and pepsin through non-competitive manner.
values but the lower Km value suggest the stronger enzymatic activity
of proteinase [20]. As shown in Table 4, both the kcat and kcat/Km values
in two enzymatic reactions were all decreased with the increasing con-
centration of AR, suggesting that AR inhibited the activities of protein-
ases through the concentration-dependent manner. When the
concentration of AR was increased to 3.6 × 10−4 mol L−1, both the kcat
and kcat/Km values of trypsin were reduced to 67.4% and 67.1% of their
initial values. Comparably, the kcat and kcat/Km values of pepsin were re-
duced to 64.1% and 63.4% of their initial values if 3.6 × 10−4 mol L−1 of
AR was present. It is obvious that AR inhibited the activity of pepsin
more efficiently, due to the longer distances between AR molecule and
the catalytic triad His-57, Asp-102, and Ser-195 of trypsin.
The catalytic constant (kcat) of proteinase can be calculated through
the equation of kcat = Vmax/[Proteinase], and the catalytic efficiency
(kcat/Km) can be used to evaluate the enzymatic ability of proteinase
[20]. Usually, the values of Km, Vmax, kcat, and kcat/Km can efficiently eval-
uate the activity of proteinase, and the higher Vmax, kcat, and kcat/Km
3.3.2. SDS-PAGE
Trypsin and pepsin can digest numerous peptides, amidos, and ester
bonds of Lys with arginine (Arg) and fluorescent amino acid residues in
proteins, thus HSA was widely used as the substrate to evaluate the
Table 4
Kinetic parameters of two enzymatic reactions with different concentrations of AR (mean value standard deviation, n = 3).
AR
Trypsin-BApNA
Pepsin-hemoglobin
(10−4 L mol−1
)
Km
Vmax
(10−5 mol L−1 min−1
kcat
kcat/Km
Km
Vmax
(10−5 mol L−1 min−1
kcat
kcat/Km
(10−4 mol L−1
)
)
(min−1
)
(105 L mol−1 min−1
)
(10−5 mol L−1
)
)
(min−1
)
(104 L mol−1 min−1
)
58.6
0.01
52.9
0.01
45.7
0.02
39.5
0.04
2.09
0.03
1.75
0.09
1.54
0.04
1.34
0.02
0
2.60
2.61
2.61
2.62
0.01
0.01
0.02
0.02
1.23
1.11
0.96
0.83
0.02
0.10
0.01
0.01
2.25
2.06
1.75
1.51
0.07
0.06
0.11
0.01
2.81
2.82
2.83
2.84
0.10
0.07
0.17
0.02
1.17
0.98
0.86
0.75
0.02
0.06
0.04
0.01
7.44
6.21
5.44
4.72
0.28
0.11
0.09
0.02
1.2
2.4
3.6
14

Q. Xiao, J. Liang, H. Luo et al.
Journal of Molecular Liquids 319 (2020) 114359
Fig. 14. (A) SDS-PAGE electropherogram demonstrating the low molecular weight marker (Line 1), HSA (Line 2), HSA-trypsin (Line 3), HSA-trypsin-AR (Lines 4 to 6), AR-trypsin (Line 7),
and trypsin (Line 8), respectively. (B) SDS-PAGE electropherogram demonstrating the low molecular weight marker (Line 1), HSA (Line 2), HSA-pepsin (Line 3), HSA-pepsin-AR (Lines 4 to
6), AR-pepsin (Line 7), and pepsin (Line 8), respectively. The concentrations of HSA, trypsin, and pepsin were 6.0 × 10−6, 2.5 × 10−5, and 1.1 × 10−5 mol L−1, respectively. The
concentration of AR (from Lines 3 to 6) was increased from 0 to 2.4 × 10−2 mol L−1 with interval of 8.0 × 10−3 mol L−1
.
activity of proteinases via SDS-PAGE [42,43]. Fig. 14 shows SDS-PAGE
electropherograms of HSA before or after the addition of proteinases
and/or AR. Lines 1 in both Fig. 14A and B showed the migration band
of the low molecular weight marker which was often used to estimate
the molecular weights of the samples. This low molecular weight
marker usually includes 97.2 KDa for phosphatase b, 66.4 KDa for bovine
serum protein, 44.3 KDa for ovalbumin, 29.0 KDa for carbonic
anhydrase, 20.1 KDa for trypsin inhibitor, and 14.3 KDa for lysozyme.
HSA showed only one characteristic migration band with the molecular
weight of around 66.4 KDa (lines 2 in both Fig. 14A and B). Trypsin and
pepsin all exhibited only one characteristic migration band (lines 7 in
both Fig. 14A and B), and 2.4 × 10−2 mol L−1 of AR did not affect the mi-
gration bands of proteinases at all (lines 8 in both Fig. 14A and B). After
the enzymatic reactions by trypsin and pepsin, HSA was digested by
proteinases to produce several small protein/peptide-rich product mix-
tures with the molecular weights of lower than 66.4 KDa, resulting in
the existences of several migration bands with smaller molecular
weights (lines 3 in both Fig. 14A and B). After the addition of
8.0 × 10−3 mol L−1 of AR, the colors of the migration bands with bigger
molecular weights became dark but the colors of the migration bands
with lower molecular weights became shallow (lines 4 in both
Fig. 14A and B). When the concentration of AR was increased from
1.6 × 10−2 to 2.4 × 10−2 mol L−1, the colors of the migration bands
with bigger molecular weights became much obvious while the colors
of the migration bands with lower molecular weights became much ob-
scure (lines 5 to 6 in both Fig. 14A and B). All these phenomena con-
firmed the inhibition ability of AR on the digestion activities of
proteinases for HSA. Most interestingly, the color of the migration
band with molecular weight of around 66.4 KDa became clearer in
AR-pepsin system, thus partial HSA was not digested by pepsin with
more significant variations in both conformational structure and enzy-
matic activity. These researches explored the potential toxicity of food
colourant AR in biological fields.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
This work was financially supported by National Natural Science
Foundation of China (21763005, 21864006), Natural Science Founda-
tion
of
Guangxi
Province
(2017GXNSFDA198034,
2017GXNSFFA198005), Scientific Research and Technology Develop-
ment Program of Guangxi (AD17195081), the Thousands of Young
Teachers Training Program of Guangxi Province (guijiaoren[2018]18),
the High-Level-Innovation Team (guijiaoren[2017]38) and Outstanding
Scholar Project of Guangxi Higher Education Institutes, and BAGUI
Scholar Program of Guangxi Province of China.
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