Journal of Hazardous Materials 453 (2023) 131389

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Journal of Hazardous Materials

journal homepage: www.elsevier.com/locate/jhazmat

Research Paper

Intrinsic mechanisms for the inhibition effect of graphene oxide on the

catalysis activity of alpha amylase
Xinwei Liu 1, Binbin Sun 1, Chunyi Xu , Tianxu Zhang , Yinqing Zhang , Lingyan Zhu *
Key Laboratory of Pollution Processes and Environmental Criteria (Ministry of Education), Tianjin Key Laboratory of Environmental Remediation and Pollution Control,
College of Environmental Science and Engineering, Nankai University, Tianjin 300350, PR China

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Binding interaction between GO and

α-amylase was firstly investigated.
• GO strongly binds with α-amylase in a
spontaneous and exothermic manner.
• GO binds with α-amylase mainly via
hydrogen bonding and van der Waals
force.
• GO quenches intrinsic fluorescence of
α-amylase by changing secondary
conformation.
• GO reduces the ability of α-amylase to
catalyze digestion of starch into glucose.

A R T I C L E I N F O A B S T R A C T

Lingxin CHEN Comprehending the interactions between graphene oxide (GO) and enzymes is critical for understanding the
toxicities of GO. In this study, the inherent interactions of GO with α-amylase as a typical enzyme, and the
Keywords: impacts of GO on the conformation and biological activities of α-amylase were systematically investigated. The
Graphene oxide results reveal that GO formed ground-state complex with α-amylase primarily via hydrogen bonding and van der
α-amylase
Waals interactions, thus quenching the intrinsic fluorescence of the protein statically. Particularly, the strong
Binding interaction
interactions altered the microenvironment of tyrosine and tryptophan residues, caused rearrangement of poly­
Conformational structure
Biological activity peptide structure, and reduced the contents of α-helices and β-sheets, thus changing the conformational structure
of α-amylase. According to molecular docking results, GO binds with the amino acid residues (i.e., His299,
Asp300, and His305) of α-amylase mainly through hydrogen bonding, which is in accordance with in vitro in­
cubation experiments. As a consequence, the ability of α-amylase to catalyze starch hydrolysis into glucose was
depressed by GO, suggesting that GO might cause dysfunction of α-amylase. This study discloses the intrinsic
binding mechanisms of GO with α-amylase and provides novel insights into the adverse effects of GO as it enters
organisms.

* Corresponding author.
E-mail address: zhuly@nankai.edu.cn (L. Zhu).
1
These authors contributed equally to this work and should be considered co-first authors.

https://doi.org/10.1016/j.jhazmat.2023.131389
Received 11 January 2023; Received in revised form 25 March 2023; Accepted 7 April 2023
Available online 8 April 2023
0304-3894/© 2023 Elsevier B.V. All rights reserved.
X. Liu et al.
1. Introduction
Because of the distinctive properties, such as large surface area, good
biocompatibilities, and strong affinities with biomacromolecules (e.g.,
enzymes), graphene oxide (GO), a kind of typical and common two-
dimensional (2D) nanomaterial, finds extensive applications in
biomedical and environmental fields [1,2]. GO has found many appli­
cations in biomedical fields, such as drug and protein delivery, bio­
sensors and bioimaging, and antimicrobial agents [3–5]. GO has higher
loading efficiency and biocompatibility with drug molecules or/and
biomolecules and better stability in physiological environment than
other therapeutic agents, such as metal nanoparticles, polymers, and
other carbon-based nanomaterials [6–8]. Large-scale applications and
improper discharges may lead to the inevitability of GO release into the
natural aquatic environment [9]. As a result, it may pose direct or in­
direct adverse effects to aquatic organisms and eventually human beings
through food chains [10,11]. Once GO enters organisms through mul­
tiple exposure pathways (e.g., oral ingestion, inhalation, or skin con­
tact), it may firstly contact with biomacromolecules widely existing in
biofluids. Understanding the interactions between GO and bio­
macromolecules at molecular level is of importance for better predicting
its biological effects. Current studies on the interactions between GO and
biomacromolecules mainly focus on serum albumin, fibrinogen, and
immunoglobulin, and the results reveal that GO may significantly
change the spatial conformation of proteins and finally influence their
physiological functions [12,13]. As another important component of
serum biomacromolecules, enzymes show different conformational
structure and biological functions, while their interactions with GO have
been largely overlooked.
As typical and widely existing biological catalysts, enzymes partici­
pate in specific metabolic reactions, such as energy transformation and
bond hydrolysis, while their structures and activities remain unchanged
before and after reactions [14,15]. Previous studies found that GO had
greater adsorption capacity for enzymes (e.g., lysozyme) than other
common nanoparticles like single-walled and multi-walled carbon
nanotubes, which may be due to the strong hydrophobic and electro­
static interactions of GO with enzymes [16,17]. The interactions be­
tween GO and enzymes might alter the conformational structure and
catalytic functions of enzymes. For example, Huang et al. discovered
that GO bound to the allosteric sites of trypsin and inhibited its activities
in a non-competitive way [18]. Conversely, the peroxidase activity of
cytochrome c was improved after binding to GO since the active sites
were slightly perturbed [19]. These discrepant results suggest that the
impacts of GO on enzymes’ catalytic activities and conformational
change are related to the physicochemical properties of enzymes, such
as surface charge, amino acid amount, molecular weight, and active
sites.
Previous research discovered that after GO treatment in the parent
zebrafish, the biosynthesis and metabolism of glucose in their offspring
zebrafish were significantly disturbed [20]. GO also downregulated the
carbohydrate metabolism in pepper fruit and markedly increased the
number of starch grains in single-celled organism Chlorella vulgaris [21,
22]. All these metabolic processes are associated with α-amylase, which
is a kind of hydrolytic enzyme that catalyzes the digestion of starch into
monosaccharides or oligosaccharides and finally influences the carbo­
hydrate metabolism [23]. α-amylase is widely distributed in animals (e.
g., salivary gland, pancreas, and blood), plants, and microorganisms
[24]. The molecular weight of pancreatic α-amylase in human body is
about 57.78 kDa, containing 511 amino acid residues in one single
oligosaccharide chain, and the detailed physicochemical properties are
shown in Table S1 [25]. The basic structure of α-amylase consists of
three structural domains (A, B, and C, respectively). The largest domain
A is a barrel of eight parallel stretches of extended chain enclosed by
eight helices [24]. α-amylase cleaves the link between the two sugars in
the starch chain with the help of three acidic groups (i.e., glutamate 233,
aspartate 197, and aspartate 300) at its active site. A chloride ion binds
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Journal of Hazardous Materials 453 (2023) 131389
underneath the active site and assists the reaction, while the enzyme
structure is stabilized by a calcium ion. Any increase or decrease in
enzymatic activity can result in a wide range of human diseases and
disorders, such as metabolic imbalance and specific enzyme mutations
[26]. α-amylase is one of the most important digestive enzymes in vivo
and the inhibitory effects on α-amylase activity may result in appetite
loss and glucose metabolism disorders, which can lead to abnormalities
in body function [27]. Dhobale et al. discovered that ZnO nanoparticles
at 20 mg/L inhibited α-amylase activity by 49% [28]. Jiang et al. found
that spherical and polygonal starch nanoparticles inhibited the enzy­
matic activity of α-amylase by impacting its spatial conformation [29].
In contrast, citrate-stabilized gold nanoparticles and silver nanoparticles
significantly enhanced the specific activity of α-amylase [30,31]. As one
typical 2D nanomaterial with plentiful functional groups, it is hypoth­
esized that GO might interact with α-amylase actively and change its
conformational structure and biological activity differently from other
nanoparticles.
Herein, the interactions of GO and α-amylase at molecular level were
systematically explored through various spectroscopic characterizations
and theoretical calculations. Enzymatic activities of α-amylase in the
absence and presence of GO were also investigated. This work would
provide novel insights into affinities of enzymes with GO and the change
trend of conformational structure and biological effects induced by GO.
2. Materials and methods
2.1. Materials
GO powder was purchased from Xianfeng Nano Materials Technol­
ogies Co., Ltd (Nanjing, China). Lyophilized powder of α-amylase
(10065, ≥98%) was provided by Sigma Aldrich company (St. Louis, MO,
USA). Thioflavin T (ThT, D844310, ≥97%) was supplied by Macklin
Biochemical Co., Ltd (Shanghai, China). Phosphate-buffered saline (PBS,
10 mmol/L, pH 7.4) was obtained through dissolving 2.9 g Na2HPO4,
0.2 g KH2PO4, 8.0 g NaCl, and 0.2 g KCl into 1 L ultrapure water.
α-amylase was dissolved in the PBS solution and stored at 4 ◦ C. Ultra­
pure water (18.2 MΩ/cm, HHitech, China) was utilized throughout the
experimental studies.
2.2. GO preparation and characterization
The GO stock solution was prepared according to our earlier research
with a few modifications [32]. Briefly, 80 mg of GO powder was mixed
with 400 mL of ultrapure water and stirred for 12 h at 300 rpm/min. The
GO suspension was ultrasonically treated in an ice bath for 6 h to obtain
a uniform stock solution. Before each set of experiment, the GO stock
solution was ultrasonicated for 15 min. Field emission scanning electron
microscopy (FE-SEM, Gemini 500, ZEISS, Germany) and high-resolution
transmission electron microscopy (HR-TEM, 2100F, JEOL, Japan) were
used to characterize the morphological feature and lateral size of GO.
The surface chemistry and elemental compositions of GO were deter­
mined using X-ray photoelectron spectrometer (XPS, PHI 5000 VersaP­
robe, ULVAC-PHI, Japan), FT-IR spectrometer (TENSOR 37, Bruker,
Germany), elemental analyzer (vario EL cube, elementar, Germany),
Raman spectra (Renishaw inVia, UK), and UV-Vis spectrometer
(UV-2600, Shimadzu, Japan). Our previous study provided a compre­
hensive description of the characterization methods [33].
2.3. Interaction between GO and α-amylase
2.3.1. Adsorption experiments
Batch experiments were conducted at room temperature to reveal the
adsorption affinity of α-amylase (from 50 to 1000 mg/L) on GO (100
mg/L). A rotary shaking incubator was used to shake the vials for 12 h,
and all experiments were carried out in triplicate. After incubation, the
suspensions were centrifuged at 8000 rpm/min for 15 min before being
X. Liu et al.
filtered through 0.22 µm nylon membrane. The filtered protein sus­
pension was placed in the microplate (NEST Biotechnology, China) and
the Bradford protein assay kit was used to determine its concentration
[34]. Langmuir and Freundlich models were utilized to fit the adsorption
isotherms to understand the adsorption mechanism.
2.3.2. Size and zeta potential measurements
The hydrodynamic diameter (Dh) and zeta potential of GO before and
after incubation with α-amylase in PBS were determined by Malvern
Zetasizer (Nano ZS90, Malvern, UK). Zeta potential would be automat­
ically obtained by the conversion of electrophoretic mobilities using the
zeta potential analyzer of Malvern Zetasizer.
2.3.3. Fluorescence spectroscopy
The fluorescence intensity changes of α-amylase caused by GO of
various concentrations (0–100 mg/L) were recorded at different incu­
bation time (0, 30, and 60 min) and temperatures (277 K, 298 K, and
310 K). Fluorescence emission spectra of α-amylase as a function of GO
concentration ranging from 290 to 500 nm were recorded by a fluo­
rescence spectrophotometer installed with Xe lamp (F-7000, Hitachi,
Japan) with the excitation wavelength of 280 nm and slit width of 2.5
nm.
Time-resolved fluorescence spectra of α-amylase and α-amylase-GO
system were recorded on a FLS1000 high sensitivity photoluminescence
spectrometer with both steady state and time-resolved mode (Edin­
burgh, England) at 298 K. Fluorescence decay trace was recorded via the
time-correlated single photon counting system using pulsed light emit­
ting diodes of the EPLED as the excitation source. The wavelengths of
excitation and emission were 280 nm and 335 nm, respectively. The
concentration of α-amylase was fixed at 300 mg/L and that of GO was
10 mg/L.
Synchronous fluorescence spectroscopy was applied to confirm
characteristic features about tyrosine (Tyr) and tryptophan (Trp) resi­
dues of α-amylase as a function of GO concentration. Wavelength in­
tervals (Δλ) were fixed as 15 and 60 nm, which represent the Tyr and Trp
residues, respectively [35].
The isometric three-dimensional (3D) fluorescence spectroscopy
could reveal the proteins’ structural information. GO concentration was
set at 10 mg/L, and that of α-amylase was at 30 mg/L in all the groups.
GO and α-amylase solution were then mixed and vortexed at 200 rpm/
min for 10 s. The excitation wavelength ranged from 200 to 500 nm,
while the emission wavelength was in the range of 250 and 550 nm with
a 5 nm interval. The scan speed was 12000 nm/min, and both the
emission and excitation slits were 5 nm.
2.3.4. UV-Vis absorption spectroscopy
Influence of GO on the UV-Vis absorption spectra of α-amylase was
recorded in the range of 200–600 nm by the UV-Vis spectrometer. GO
itself was used as the background absorbance. The α-amylase concen­
tration was kept constant at 300 mg/L, while the GO concentration
varied between 0 and 75 mg/L.
2.3.5. FT-IR spectroscopy
First, 100 and 600 mg/L of α-amylase were added into the GO sus­
pensions (200 mg/L) and the vials were shaken in a rotary shaking
incubator at 250 rpm/min for 12 h at room temperature. The suspen­
sions were then centrifuged at 10000 rpm/min for 15 min and the
precipitates were freeze dried, which were later characterized by FT-IR.
FT-IR spectra in the 4000–400 cm− 1 wave number range were collected.
The amide I region (1700–1600 cm− 1) that originates from the
stretching vibrations of C-O bond in the FT-IR spectra of the proteins was
analyzed by Peakfit 4.12 software. The second derivative spectra of the
amide I region were obtained using a 9-point Savitsky-Galey function,
which were baseline corrected and fitted to separate overlapping peaks
based on the maximum absorption intensity, band frequency, and
bandwidth from the second derivative (R2 > 0.999) [36].
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Journal of Hazardous Materials 453 (2023) 131389
2.3.6. Circular dichroism (CD)
The far-UV CD spectra of α-amylase and GO complexes were ob­
tained by Spectrometer J-810 (Jasco, Japan) using a quartz cell with a
path length of 0.1 cm. An accumulation of three scans with 200 nm/min
speed was performed and spectra were obtained from 185 to 260 nm
with a band width of 2 nm. α-amylase concentration was fixed at 3000
mg/L, while that of GO varied between 0 and 75 mg/L. Appropriate
background corrections were applied to all spectra.
2.3.7. Fluorescent dying experiment with ThT
The conformational information of α-amylase was characterized
using the fluorescent dye ThT [37]. α-amylase at 300 mg/L was firstly
incubated with various concentrations of GO (from 0 to 100 mg/L) for
0.5 h; 120 μM ThT was then added into the suspensions and further
incubated for another 0.5 h. The fluorescence spectra of α-amylase-ThT
conjugates with and without GO were measured with an excitation
wavelength of 440 nm and slit width of 5 nm in the range from 400 to
600 nm.
2.3.8. Assay of α-amylase activity
The starch hydrolytic activity of α-amylase with and without GO was
evaluated by monitoring the sugar release utilizing the 3, 5-dinitrosali­
cylic acid (DNS) method in accordance with the procedure reported by
Bernfeld et al. [38]. The α-amylase assay kits (Cat#BC0610) were ob­
tained from Solarbio Science & Technology Co., Ltd (Beijing, China).
The α-amylase-GO complex was prepared by mixing 250 μL of appro­
priately diluted α-amylase (10 mg/L) and 250 μL of GO suspensions
(from 0 to 100 mg/L). The reaction was started by adding 500 μL of
gelatinized starch (1%) solution to the prepared complex solution. The
solution was then incubated at 40 ◦ C for 5 min in a thermostatic water
bath shaker to avoid the effect of temperature change on enzyme ac­
tivity. And 1.0 mL of DNS reagent was added to stop the reaction. To
develop color, the solution was incubated in boiling water for 10 min
and cooled at room temperature. To remove the effect of the absorbance
of GO itself, 0.22 µm filter membranes were used to remove the GO from
the mixed suspensions before measuring the UV-Vis absorbance. The
UV-Vis absorbance was measured at 540 nm using Shimadzu UV-2600
spectrophotometer. To figure out the amount of reducing sugars
released in the reaction, a standard curve was prepared that plotted
absorbance against amount of reducing sugars. The control set of each
experiment was added ultrapure water instead of GO.
The percent relative activities of α-amylase and α-amylase-GO
complexes were calculated by the following equation [39]:
Relative activity (%) = (A1/A0)×100
where A1 and A0 are the absorbances at 540 nm with and without GO,
respectively.
2.3.9. Molecular docking analysis
Molecular docking between GO and α-amylase was conducted to
analyze their binding affinities and modes of interaction using Autodock
(http://autodock.scripps.edu/), a silico protein-ligand docking software
[40]. The 3D coordinates of α-amylase crystal structure (PDB ID, 1C8Q;
resolution, 2.30 Å) was obtained from the RCSB PDB database
(https://www.rcsb.org/). GO structure (PubChem CID: 163320950) was
determined from the PubChem database (https://pubchem.ncbi.nlm.
nih.gov) [41]. For the crystal structure of α-amylase, all water molecules
were excluded, and polar hydrogen atoms were added. After optimizing
the energy and adjusting the parameters of the force field, the
low-energy conformations satisfying the ligand structure were selected.
Finally, the docking results were analyzed and visualized using Dis­
covery Studio Visualizer and PyMOL software.
X. Liu et al.
3. Results and discussions
3.1. GO characterization
As illustrated in the HR-TEM and FE-SEM images in Fig. S1, the GO
material was consisted of wrinkled nanosheets with a thickness of single-
atom layer and a lateral size of about 100.0–2000 nm. As shown in
Fig. S2, the mean lateral size of GO measured by FE-SEM was 375.1 ±
137.9 nm. The average Dh of GO in pure water was 242.0 ± 1.400 nm
and slightly increased to 263.0 ± 7.400 nm in the PBS solution without
α-amylase. The zeta potential of the GO in pure water was − 48.20 ±
0.3000 mV and increased to − 28.30 ± 0.8000 mV in the PBS solution,
implying that GO was still well dispersed in the PBS solution. Fig. 1(A)
illustrates that the maximum UV-Vis absorption of GO was at 227 nm,
which corresponds to the π-π * transition within GO [42]. According to
Fig. 1(B), the value of ID/IG (the ratio of the intensity of D peak at 1343
cm− 1 to the intensity of G peak at 1576 cm− 1) in the Raman spectra was
0.9431. This ratio was lower than those of preceding studies
(1.550–1.780), demonstrating that the GO material prepared in this
study has a relatively higher graphitization level [43,44]. The FT-IR
spectrum of GO (Fig. 1(C)) reveals that there are abundant reactive
functional groups on its surface, including C– – O (1727 cm− 1), C– –C
(1624 cm− 1), C-OH (1400 cm− 1), and C-O-C (1050 cm− 1) [45,46]. The
H2O molecules adsorbed on the GO surface may be responsible for the
relatively wide absorption peak at 3425 cm− 1 in the FT-IR spectrum of
GO. The XPS spectrum of GO shows that the content ratio of C to O was
2.02. The C 1s spectrum (Fig. 1(D)) was convolved into 4 peaks with
binding energies of 284.4, 286.5, 287.8, and 288.8 eV, corresponding to
the C-C/C– – C, C-O-C/C-O, C– – O, and O-C– – O groups, respectively [47,
48], and their contents are listed in Table S2. The oxygen-containing
functional groups (i.e., C-O-C/C-O, C– – O, and O-C– – O) were predomi­
nant with a contribution of 52.36%. As determined by the elemental
Fig. 1. Characterization of GO: (A) UV-Vis spectrum of GO (20 mg/L in water). (B) Raman spectrum, (C) FT-IR spectrum, and (D) XPS spectrum of GO powder.
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Journal of Hazardous Materials 453 (2023) 131389
analyzer, the content of O (51.99%) was the highest among the elements
of GO, fortifying the high oxidation degree of GO (Table S3).
3.2. Adsorption of α-amylase on GO and interactions between them
Protein stability is critical for preserving its conformational structure
and biological function [49,50]. The respective UV-Vis and fluorescent
spectra of α-amylase in PBS did not change significantly compared with
those in water (Figs. S3 and S4), indicating that the protein was stable
and did not decompose under the testing conditions. The HR-TEM im­
ages (Fig. S5) also reveal that GO nanosheets were well dispersed in the
presence of 300 mg/L α-amylase. The calculated thickness of protein
adsorption layer on GO (Table S4) according to Ohshima’s soft particle
theory was 3.506 nm, implying a monolayer adsorption of α-amylase on
GO surface [51].
The pH did not change distinctly (~7.4) after adsorption, which
might be due to the buffering capacity of PBS. As shown in Fig. 2(A),
adsorption of α-amylase on GO surface reached saturation when
α-amylase concentration was above 500 mg/L, and the largest adsorp­
tion amount was about 2000 mg/g. The adsorption isotherms are illus­
trated in Fig. 2(B). The fitted isotherms of Langmuir and Freundlich
models are presented in Fig. 2(B) and S6 and the fitting parameters are
listed in Table S5. The fitting by Langmuir model (R2 = 0.9180) was
better than that by Freundlich model (R2 = 0.8625). This suggests that
the adsorption process mainly occurs via foration of monomolecular
layer on a homogeneous adsorbent surface [52], which is in agreement
with the calculated thickness of the protein adsorption layer. The
calculated maximum adsorption capacity (Qe, 2317 mg/g) fitted by
Langmuir model was consistent with the measured result
(~2000 mg/g). The Qe of α-amylase was much higher than that of other
proteins (e.g., 100.0 mg/g for trypsin and ~750.0 mg/g for BSA) at
similar pH and temperature, revealing that GO has stronger binding
X. Liu et al.
Fig. 2. (A) Adsorption of α-amylase on GO at 298 K. GO concentration was fixed at 100 mg/L and the initial α-amylase concentration varied from 50 to 1000 mg/L.
pH = 7.4. (B) Fitting adsorption isotherm of α-amylase on GO using Langmuir model. (C) Dh and (D) zeta potential of GO with and without α-amylase.
affinity with α-amylase [53,54]. Additionally, GO also exhibited much
higher adsorption capacity to α-amylase than other nanoparticles
(427.8 mg/g for ZnO and 276.2 mg/g for Fe3O4 nanoparticles) [55].
Hydrophobic interaction, hydrogen bonding, van der Waals force, and
electrostatic interaction force might contribute to the strong interactions
between them due to the amphipaths of both GO and α-amylase [56].
Besides, the aromatic functional groups of both GO and α-amylase could
induce π-π stacking between them [57]. With rising concentrations of
α-amylase, the Dh of GO increased slightly, while the GO zeta potential
became less negative (Fig. 2(C)(D)). Under the experimental pH (7.4),
α-amylase is negatively charged, but it still contains a certain amount of
positively charged amino acid residues (e.g., lysine) [58], which may
bind to the negatively charged functional groups of GO via electrostatic
attraction, lowering the absolute zeta potential of GO [59].
Fluorescence spectra were used to study the interaction between GO
and α-amylase, and the results are depicted in Fig. S7 and Fig. S8. The
maximum fluorescence emission peak of α-amylase was at 340 nm, and
its fluorescence spectral shape and maximum emission peak position did
not change after incubation with GO. GO showed negligible fluorescence
intensity, and thus the fluorescence of α-amylase-GO conjugates was
primarily originated from α-amylase [18]. A decline of fluorescence
intensity was observed as the GO concentrations rose under different
temperatures (277 K, 298 K, and 310 K), demonstrating that the
intrinsic fluorescence of α-amylase was quenched by GO. The F/F0 of
α-amylase with the addition of GO at the same concentration increased
with increasing temperatures (Fig. 3(A)), implying that the interaction
was inhibited at higher temperatures. As shown in Fig. S8(D), the
variation curves of F/F0 were similar at different incubation durations,
suggesting that the interaction of GO and α-amylase was strong and
quick.
The modified Stern-Volmer graphs (described in the Supplementary
Information) of α-amylase-GO conjugates at different temperatures are
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Journal of Hazardous Materials 453 (2023) 131389
depicted in Fig. 3(B). As listed in Table S6, the correlation constants of
the modified Stern-Volmer equation (Ka) decreased with increasing
temperatures, suggesting that α-amylase fluorescence was statically
quenched by GO and they formed a non-fluorescent stable ground-state
complex [60].
Time-resolved fluorescence spectroscopy was further conducted to
investigate the fluorescence quenching mechanism of α-amylase caused
by GO. Fluorescence decay traces of α-amylase in the absence and
presence of GO are presented in Fig. 3(C) and the fluorescence lifetime of
α-amylase was calculated using biexponential function [61]:
< τ >= b1 τ1 + b2 τ2
where < τ > is the average lifetime of the protein; τi and bi are the fitting
fluorescence lifetime and corresponding weighted proportion of fluo­
rescence quantum yield (Σbi = 1). The calculated τ values are listed in
the inset table in Fig. 3(C). In dynamic fluorescence quenching, the
compound elastically collides with fluorophore at its excited state and
the lifetime of fluorophore changes. On the contrary, the fluorescence
lifetime of fluorophore remains unchanged if static quenching occurs
and ground-state complex is formed between fluorophore and com­
pound [62]. The average fluorescence lifetime of α-amylase without and
with GO was similar, which directly reveals that the fluorescence of
α-amylase was statically quenched by GO and they formed stable
ground-state complex. This result was in good agreement with the re­
sults obtained from the modified Stern-Volmer equation [63].
Energy transfer between GO and α-amylase was calculated on the
basis of fluorescence resonance energy transfer (FRET) theory [64]. The
energy transfer efficiency (E), the binding distance (r) and binding dis­
tance taken at E = 50% (R0) were obtained by calculating the overlap
integral (J) of the GO absorption spectra and the α-amylase fluorescence
emission spectra (Fig. S9), and the results are shown in Table S7. The
X. Liu et al. Journal of Hazardous Materials 453 (2023) 131389

Fig. 3. (A) F/F0 of α-amylase with increasing GO concentration at different temperatures. (B) Modified Stern-Volmer plots of α-amylase-GO system. (C) Fluorescence
decay traces of α-amylase with and without GO. τ is the fluorescent lifetime of the protein and b is the normalized pre-exponential factor. The concentrations of
α-amylase and GO were 300 mg/L and 10 mg/L, respectively. (D) Van’t Hoff plot of α-amylase-GO system.

UV-Vis spectra of GO (Fig. S9(D)) remained almost the same under The interactions of GO and α-amylase were further inspected by UV-
different temperatures (277 K, 298 K, and 310 K). If the binding dis­ Vis absorption spectra, and the adsorption spectra of α-amylase as a
tance (r) is larger than the critical distance (~10 nm), the energy function of GO concentration are displayed in Fig. S10. The obvious
transfer efficiency is too low, and the energy transfer process is hardly to peaks at 205 nm and 280 nm of α-amylase corresponded to the main
occur. The r values in this study were all below 10 nm, magnifying that chain structures of the protein and the double bonds of the Tyr and Trp
the interaction of GO with α-amylase was strong enough to allow energy residues, respectively [70,71]. The absorption intensities of α-amylase at
transfer between them and also verified fluorescence quenching 205 and 280 nm were enhanced with the increase of GO concentration,
occurred between GO and α-amylase [65]. The binding distance which verified the formation of ground-state complex between GO and
(Table S7) was positively correlated with the incubation temperatures, α-amylase and the dominance of static quenching [72].
suggesting that the energy transfer was inhibited at higher tempera­
tures. These results reveal that the α-amylase-GO complex became less
3.3. Conformational structure change of α-amylase after interaction with
stable with increasing temperatures, and energy transfer mainly derived
GO
from static quenching [66].
The Van’t Hoff equation and thermodynamic equation (described in
FT-IR spectrometry could well reveal the functional group change of
the Supplementary Information) were used to calculate the enthalpy
α-amylase before and after incubated with GO (Fig. 4(A)). After incu­
change (ΔH), free-energy change (ΔG), and entropy change (ΔS) of GO
bation of GO with α-amylase, all the peaks migrated to longer wave­
complexation with α-amylase (Fig. 3(D)), and the results are listed in
numbers and the transmittance decreased, further magnifying the
Table S8. The negative values of ΔG (− 26.71, − 26.48, and − 26.34 KJ/
formation of ground-state complex [18]. The pure α-amylase displayed
mol) and ΔH (− 29.76 KJ/mol) reveal that the interaction of α-amylase
amide A band at 3388 cm− 1, amide B band at 2929 cm− 1, amide I band
and GO was spontaneous and exothermic. The ΔH, ΔG, and ΔS
at 1653 cm− 1, and amide II band at 1536 cm− 1, representing the
(− 11.02 J mol− 1 K− 1) were all negative, indicating that hydrogen
stretching vibrations of O-H superimposed on N-H, C-H, C– – O, and C-N
bonding and van der Waals force dominated the interaction of GO and
superimposed on N-H, respectively. After incubation with GO, the amide
α-amylase [67]. The negative values of ΔG (− 26.71, − 26.48, and
B and II band at 2929 and 1536 cm− 1 in the spectrum of α-amylase
− 26.34 KJ/mol) also indicate that the binding between GO and
became smooth and even disappeared, suggesting that GO might cover
α-amylase is spontaneous and stronger than that between GO and other
the surface of α-amylase and induce rearrangement of its polypeptide
proteins (ΔG= − 0.5499 and − 0.6662 KJ/mol for HSA, ΔG=
structure [18,73,74]. When the mass ratio of GO to α-amylase increased
− 23.14 KJ/mol for human hemoglobin) [68,69].
from 1:3 to 2:1, the band at 3388 cm− 1 of α-amylase shifted obviously to

6
X. Liu et al.
Fig. 4. (A) FT-IR spectra of GO, α-amylase, and α-amylase-GO conjugates. The concentrations of α-amylase were 50 and 300 mg/L, while the GO concentration was
fixed at 100 mg/L. (B) F/F0 in the synchronous fluorescence spectra of α-amylase (30 mg/L) as a function of GO concentration (0, 0.5, 1, 2.5, 5, 10, 25, and 50 mg/L).
The isometric three-dimensional contour spectra of (C) α-amylase and (D) α-amylase-GO conjugates. Concentrations of GO and α-amylase in the isometric three-
dimensional contour spectra were 10 and 30 mg/L, respectively.
longer wavenumber (3419 and 3420 cm− 1); while the band at
1653 cm− 1 in the α-amylase spectrum shifted to lower wavenumber
(1637 and 1628 cm− 1). The positions of both bands shifted, implying
that the peptide bonds of α-amylase might participate in the interaction
with GO.
Moreover, FT-IR as a semi-quantitative method was used to deter­
mine the variations in the secondary structure of protein before and after
incubated with GO, where the amide I region (1700–1600 cm− 1) cor­
responds to the secondary structure of the protein [32]. After decon­
volution and fitting, β-sheets (1640–1610 cm− 1), random coils
(1650–1640 cm− 1), α-helices (1660–1650 cm− 1), and β-turns
(~1700–1660 cm− 1) were observed [75]. After incubation of α-amylase
with GO, the total relative content of α-helices decreased from 31.17%
to 15.52%; that of β-sheets also declined from 39.15% to 29.49%
(Fig. S11 and Table S9). In contrast, the relative abundance of random
coils increased from 16.06% to 28.84%. These results suggest that
α-amylase transformed into a less compact and more flexible structure,
which might be attributed to its strong affinity with GO.
Synchronous fluorescence spectra of α-amylase as functions of GO
concentrations are illustrated in Fig. 4(B). The peak at 277.6 nm in the
spectrum of α-amylase without GO represented the Trp residue
(Δλ=60 nm) (Fig. S12(A)), whereas that at 288.8 nm corresponded to
the Tyr residue (Δλ=15 nm) (Fig. S12(B)). Both peak intensities of
α-amylase decreased with increasing GO concentration, suggesting that
GO could bind with both amino acid residues strongly. The fluorescence
7
Journal of Hazardous Materials 453 (2023) 131389
intensity of Trp residue was larger than that of Tyr residues, signifying
that Trp was accountable for the majority of the α-amylase intrinsic
fluorescence. The fluorescence intensity of Trp after binding with GO
decreased to a greater extent (from 6441 to 382.1) than that of Tyr (from
1090 to 80.34), implying that the binding sites were closer to Trp or/and
GO had stronger affinity with Trp than Tyr [76]. The emission spectra
peak of Tyr and Trp residues both displayed a slight red shift with the
increase of GO concentration, revealing that GO might cause change of
α-amylase conformational structure and reduce the microenvironment
hydrophobicity around Trp and Tyr residues [77].
3D fluorescence spectra can display visual conformational changes of
α-amylase after incubation with GO. Fig. S13(A) reveals that GO showed
little intensity of 3D fluorescence spectra. The isometric 3D contour
spectra and projections of α-amylase with and without GO are depicted
in Fig. 4 and Fig. S13, respectively. The wavelengths of the fluorescence
peaks and the corresponding fluorescence intensities are listed in
Table S10. Characteristic fluorescence peak 1 (λex/λem = 225/330) and
peak 2 (λex/λem = 275/335) in the spectra of pure α-amylase correspond
to Trp residue and peptide backbone structure of α-amylase, respectively
[78]. The intensity of peak 1 decreased significantly from 6586 to 2961
after addition of GO, while the decrease of peak 2 was from 6455 to
3516. The obvious decreases of peak 1 and 2 intensities reveal signifi­
cant changes in tertiary and secondary structures of α-amylase. Stokes
shift of peak 2 remained unchanged, showing that GO did not influence
the backbone of the protein distinctly. The larger Stokes shift of peak 1
X. Liu et al.
after incubation with GO suggested that a red shift of the emission peak
occurred and the microenvironment of Trp residues was altered by GO,
which was consistent with the results of synchronous fluorescence
analysis.
CD is an effective and sensitive method for investigating the
conformational changes of proteins. According to Fig. 5(A), two nega­
tive characteristic peaks were observed around 208 and 222 nm in all
CD spectra of α-amylase with addition of GO at different concentrations.
These two peaks come from n-π * and π-π * transitions of the peptide
bond inside the α-helix [79], and their absolute intensities were reduced
distinctly, implying the α-helix content of α-amylase decreased. The
negative band around 217 nm representing β-sheet also exhibited a shift
and decreased as the GO concentration increased from 0 to 75 mg/L.
The secondary structure variations obtained from the CD results are
consistent with the FT-IR fitting results in the amide I region. After in­
cubation with GO, the α-helical structure of α-amylase decreased by
~11.57%, and the β-sheet content declined by ~10.02%. The results of
FT-IR and CD both reveal that the α-helix and β-sheet contents of
α-amylase decreased due to interaction with GO. The declines of both
α-helix and β-sheet structure suggest that GO altered the secondary
structure and caused a much looser conformation of α-amylase.
ThT is a kind of cationic benzothiazole fluorescent dye with good
water solubility, high sensitivity, and stable affinity [80]. ThT binds
specifically with the aromatic amino acid of β-sheets to generate a
characteristic emission peak at 484 nm with the excitation wavelength
of 440 nm. As displayed in Fig. 5(B), the ThT fluorescence intensity of
α-amylase-GO conjugate at 484 nm decreased gradually as the concen­
tration of GO increased from 0 to 100 mg/L, implying that binding with
GO reduced the total content of β-sheet structure [81].
Conformational structure is the basis of protein function and
Fig. 5. (A) CD spectra of α-amylase (3000 mg/L) in the presence and absence of GO (0, 5, 40, and 75 mg/L). (B) ThT fluorescence of α-amylase (300 mg/L) as a
function of GO concentration (0–100 mg/L). (C) relative inhibition of enzymatic activity of α-amylase (10 mg/L) induced by GO with varying concentrations (20, 50,
and 100 mg/L).
8
Journal of Hazardous Materials 453 (2023) 131389
determines the biological activity of enzymes. If the secondary and
tertiary structure of α-amylase is destroyed, its catalytic activity may be
depressed. The effect of GO on the activity of α-amylase was explored by
in vitro incubation at physiological pH of 7.4. Fig. 5(C) demonstrates
that as the concentration of GO increased, the α-amylase activity became
lower. The enzymatic activity of α-amylase decreased by 9.420%,
28.91%, and 47.02% after incubation with GO at 20, 50, and 100 mg/L,
respectively. The hydrolysis of starch catalyzed by α-amylase is related
to its conformational structure. Therefore, the inhibited enzymatic ac­
tivity might be due to the significant structural change of α-amylase
[82]. Active amino residues of α-amylase are observed on the β-strands
of the β-barrel or on loops that connect a β-strand to the surrounding
helix [83]. The decreased contents of α-helix and β-sheet may imply the
destroy of α-amylase active sites and thus the loss of enzymatic activity.
Moreover, less α-helix and β-sheet contents may also lead to looser
structure of α-amylase. As a result, GO is more likely to competitively
cover the binding sites or active sites of α-amylase and substrate, pre­
venting subsequent substrate from binding reaction and decreasing
apparent enzyme activity [84].
3.4. Molecular docking analysis
The binding poses and interactions of GO (Fig. S14) with the protein
are obtained with Autodock software, and the corresponding binding
energy was calculated. The docking pocket is a hydrophilic pocket based
on the interaction modes of protein and GO. Formation of hydrogen
bonding was observed between the amino acid residue of α-amylase (i.
e., His299, Asp300, and His305) and the oxygen or hydrogen atoms of
GO, with the lowest binding energy (Fig. 6). The lowest binding energy
between α-amylase and GO was − 13.30 kcal/mol, implying that the
X. Liu et al.
Fig. 6. The lowest binding energy conformation of the surface binding mode of GO with α-amylase in molecular docking analysis.
binding interaction was spontaneous and a highly stable complex was
formed. The calculated binding energy was much lower than that be­
tween polystyrene nanoplastics and α-amylase (− 4.960 kcal/mol) and
between GO and trypsin (− 10.41 kcal/mol) [18,85], revealing there is a
very strong binding between GO and α-amylase. Fig. 6(D) illustrates that
there is a π-cation interaction between the aromatic benzene ring of GO
with His305. Moreover, π-π stacking and π-π T-shaped interactions were
also witnessed between the aromatic benzene rings of GO with Tyr151
and Trp59, respectively. It was reported that Glu233, Asp197, and
Asp300 residues were catalytic active sites of α-amylase [86]. The
interaction between Asp300 of α-amylase and GO indicates that the
catalytic activity of α-amylase was affected by GO. Trp59 and Asp300
are located in the α-helix secondary structure of α-amylase and their
binding with GO nanosheet verified the decline of α-helix content in
α-amylase after incubated with GO. In summary, both experimental and
computerized results magnify that GO binds with α-amylase strongly
primarily via hydrogen bonding, which significantly alters the biological
activity and conformational structure of α-amylase.
4. Conclusions
The interactions between GO and α-amylase, as well as the confor­
mation and biological activity changes of α-amylase induced by GO,
were thoroughly investigated in this study. GO could interact with
α-amylase strongly to produce ground-state complex, and thus quench
the intrinsic fluorescence of α-amylase in a static manner. The in vitro
and in silico findings reveal that GO interacts with α-amylase mainly via
hydrogen bonding, van der Waals force, and electrostatic interaction
spontaneously. The biological activity of α-amylase was strongly
inhibited by GO because of binding interaction and conformational
change. This research provides novel insight into the interaction be­
tween GO and α-amylase, which reveals the potential toxicity of GO in
physiological environment and is beneficial for risk assessment of GO in
biomedical fields.
9
Journal of Hazardous Materials 453 (2023) 131389
Environmental implication
Graphene oxide (GO), as a kind of typical two-dimensional nano­
material, is potential to accumulate in organisms and interact with
biomacromolecules. Our study found that GO forms ground-state com­
plex with α-amylase mainly through hydrogen bonding and van der
Waals force and quenches the intrinsic fluorescence of α-amylase.
Consequently, the secondary conformational structure of α-amylase was
altered, and its enzymatic activity was inhibited. The results imply that
GO might greatly affect the energy metabolism and cause subsequent
adverse effects to organisms.
Supplementary information
Additional methods and equations; HR-TEM and FE-SEM images of
GO (Fig. S1); GO lateral size distribution (Fig. S2); UV-Vis spectra of
α-amylase in PBS and water (Fig. S3); steady-state fluorescence spectra
of α-amylase in PBS and water (Fig. S4); HR-TEM images of GO with
α-amylase (Fig. S5); fitted results of Freundlich adsorption isotherm
model (Fig. S6); fluorescence spectra of α-amylase as a function of GO
concentration at different temperatures (Fig. S7); fluorescence spectra of
α-amylase as a function of GO concentration at different incubation
duration (Fig. S8); spectral overlap of GO absorption spectra and
α-amylase emission spectra (Fig. S9); UV-Vis difference adsorption
spectra (Fig. S10); curve-fitted amide I region (1700–1600 cm− 1) for
α-amylase (Fig. S11); the synchronous fluorescence spectra of α-amylase
with and without GO (Fig. S12); the isometric 3D contour spectra of GO
and the isometric 3D projections of α-amylase and α-amylase-GO system
(Fig. S13); 2D structure of GO (Fig. S14); typical physiochemical prop­
erties of α-amylase (Table S1); results of XPS (Table S2); results of
elemental analysis (Table S3); calculated adsorption layer (Table S4);
fitting parameters of Langmuir and Freundlich models (Table S5); Ka of
modified Stern-Volmer equation (Table S6); FRET parameters
(Table S7); thermodynamic parameters of different temperatures
X. Liu et al.
(Table S8); variation of relative content of secondary structures in
α-amylase (Table S9); results of the isometric 3D fluorescence spec­
troscopy (Table S10).
CRediT authorship contribution statement
Xinwei Liu: Investigation, Data curation, Writing - original draft.
Binbin Sun: Conceptualization, Methodology. Chunyi Xu: Visualiza­
tion, Software. Yinqing Zhang: Resources, Supervision. Lingyan Zhu:
Funding acquisition, Supervision, Writing - review & editing. Tianxu
Zhang: Visualization, Data curation.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgments
The authors gratefully acknowledge the National Natural Science
Foundation of China (grants 41991313, 42207435), Ministry of Science
and Technology (2022YFC3703200), the 111 program, Ministry of Ed­
ucation, China (T2017002), and the China Postdoctoral Science Foun­
dation (2020M680868). The authors would like to thank Li Meiyu from
Shiyanjia Lab (www.shiyanjia.com) for time-resolved fluorescence
spectroscopy test.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.jhazmat.2023.131389.
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