Research article Received: 11 August 2014,
Revised: 27 October 2014,
Accepted: 30 November 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2841
Inhibition effect of graphene oxide on the catalytic activity of acetylcholinesterase enzyme Yong Wang,a,b Yao Gu,b Yongnian Nia,b* and Serge Kokotc ABSTRACT: Variations in the enzyme activity of acetylcholinesterase (AChE) in the presence of the nano-material, graphene oxide (GO), were investigated with the use of molecular spectroscopy UV-visible and fluorescence methods. From these studies, important kinetic parameters of the enzyme were extracted; these were the maximum reaction rate, Vm, and the Michaelis constant, Km. A comparison of these parameters indicated that GO inhibited the catalytic activity of the AChE because of the presence of the AChE–GO complex. The formation of this complex was confirmed with the use of fluorescence data, which was resolved with the use of the MCR-ALS chemometrics method. Furthermore, it was found that the resonance light-scattering (RLS) intensity of AChE changed in the presence of GO. On this basis, it was demonstrated that the relationship between AChE and GO was linear and such models were used for quantitative analyses of GO. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: acetylcholinesterase; graphene oxide; catalysis; inhibition; molecular spectroscopy
Introduction Nano-particles and nano-materials are well known to interact with small organic molecules and bio-polymers. They also can penetrate cells,1 but differently prepared particles can react differently with bio-molecules (1,2). In this regard, nano-particles and nano-materials can regulate bio-molecular activity of proteins, e.g. serum albumin (3,4), and some enzymes (5–8). Such regulatory effects may ultimately influence cellular DNA. Consequently, investigations of the interactions of nano-particles and nano-materials with enzymes remain of considerable interest when carried out with bio–molecules in general, and biopolymers in particular. Acetylcholinesterase (AChE, a serine hydrolase) and acetylcholine (ACh) are both important neurological substances found in humans, and ACh can be catalyzed to choline (Ch) by AChE, and this catalytic reaction has been utilized for the development of a spectroscopic method for quantitative analysis of pesticides (9–11). The AChE catalyst has also been investigated in the presence of graphene oxide (GO), and its biological role was found to involve the termination of impulse transmissions at cholinergic synapses within the nervous system; this was achieved by hydrolysing the neurotransmitter, ACh. This AChE investigation has led to further studies involving therapeutic effects (12,13), the underpinning chemistry (14,15), as well as pesticides and plant protection (16). Also, an interesting finding in this work was that the catalysis of AChE controls the amount of the Ach available in the nervous system. Such regulation of this substance may have some influence on the progress of the Alzheimer’s disease (17,18). Recently, sensitive amperometric biosensors, developed for pesticide analysis, were constructed on the basis of immobilization of the AChE on a glassy carbon electrode (GCE), and Wang et al. (19) developed a novel AChE biosensor based on the deposition of zinc oxide nano-particles on carboxylic graphene. This
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electrode was able to analyse chlorpyrifos and carbofuran in the ranges of 1 × 10–8 to 1.0 × 10–13 M and 1.0 × 10–8 to 1.0 × 10–12 M, respectively, and their corresponding detection limits (DLs) were 5.0 × 10–14 M and 5.2 × 10–13. Li et al. (20) made a sensitive amperometric biosensor, which was fabricated by modifying a GCE that was coated with AChE immobilized on porous, reduced GO. This sensor successfully analysed carbaryl in the range of 0.001–0.050 μg mL–1 (DL: 0.5 ng mL–1). Zhai et al. (21) developed a novel AChE-based biosensor by immobilizing this enzyme on a GCE with the aid of Cu–Mg–Al double-layered hydroxide (CLDH). The biosensor detected chlorpyrifos in the concentration range of 0.05–150 μg L–1 (DL: 0.05 μg L–1). In this work, a Lineweaver–Burk plot was obtained with the use of spectrofluorimetry and spectrophotometry in order to study the AChE catalysis and to estimate the enzymatic activity in the presence of GO. Furthermore, two important kinetic parameters were calculated so as to investigate whether the activity of AChE can change in the presence or absence of GO. Additionally, to investigate the formation of AChE in the presence of GO, and to establish if there was any change in the
* Correspondence to: Yongnian Ni, Department of Chemistry, Nanchang University, Nanchang 330031, China. E-mail: [email protected] a
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China
b
Department of Chemistry, Nanchang University, Nanchang 330031, China
c
School of Chemistry, Physics and Mechanical Engineering, Queensland University of Technology, Brisbane 4001, Australia Abbreviations: AFM, atomic force microscope; DL, detection limits; DTNB, 5,5′-dithiobis(2-nitrobenzonic acid); EFA, evolving factor analysis; GCE, glassy carbon electrode; GO, graphene oxide; ITO, indium tin oxide; RLS, resonance light-scattering; SEM, scanning electron microscopy.
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Y. Gu et al. secondary structure of the protein, a titration method involving the addition of GO was investigated. The obtained fluorescence data were resolved with the aid of a powerful chemometrics method, i.e. the multivariate curve resolution–alternative least squares (MCR–ALS). The obtained fluorescence spectra and the concentration change of the reaction were used to study the formation process of the AChE–GO complex. Scanning electron microscopy (SEM) and resonance light-scattering techniques (RLS) were utilized to investigate any changes in the spectral profiles of AChE in the presence of GO; further, the effect of GO on AChE was quantitatively investigated with the use of RLS. AChE and the associated catalysis mechanism – background to the extraction and interpretation of the related kinetic parameters AChE is a powerful reagent that can catalyze the hydrolysis of ACh or, in this work, ATChI (acetylthiocholine iodide). The interaction between the above two reagents is illustrated by eqn 1: AChE þ ATChI→TCh þ CH3 COO– þ I–
(1)
The hydrolysis product of the above reaction is thiocholine, TCh, and it can react with the chromogenic reagent, 5,5′dithiobis(2-nitrobenzonic acid) (DTNB), to give a yellow product, which has a maximum absorbance at 412 nm: TCh þ DTNB→TNBðyellowÞ
(2)
Enzyme activity is expressed as the number of moles of substrate converted per unit time, and this can be used as a quantitative estimate of the amount of the active enzyme. Reaction yield is a function of the experimental conditions (22). In this work, the activity of AChE was reported in moles of ATChI, which can be hydrolysed by one mmol AChE per minute; this may be established from the amount of the formed yellow product, i.e. TNB (23). Two other important enzymatic, kinetic parameters are the maximum reaction rate, Vm, and the Michaelis-Menten constant, Km. They are defined by the Michaelis–Menten kinetics (24) – Km refers to the concentration of the substrate, i.e. ATChI, at which the rate of the enzymatic reaction is half the Vm. The above two parameters, Km, and Vm, can be calculated by the Lineweaver–Burk method: 1=V ¼ 1=V m þ ðK m =V m Þ=½ATChI
(3)
where V is the initial reaction rate and [ATChI] is the equilibrium concentration of the substrate. If 1/V versus [ATChI] graph is plotted, the intercept corresponds to 1/Vm, and the slope corresponds to Km / Vm. It is useful to note that Vm is related to the concentration of the enzyme, while Km is independent of this concentration. As an equilibrium parameter, Km, indicates the stability of the enzyme–substrate complex, i.e. the larger the value of Km, the more easily the complex decomposes; this implies that the affinity between the enzyme and the substrate is poor. For an inhibited enzyme reaction, the type of the inhibition can be inferred from a comparison of the Km and Vm values. Increasing Km values at a constant Vm indicates a competitive
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inhibition, i.e. the active site is occupied by the inhibiting reagent, while a constant Km and lower Vm values indicate uncompetitive inhibition.
Experimental Reagents and instrumentation Graphene oxide (oxygen = 20%) was purchased from Graphene Supermarket (Graphene Laboratories Inc. NY, U.S.). Acetylcholinesterase solution (AChE), acetylthiocholine iodide (ATChI) and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) were purchased from Asian Technology Development Co., Xiamen, China. Kolthoff buffer (25) was prepared by mixing a KH2PO4 solution (0.01 mol L–1) with boric acid (0.02 mol L–1) in a suitable volume ratio. AChE (500 U mg–1) was prepared from the AChE solution as provided, and this was stored at 5 °C. The activity of this solution was determined by the Ellman method (26). ATChI solution (8.99 mmol L–1) was prepared by dissolving the appropriate amount of the ATChI crystals in 10 mL double-distilled water. The DTNB solution (4.30 mmol L–1) was prepared by dissolving the appropriate amount of DTNB powder (42.6 g) in 25.0 mL Kolthoff buffer (pH 8.1). Ultraviolet–visible (UV–vis) spectra were collected on an Agilent 8453 UV–vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA); fluorescence spectra were collected on an LS-55 luminescence spectrometer (Perkin-Elmer Co., MA, USA) equipped with a thermostatic bath (Model ZC-10, Ningbo Tianheng Instruments Factory, China). The collected spectra were loaded onto FL-WinLab software (Perkin-Elmer). The resonance light-scattering spectra (RLS) were collected with the use of the Hitachi fluorescence spectrophotometer F-7000 (Hitachi Ltd, Tokyo, Japan). The SEM images were obtained by the JSM-6701 F field emission–scanning electron microscope (JEOL, Japan). Samples for SEM imaging were prepared by depositing the GO solution on indium tin oxide (ITO) electrodes, and then drying them in air. The operational power was 2.0 kV, and the sample magnification was × 50,000. The atomic force microscope (AFM) images were obtained with the use of an AJ-ΙΙΙ instrument (Shanghai Aijian Nanotechnology, China) in the tapping mode. Also, this set up produced simultaneously the intensity and phase data. Standard silicon cantilevers (spring constant: 0.6–6 N/m) were used at their resonance frequencies (typically 60–150 kHz). Samples for the AFM imaging were prepared by depositing 10 μL of the GO solution on freshly cleaved mica plates, which were dried overnight. The program for multivariate curve resolution was run on MATLAB (Version 6.5, Mathworks) (27).
Kinetic analysis of the enzymatic activity of AChE Enzyme activity estimation. A modified Ellman colorimetric method (26) was used to collect the measured data. The modification involved the concentration ratio, DTNB/ATChI, which should not be too high because otherwise the enzyme activity could be inhibited. Thus, AChE (20.0 μL) and DTNB (100.0 μL) solutions were added to a quartz cuvette containing 3.0 mL pH 8.1 buffer. The sample was then allowed to stand at 45 °C for 2 min, and 20.0 μL 8.99 mmol L–1 ATChI solution were added to the cuvette. The absorbance at 412 nm was recorded from 0 to 5 min. The catalytic reaction was also carried out under the same
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Inhibition effect of graphene oxide on catalytic activity of enzyme conditions, but in the presence of GO (11.0 μg mL–1), and the absorbance at 412 nm was recorded in the same range. A bulk sample was subsampled five times, and then each lot was treated with an aliquot of the AChE solution (100.0 μL), DTNB solution (200.0 μL), and 3.0 mL pH 8.1 buffer, respectively. The samples were kept at 45 °C for 2 min, and then, different amounts of ATChI were added to them. Thus, the initial concentrations of ATChI in the samples were 0.04, 0.08, 0.12, 0.16, and 0.20 mmol L–1, respectively. The absorbance of each of these samples was recorded in the range of 0–15 min. Also, a similar investigation was carried out with the same solutions but, in addition, each sample contained 11.0 mg L–1 GO. Enzyme kinetic parameters estimation.
Fluorescence analysis AChE solution (50 μL) and 3.0 mL Kolthoff buffer were added to a quartz cuvette. A GO solution (275.0 mg L–1) was added to the cuvette sequentially run-after-run; there were nine aliquots of 4.0 μL at a time. Thus, a total of 10 solutions was obtained with the GO concentrations of 0, 0.367, 0.733, …, 3.300 mg L–1, respectively. The resulting fluorescence emission spectra of each sample was recorded between 274.5–399.5 nm at 0.5 nm intervals resulting in a total 251 points, and also, between 293– 444.5 nm (total points: 303). The corresponding excitation wavelengths were 257 nm and 280 nm, respectively. The temperature was kept at 25 °C throughout the experiments, the slit width was set at 10 nm and the scanning rate was 800 nm min–1.
Results and discussion Optimisation of experimental conditions for the catalytic reaction Important variables for the catalytic reaction under investigation included enzyme’s (AChE) volume, the concentrations of the substrate (ATChI) and that of the chromogenic agent (DTNB), as well as the reaction temperature and pH of the reaction medium. Each of these factors was investigated individually while the others were kept constant. The results indicated that the catalyzed reaction increased almost linearly as a function of the increasing volume of the AChE (Fig. 1A). Also, the same reaction in the relatively narrow concentration range until about 1.0 × 10–4 mol L–1 ATChI increased linearly, then the plot began to curve until about 2 × 10–4 mol L–1 and finally, the reaction slowed down such that it reached equilibrium at about 4.0 × 10–4 mol L–1 enzyme (Fig. 1B). Conversely, similar changes in concentration of the chromogenic agent (DTNB) made no significant difference on the catalytic reaction range (Fig. 1C). In general, the effect of temperature between 35–55 °C was quite small with arguably the maximum absorbance value being recorded at 45 °C. However, after 50 °C, the absorbance fell away rather sharply suggesting that the most productive reaction temperature was at 45 °C (Fig. 1D). The change in absorption as a function of pH suggested that the reaction may be best performed at about pH 9 but this would be counterproductive for the solubility of another important reaction component, i.e. GO, which would actually be more soluble at somewhat lower pH. Consequently, reaction conditions for this work were chosen to be: volume AChE – 100.0 μL, concentration ATChI – 0.11 mmol L–1, concentration DTNB – 0.26 mmol L–1, temperature – 45 °C, and pH 8.1.
UV–vis and resonance light-scattering spectroscopic analyses The UV–vis spectra were collected from the AChE samples, which contained GO at different concentrations. The compositions of the samples were the same as those in the samples prepared for the fluorescence spectroscopy measurements (Section: Fluorescence analysis). These samples were also analysed with the use of the RLS measurements. These involved simultaneous scanning of the excitation and emission monochromators, i.e. Δλ = 0 nm). For these measurements, the excitation wavelength was: between 300–500 nm, the slit width – 10 nm, and the scanning speed – 800 nm min–1.
Inhibition of catalysis induced by GO Kinetic behavior of the catalytic reaction in the presence or absence of GO was investigated (see section of Enzyme kinetic parameters estimation). The dashed line plots (with GO; Fig. 2A) are relatively close together and their slopes are clearly similar or flatter than those of the solid lines (without GO). The latter lines clearly have higher slopes and are further apart from each other. Apparently, in the presence GO, the incremental change of the catalytic reaction, measured as ΔAbsorbance (versus time), was lower. This indicated that GO
Figure 1. Influence of the different conditions of the catalytic reaction on the absorbance values: (A) volume AChE, (B) concentration ATChI, (C) concentration DTNB, (D) temperature (°C), and (E) pH.
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Y. Gu et al. The effect of GO on the activity of the enzyme Tryptophan, tyrosine, phenylalanine, and other common amino acids, except CysH and CyS–SCy, do not contain sulfur, and all absorb in the 230–310 nm UV range. In the context of this work, such spectra of the AChE indicated that this enzyme absorbed at 220 and 268 nm, while GO absorbed at 231 nm. When GO was added to the AChE solution, the peak at 268 nm showed a blue shift and slowly disappeared (see Fig 3). This peak (268 nm) was the result of π–π* and n–π* transitions, which suggested that the GO molecule interacted with the AChE molecule. Additionally, fluorescent measurements provided further supporting information for the above conclusions from the UV–vis spectra. It was found that when a solution just containing AChE, was irradiated at 270 nm, a broad fluorescence band at 305 nm with a shoulder at 350 nm the presence of was observed (Figure not included). This may be due to the fluorescing substances present, including the amino acids in the AChE solution. However, no fluorescence
Figure 2. (A) Kinetic curves of the AChE catalytic reaction in the presence of (blue dashed lines) and the absence (red solid lines) of GO; (B) the corresponding Lineweaver–Burk plots, and the intercept of 1/Vm = 0.89 for both cases. Note that the concentrations of ATChI in the samples (from left to right) were 0.20, 0.16, –1 0.12, 0.08 and 0.04 mmol L , respectively.
Figure 3. UV–vis spectra of GO (black, dashed) and AChE with the addition of GO at different concentrations.
Table 1. Kinetic parameters of the AChE reaction in the absence and presence of GO System AChE AChE + GO
Activity (mmol min–1 μL–1) 0.31 0.21
Km (4) 0.73 1.30
Vm (10–3) 1.12 1.12
suppressed the catalytic reaction. This interpretation was supported by the slope values of the plots, which were regarded as the reaction rates, and these were clearly influenced by the presence of GO. Thus, the activities of the AChE for the two reactions involved, were calculated (Table 1) from the amount of ATChI (moles) hydrolysed by 1 μL AChE during a 1 min reaction time (mmol min–1 μL–1). These activity values indicated that in the presence of GO, the reaction rate decreased, i.e. the reaction was inhibited. The kinetic parameters, Km and Vm, were calculated with the use of the Lineweaver–Burk plot (data as in Fig. 2A). Results in Table 1 and those from the Lineweaver–Burk plot (Fig. 2B) showed that when GO was present in the catalyzed reaction, Km increased. This indicated that GO facilitated the formation and stability of the AChE– ATChI intermediate product. However, from Fig. 2B, the parameter, Vm (1/Vm = 0.89, and Vm = 1.12), for both lines indicated that the inhibition effects were competitive, i.e. GO occupied the active site of AChE in the catalytic process in such a way so as to prevent the formation of the AChE–ATChI intermediate.
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Figure 4. Emission fluorescence spectra of the AChE in the presence of GO with different concentrations, excited at (A) 257 nm and (B) 280 nm.
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Inhibition effect of graphene oxide on catalytic activity of enzyme
Figure 5. (A) Trends of the concentration changes (square and circle represent the concentrations of the AChE–GO and AChE, respectively). (B) The fluorescence spectral profiles recovered by the MCR-ALS from the fluorescence data of AChE in the presence of GO, with increasing concentration. Excitation wavelengths: 257 nm and 280 nm (blue profile corresponds to the AChE–GO samples, while the red profile corresponds to the AChE samples. Note: the dashed curve (real spectra) overlaps the solid one (recovered spectra) for AChE.
peak could be associated with GO because its fluorescence emission occurs usually at about 400 nm (28). For amino acids in general, the usual fluorescence excitation wavelength for phenylalanine is 257 nm, and for both tyrosine and tryptophan it is about 280 nm (29,30). The AChE samples with and without GO, were excited at these two wavelengths to obtain the corresponding emission spectra. From Figure 4(A, B), only the overlapped fluorescence spectra of these three amino acids can be observed. The peak at 302 nm (Fig. 4A) and that at 308 nm (Fig. 4B) mainly contained spectral contributions from tyrosine, and the 6 nm difference between them may have resulted from the differences in the excitation wavelengths (phenylalanine – 257 nm; tyrosine – 280 nm). Also, as the optimal excited wavelength for tryptophan is 280 nm, then only a shoulder peak at 350 nm (maximum emission) was observed (Fig. 4B). It was somewhat stronger than the one in Fig. 4A. Spectra from the 10 samples with different GO concentrations were collected, and thus, two fluorescence data matrices could be obtained: F257 (10 × 251, 10 – number of samples and 251 – number of emission wavelengths) and F280 (10 × 303, 10 – number of samples and 303 – number of the emission wavelengths). These spectra (Fig. 4) demonstrated that on addition of GO, the fluorescence intensity was quenched as a function of the amount of GO added. The matrices, F257 and F280, were combined (row-wise augmented) to form a new matrix, [F257 F280] (10 × 554), which was resolved with the use of the multivariate curve resolutionalternative least squares (MCR-ALS) method. The Evolving factor analysis (EFA) was used to extract the number of the fluorescence components in the system, and it was two. The fluorescent spectra and the concentration profiles of the two fluorescent components were recovered by iterative calculations
Figure 6. SEM images of the (A) AChE; (B) GO; and (C) AChE–GO; and tapping mode AFM image of GO sheet deposited on mica substrate.
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Y. Gu et al. (Fig. 5). The concentration trend of the two fluorescent components in the titration process (Fig. 5A), and the emission fluorescence spectra of the two fluorescent components were also recovered (Fig. 5B). The concentration of one component (red line) decreased in concentration and therefore, was most likely to represent AChE, while the other component was inferred to be the AChE–GO (blue line). The emission spectra of AChE–GO were similar in appearance to that from AChE. However, the width of the fluorescence peak of AChE was larger than that of AChE–GO. Also, a spectrum of an actual sample of AChE was obtained (Fig. 5B), and this spectrum was very similar to the recovered one. Consequently, it can be concluded that GO may bind with AChE and form a complex, i.e. AChE–GO, and the binding site was the active site of the AChE enzyme. Scanning electron microscopy (SEM) images were collected from AChE, GO, and AChE + GO samples (Fig. 6). The AChE image (Fig. 6A) displayed somewhat dispersed and quite globular structures, while the GO image appeared to show a relatively thick, wrinkly material, which seemed to form roughly circular inter-connected structures with wrinkled edges (Fig. 6B). The thickness and size of the GO sheet were estimated to be about 0.93 nm and 1.2 μm, respectively, by AFM imaging (Fig. 6D). When GO and AChE were combined to form the AChE–GO composite (Fig. 6C), the overall structure appeared to be somewhat less disorderly and less thick than that with the GO-only structures. In this case, the globular shaped material seemed to be clearly imbedded in a relatively flowing structure. The appearance suggested that the AChE particles were embedded in a refined mesh of the GO polymer.
Quantitative analysis of GO Firstly, the RLS analysis was applied to study the interaction between small molecules and the bio-macromolecule (31). The RLS is a useful technique for the investigation of the nature of solutions, especially the structure and the size of the solutes (32–34). The RLS intensity versus emission wavelength plot (Fig. 7) of AChE/GO solutions was characterized by a growing peak at 375 nm, but there was no evidence of a GO peak. Peak growth suggested the formation of an AChE–GO complex. The formation of the growing band was described by a linear model involving the RLS band intensity and the concentration of GO, i.e. RLS intensity = 168.3cGO + 437.06. Thus, the concentration of GO can be established. The associated DL was 0.69 mg L–1, and the regression coefficient was 0.9777.
Figure 7. Resonance light-scattering spectra of the AChE samples in the presence of GO at different concentrations.
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Conclusions Initially, the optimum conditions for the enzymatic reaction were established i.e. volume (AChE) – 100.0 μL, concentration (ATChI) – 0.11 mmol L–1, concentration (DTNB) – 0.26 mmol L–1, temperature – 45 °C, and pH – 8.1. Under these conditions the estimated enzyme activity was 0.31 mmol min–1 μL–1. The enzymatic reactions were carried out with or without the inclusion of GO, and the enzyme activity and kinetic parameters were calculated and compared accordingly. The key finding was that the activity of AChE was inhibited by GO, and the likely reason for that observation was that the active site of the enzyme was occupied by GO. Spectroscopic techniques (UV–vis and fluorescence) were used to investigate the role of AChE in catalized reactions, and in this context, the role of some fluorescing amino acid residuals as well as changes in the protein secondary structure were discussed. This led to an investigation of the complex formation, AChE–GO, with the use of a titration method, and the formation of this complex was confirmed. Furthermore, the use of the SEM and RLS techniques confirmed that GO does inhibit the AChE activity by occupying the active site of AChE. In addition, it was also shown that the RLS technique could be successfully used for quantitative analysis of GO. Acknowledgements This work was supported by the National Natural Science Foundation of China (NSFC-21065007 and NSFC-21305061), the Natural Science Foundation of Jiangxi Province (20132BAB213011), the Jiangxi Provincial Department of Education (GJJ13026), the State Key Laboratory of Chemo/Biosensing and Chemometrics of Hunan University (SKLCBC-2013010) and the State Key Laboratory of Food Science and Technology of Nanchang University (SKLF-ZZA201302 and SKLF-ZZB201303).
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22. Nomenclature Committee of the International Union of Biochemistry (NC-IUB). Units of enzyme activity. Eur J Biochem 1979; 97: 319–20. 23. Ellman GL, Courtney KD Jr, Andres V Featherstone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961; 7: 88–95. 24. Michaelis L, Menten ML. Die kinetik der invertinwirkung. Biochem Z 1913; 49: 333–69. 25. Kolthoff IM. A new set of buffer mixtures that can be prepared without the use of standardized acid or base. J Biol Chem 1925; 63: 135–41. 26. Komersova A, Komers K Cegan A. New findings about Ellman’s method to determine cholinesterase activity. Z Naturforsch 2007; 62(1–2): 150–4. 27. Multivariate Curve Resolution Homepage, http://www.cid.csic.es/homes/ rtaqam/tmp/WEB_MCR/welcome.htm, (accessed June 18, 2014). 28. Loh KP, Bao QL, Eda G Chhowalla M. Graphene oxide as a chemically tunable platform for optical applications. Nat Chem 2010; 2: 1015–24. 29. Zhang ZY, Yang JF, Ren Y, Gao LY. Simultaneous determination of tryptophan, phenylalanine and tyrosine by Kalman filtering in fluorospectrophotometry. Spectros Spectral Anal 1994;14:5–8 (in Chinese). 30. Kan C, Wu HL, Xie LX, Xiang SX Yu RQ. Direct quantitative analysis of aromatic amino acids in human plasma by four-way calibration using intrinsic fluorescence: Exploration of third-order advantages. Talanta 2014; 122: 293–301. 31. Pasternack RF, Bustamante C, Collings PJ, Giannetto A Gibbs EJ. Porphyrin assemblies on DNA as studied by a resonance light-scattering technique. J Am Chem Soc 1993; 115: 5393–9. 32. Qi HB, Bi N, Chen YH, Zheng X, Zhang HQ, Chen Y Tian Y. A DNA biosensor based on resonance light scattering using unmodified gold bipyramids. Mikrochim Acta 2012; 178: 131–7. 33. Bi SY, Wang Y, Pang B, Yan LL Wang TJ. An investigation on the interaction of DNA with hesperetin/apigenin in the presence of CTAB by resonance Rayleigh light scattering technique and its analytical application. Spectrochim Acta A 2012; 90: 158–64. 34. Wang F, Huang W, Jiang LY Tang B. Quantitative determination of protein based on strong fluorescence enhancement in curcuminchitosan-proteins systems. J Fluoresc 2012; 22: 615–22.
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Revised: 27 October 2014,
Accepted: 30 November 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2841
Inhibition effect of graphene oxide on the catalytic activity of acetylcholinesterase enzyme Yong Wang,a,b Yao Gu,b Yongnian Nia,b* and Serge Kokotc ABSTRACT: Variations in the enzyme activity of acetylcholinesterase (AChE) in the presence of the nano-material, graphene oxide (GO), were investigated with the use of molecular spectroscopy UV-visible and fluorescence methods. From these studies, important kinetic parameters of the enzyme were extracted; these were the maximum reaction rate, Vm, and the Michaelis constant, Km. A comparison of these parameters indicated that GO inhibited the catalytic activity of the AChE because of the presence of the AChE–GO complex. The formation of this complex was confirmed with the use of fluorescence data, which was resolved with the use of the MCR-ALS chemometrics method. Furthermore, it was found that the resonance light-scattering (RLS) intensity of AChE changed in the presence of GO. On this basis, it was demonstrated that the relationship between AChE and GO was linear and such models were used for quantitative analyses of GO. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: acetylcholinesterase; graphene oxide; catalysis; inhibition; molecular spectroscopy
Introduction Nano-particles and nano-materials are well known to interact with small organic molecules and bio-polymers. They also can penetrate cells,1 but differently prepared particles can react differently with bio-molecules (1,2). In this regard, nano-particles and nano-materials can regulate bio-molecular activity of proteins, e.g. serum albumin (3,4), and some enzymes (5–8). Such regulatory effects may ultimately influence cellular DNA. Consequently, investigations of the interactions of nano-particles and nano-materials with enzymes remain of considerable interest when carried out with bio–molecules in general, and biopolymers in particular. Acetylcholinesterase (AChE, a serine hydrolase) and acetylcholine (ACh) are both important neurological substances found in humans, and ACh can be catalyzed to choline (Ch) by AChE, and this catalytic reaction has been utilized for the development of a spectroscopic method for quantitative analysis of pesticides (9–11). The AChE catalyst has also been investigated in the presence of graphene oxide (GO), and its biological role was found to involve the termination of impulse transmissions at cholinergic synapses within the nervous system; this was achieved by hydrolysing the neurotransmitter, ACh. This AChE investigation has led to further studies involving therapeutic effects (12,13), the underpinning chemistry (14,15), as well as pesticides and plant protection (16). Also, an interesting finding in this work was that the catalysis of AChE controls the amount of the Ach available in the nervous system. Such regulation of this substance may have some influence on the progress of the Alzheimer’s disease (17,18). Recently, sensitive amperometric biosensors, developed for pesticide analysis, were constructed on the basis of immobilization of the AChE on a glassy carbon electrode (GCE), and Wang et al. (19) developed a novel AChE biosensor based on the deposition of zinc oxide nano-particles on carboxylic graphene. This
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electrode was able to analyse chlorpyrifos and carbofuran in the ranges of 1 × 10–8 to 1.0 × 10–13 M and 1.0 × 10–8 to 1.0 × 10–12 M, respectively, and their corresponding detection limits (DLs) were 5.0 × 10–14 M and 5.2 × 10–13. Li et al. (20) made a sensitive amperometric biosensor, which was fabricated by modifying a GCE that was coated with AChE immobilized on porous, reduced GO. This sensor successfully analysed carbaryl in the range of 0.001–0.050 μg mL–1 (DL: 0.5 ng mL–1). Zhai et al. (21) developed a novel AChE-based biosensor by immobilizing this enzyme on a GCE with the aid of Cu–Mg–Al double-layered hydroxide (CLDH). The biosensor detected chlorpyrifos in the concentration range of 0.05–150 μg L–1 (DL: 0.05 μg L–1). In this work, a Lineweaver–Burk plot was obtained with the use of spectrofluorimetry and spectrophotometry in order to study the AChE catalysis and to estimate the enzymatic activity in the presence of GO. Furthermore, two important kinetic parameters were calculated so as to investigate whether the activity of AChE can change in the presence or absence of GO. Additionally, to investigate the formation of AChE in the presence of GO, and to establish if there was any change in the
* Correspondence to: Yongnian Ni, Department of Chemistry, Nanchang University, Nanchang 330031, China. E-mail: [email protected] a
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China
b
Department of Chemistry, Nanchang University, Nanchang 330031, China
c
School of Chemistry, Physics and Mechanical Engineering, Queensland University of Technology, Brisbane 4001, Australia Abbreviations: AFM, atomic force microscope; DL, detection limits; DTNB, 5,5′-dithiobis(2-nitrobenzonic acid); EFA, evolving factor analysis; GCE, glassy carbon electrode; GO, graphene oxide; ITO, indium tin oxide; RLS, resonance light-scattering; SEM, scanning electron microscopy.
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Y. Gu et al. secondary structure of the protein, a titration method involving the addition of GO was investigated. The obtained fluorescence data were resolved with the aid of a powerful chemometrics method, i.e. the multivariate curve resolution–alternative least squares (MCR–ALS). The obtained fluorescence spectra and the concentration change of the reaction were used to study the formation process of the AChE–GO complex. Scanning electron microscopy (SEM) and resonance light-scattering techniques (RLS) were utilized to investigate any changes in the spectral profiles of AChE in the presence of GO; further, the effect of GO on AChE was quantitatively investigated with the use of RLS. AChE and the associated catalysis mechanism – background to the extraction and interpretation of the related kinetic parameters AChE is a powerful reagent that can catalyze the hydrolysis of ACh or, in this work, ATChI (acetylthiocholine iodide). The interaction between the above two reagents is illustrated by eqn 1: AChE þ ATChI→TCh þ CH3 COO– þ I–
(1)
The hydrolysis product of the above reaction is thiocholine, TCh, and it can react with the chromogenic reagent, 5,5′dithiobis(2-nitrobenzonic acid) (DTNB), to give a yellow product, which has a maximum absorbance at 412 nm: TCh þ DTNB→TNBðyellowÞ
(2)
Enzyme activity is expressed as the number of moles of substrate converted per unit time, and this can be used as a quantitative estimate of the amount of the active enzyme. Reaction yield is a function of the experimental conditions (22). In this work, the activity of AChE was reported in moles of ATChI, which can be hydrolysed by one mmol AChE per minute; this may be established from the amount of the formed yellow product, i.e. TNB (23). Two other important enzymatic, kinetic parameters are the maximum reaction rate, Vm, and the Michaelis-Menten constant, Km. They are defined by the Michaelis–Menten kinetics (24) – Km refers to the concentration of the substrate, i.e. ATChI, at which the rate of the enzymatic reaction is half the Vm. The above two parameters, Km, and Vm, can be calculated by the Lineweaver–Burk method: 1=V ¼ 1=V m þ ðK m =V m Þ=½ATChI
(3)
where V is the initial reaction rate and [ATChI] is the equilibrium concentration of the substrate. If 1/V versus [ATChI] graph is plotted, the intercept corresponds to 1/Vm, and the slope corresponds to Km / Vm. It is useful to note that Vm is related to the concentration of the enzyme, while Km is independent of this concentration. As an equilibrium parameter, Km, indicates the stability of the enzyme–substrate complex, i.e. the larger the value of Km, the more easily the complex decomposes; this implies that the affinity between the enzyme and the substrate is poor. For an inhibited enzyme reaction, the type of the inhibition can be inferred from a comparison of the Km and Vm values. Increasing Km values at a constant Vm indicates a competitive
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inhibition, i.e. the active site is occupied by the inhibiting reagent, while a constant Km and lower Vm values indicate uncompetitive inhibition.
Experimental Reagents and instrumentation Graphene oxide (oxygen = 20%) was purchased from Graphene Supermarket (Graphene Laboratories Inc. NY, U.S.). Acetylcholinesterase solution (AChE), acetylthiocholine iodide (ATChI) and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) were purchased from Asian Technology Development Co., Xiamen, China. Kolthoff buffer (25) was prepared by mixing a KH2PO4 solution (0.01 mol L–1) with boric acid (0.02 mol L–1) in a suitable volume ratio. AChE (500 U mg–1) was prepared from the AChE solution as provided, and this was stored at 5 °C. The activity of this solution was determined by the Ellman method (26). ATChI solution (8.99 mmol L–1) was prepared by dissolving the appropriate amount of the ATChI crystals in 10 mL double-distilled water. The DTNB solution (4.30 mmol L–1) was prepared by dissolving the appropriate amount of DTNB powder (42.6 g) in 25.0 mL Kolthoff buffer (pH 8.1). Ultraviolet–visible (UV–vis) spectra were collected on an Agilent 8453 UV–vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA); fluorescence spectra were collected on an LS-55 luminescence spectrometer (Perkin-Elmer Co., MA, USA) equipped with a thermostatic bath (Model ZC-10, Ningbo Tianheng Instruments Factory, China). The collected spectra were loaded onto FL-WinLab software (Perkin-Elmer). The resonance light-scattering spectra (RLS) were collected with the use of the Hitachi fluorescence spectrophotometer F-7000 (Hitachi Ltd, Tokyo, Japan). The SEM images were obtained by the JSM-6701 F field emission–scanning electron microscope (JEOL, Japan). Samples for SEM imaging were prepared by depositing the GO solution on indium tin oxide (ITO) electrodes, and then drying them in air. The operational power was 2.0 kV, and the sample magnification was × 50,000. The atomic force microscope (AFM) images were obtained with the use of an AJ-ΙΙΙ instrument (Shanghai Aijian Nanotechnology, China) in the tapping mode. Also, this set up produced simultaneously the intensity and phase data. Standard silicon cantilevers (spring constant: 0.6–6 N/m) were used at their resonance frequencies (typically 60–150 kHz). Samples for the AFM imaging were prepared by depositing 10 μL of the GO solution on freshly cleaved mica plates, which were dried overnight. The program for multivariate curve resolution was run on MATLAB (Version 6.5, Mathworks) (27).
Kinetic analysis of the enzymatic activity of AChE Enzyme activity estimation. A modified Ellman colorimetric method (26) was used to collect the measured data. The modification involved the concentration ratio, DTNB/ATChI, which should not be too high because otherwise the enzyme activity could be inhibited. Thus, AChE (20.0 μL) and DTNB (100.0 μL) solutions were added to a quartz cuvette containing 3.0 mL pH 8.1 buffer. The sample was then allowed to stand at 45 °C for 2 min, and 20.0 μL 8.99 mmol L–1 ATChI solution were added to the cuvette. The absorbance at 412 nm was recorded from 0 to 5 min. The catalytic reaction was also carried out under the same
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Inhibition effect of graphene oxide on catalytic activity of enzyme conditions, but in the presence of GO (11.0 μg mL–1), and the absorbance at 412 nm was recorded in the same range. A bulk sample was subsampled five times, and then each lot was treated with an aliquot of the AChE solution (100.0 μL), DTNB solution (200.0 μL), and 3.0 mL pH 8.1 buffer, respectively. The samples were kept at 45 °C for 2 min, and then, different amounts of ATChI were added to them. Thus, the initial concentrations of ATChI in the samples were 0.04, 0.08, 0.12, 0.16, and 0.20 mmol L–1, respectively. The absorbance of each of these samples was recorded in the range of 0–15 min. Also, a similar investigation was carried out with the same solutions but, in addition, each sample contained 11.0 mg L–1 GO. Enzyme kinetic parameters estimation.
Fluorescence analysis AChE solution (50 μL) and 3.0 mL Kolthoff buffer were added to a quartz cuvette. A GO solution (275.0 mg L–1) was added to the cuvette sequentially run-after-run; there were nine aliquots of 4.0 μL at a time. Thus, a total of 10 solutions was obtained with the GO concentrations of 0, 0.367, 0.733, …, 3.300 mg L–1, respectively. The resulting fluorescence emission spectra of each sample was recorded between 274.5–399.5 nm at 0.5 nm intervals resulting in a total 251 points, and also, between 293– 444.5 nm (total points: 303). The corresponding excitation wavelengths were 257 nm and 280 nm, respectively. The temperature was kept at 25 °C throughout the experiments, the slit width was set at 10 nm and the scanning rate was 800 nm min–1.
Results and discussion Optimisation of experimental conditions for the catalytic reaction Important variables for the catalytic reaction under investigation included enzyme’s (AChE) volume, the concentrations of the substrate (ATChI) and that of the chromogenic agent (DTNB), as well as the reaction temperature and pH of the reaction medium. Each of these factors was investigated individually while the others were kept constant. The results indicated that the catalyzed reaction increased almost linearly as a function of the increasing volume of the AChE (Fig. 1A). Also, the same reaction in the relatively narrow concentration range until about 1.0 × 10–4 mol L–1 ATChI increased linearly, then the plot began to curve until about 2 × 10–4 mol L–1 and finally, the reaction slowed down such that it reached equilibrium at about 4.0 × 10–4 mol L–1 enzyme (Fig. 1B). Conversely, similar changes in concentration of the chromogenic agent (DTNB) made no significant difference on the catalytic reaction range (Fig. 1C). In general, the effect of temperature between 35–55 °C was quite small with arguably the maximum absorbance value being recorded at 45 °C. However, after 50 °C, the absorbance fell away rather sharply suggesting that the most productive reaction temperature was at 45 °C (Fig. 1D). The change in absorption as a function of pH suggested that the reaction may be best performed at about pH 9 but this would be counterproductive for the solubility of another important reaction component, i.e. GO, which would actually be more soluble at somewhat lower pH. Consequently, reaction conditions for this work were chosen to be: volume AChE – 100.0 μL, concentration ATChI – 0.11 mmol L–1, concentration DTNB – 0.26 mmol L–1, temperature – 45 °C, and pH 8.1.
UV–vis and resonance light-scattering spectroscopic analyses The UV–vis spectra were collected from the AChE samples, which contained GO at different concentrations. The compositions of the samples were the same as those in the samples prepared for the fluorescence spectroscopy measurements (Section: Fluorescence analysis). These samples were also analysed with the use of the RLS measurements. These involved simultaneous scanning of the excitation and emission monochromators, i.e. Δλ = 0 nm). For these measurements, the excitation wavelength was: between 300–500 nm, the slit width – 10 nm, and the scanning speed – 800 nm min–1.
Inhibition of catalysis induced by GO Kinetic behavior of the catalytic reaction in the presence or absence of GO was investigated (see section of Enzyme kinetic parameters estimation). The dashed line plots (with GO; Fig. 2A) are relatively close together and their slopes are clearly similar or flatter than those of the solid lines (without GO). The latter lines clearly have higher slopes and are further apart from each other. Apparently, in the presence GO, the incremental change of the catalytic reaction, measured as ΔAbsorbance (versus time), was lower. This indicated that GO
Figure 1. Influence of the different conditions of the catalytic reaction on the absorbance values: (A) volume AChE, (B) concentration ATChI, (C) concentration DTNB, (D) temperature (°C), and (E) pH.
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Y. Gu et al. The effect of GO on the activity of the enzyme Tryptophan, tyrosine, phenylalanine, and other common amino acids, except CysH and CyS–SCy, do not contain sulfur, and all absorb in the 230–310 nm UV range. In the context of this work, such spectra of the AChE indicated that this enzyme absorbed at 220 and 268 nm, while GO absorbed at 231 nm. When GO was added to the AChE solution, the peak at 268 nm showed a blue shift and slowly disappeared (see Fig 3). This peak (268 nm) was the result of π–π* and n–π* transitions, which suggested that the GO molecule interacted with the AChE molecule. Additionally, fluorescent measurements provided further supporting information for the above conclusions from the UV–vis spectra. It was found that when a solution just containing AChE, was irradiated at 270 nm, a broad fluorescence band at 305 nm with a shoulder at 350 nm the presence of was observed (Figure not included). This may be due to the fluorescing substances present, including the amino acids in the AChE solution. However, no fluorescence
Figure 2. (A) Kinetic curves of the AChE catalytic reaction in the presence of (blue dashed lines) and the absence (red solid lines) of GO; (B) the corresponding Lineweaver–Burk plots, and the intercept of 1/Vm = 0.89 for both cases. Note that the concentrations of ATChI in the samples (from left to right) were 0.20, 0.16, –1 0.12, 0.08 and 0.04 mmol L , respectively.
Figure 3. UV–vis spectra of GO (black, dashed) and AChE with the addition of GO at different concentrations.
Table 1. Kinetic parameters of the AChE reaction in the absence and presence of GO System AChE AChE + GO
Activity (mmol min–1 μL–1) 0.31 0.21
Km (4) 0.73 1.30
Vm (10–3) 1.12 1.12
suppressed the catalytic reaction. This interpretation was supported by the slope values of the plots, which were regarded as the reaction rates, and these were clearly influenced by the presence of GO. Thus, the activities of the AChE for the two reactions involved, were calculated (Table 1) from the amount of ATChI (moles) hydrolysed by 1 μL AChE during a 1 min reaction time (mmol min–1 μL–1). These activity values indicated that in the presence of GO, the reaction rate decreased, i.e. the reaction was inhibited. The kinetic parameters, Km and Vm, were calculated with the use of the Lineweaver–Burk plot (data as in Fig. 2A). Results in Table 1 and those from the Lineweaver–Burk plot (Fig. 2B) showed that when GO was present in the catalyzed reaction, Km increased. This indicated that GO facilitated the formation and stability of the AChE– ATChI intermediate product. However, from Fig. 2B, the parameter, Vm (1/Vm = 0.89, and Vm = 1.12), for both lines indicated that the inhibition effects were competitive, i.e. GO occupied the active site of AChE in the catalytic process in such a way so as to prevent the formation of the AChE–ATChI intermediate.
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Figure 4. Emission fluorescence spectra of the AChE in the presence of GO with different concentrations, excited at (A) 257 nm and (B) 280 nm.
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Inhibition effect of graphene oxide on catalytic activity of enzyme
Figure 5. (A) Trends of the concentration changes (square and circle represent the concentrations of the AChE–GO and AChE, respectively). (B) The fluorescence spectral profiles recovered by the MCR-ALS from the fluorescence data of AChE in the presence of GO, with increasing concentration. Excitation wavelengths: 257 nm and 280 nm (blue profile corresponds to the AChE–GO samples, while the red profile corresponds to the AChE samples. Note: the dashed curve (real spectra) overlaps the solid one (recovered spectra) for AChE.
peak could be associated with GO because its fluorescence emission occurs usually at about 400 nm (28). For amino acids in general, the usual fluorescence excitation wavelength for phenylalanine is 257 nm, and for both tyrosine and tryptophan it is about 280 nm (29,30). The AChE samples with and without GO, were excited at these two wavelengths to obtain the corresponding emission spectra. From Figure 4(A, B), only the overlapped fluorescence spectra of these three amino acids can be observed. The peak at 302 nm (Fig. 4A) and that at 308 nm (Fig. 4B) mainly contained spectral contributions from tyrosine, and the 6 nm difference between them may have resulted from the differences in the excitation wavelengths (phenylalanine – 257 nm; tyrosine – 280 nm). Also, as the optimal excited wavelength for tryptophan is 280 nm, then only a shoulder peak at 350 nm (maximum emission) was observed (Fig. 4B). It was somewhat stronger than the one in Fig. 4A. Spectra from the 10 samples with different GO concentrations were collected, and thus, two fluorescence data matrices could be obtained: F257 (10 × 251, 10 – number of samples and 251 – number of emission wavelengths) and F280 (10 × 303, 10 – number of samples and 303 – number of the emission wavelengths). These spectra (Fig. 4) demonstrated that on addition of GO, the fluorescence intensity was quenched as a function of the amount of GO added. The matrices, F257 and F280, were combined (row-wise augmented) to form a new matrix, [F257 F280] (10 × 554), which was resolved with the use of the multivariate curve resolutionalternative least squares (MCR-ALS) method. The Evolving factor analysis (EFA) was used to extract the number of the fluorescence components in the system, and it was two. The fluorescent spectra and the concentration profiles of the two fluorescent components were recovered by iterative calculations
Figure 6. SEM images of the (A) AChE; (B) GO; and (C) AChE–GO; and tapping mode AFM image of GO sheet deposited on mica substrate.
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Y. Gu et al. (Fig. 5). The concentration trend of the two fluorescent components in the titration process (Fig. 5A), and the emission fluorescence spectra of the two fluorescent components were also recovered (Fig. 5B). The concentration of one component (red line) decreased in concentration and therefore, was most likely to represent AChE, while the other component was inferred to be the AChE–GO (blue line). The emission spectra of AChE–GO were similar in appearance to that from AChE. However, the width of the fluorescence peak of AChE was larger than that of AChE–GO. Also, a spectrum of an actual sample of AChE was obtained (Fig. 5B), and this spectrum was very similar to the recovered one. Consequently, it can be concluded that GO may bind with AChE and form a complex, i.e. AChE–GO, and the binding site was the active site of the AChE enzyme. Scanning electron microscopy (SEM) images were collected from AChE, GO, and AChE + GO samples (Fig. 6). The AChE image (Fig. 6A) displayed somewhat dispersed and quite globular structures, while the GO image appeared to show a relatively thick, wrinkly material, which seemed to form roughly circular inter-connected structures with wrinkled edges (Fig. 6B). The thickness and size of the GO sheet were estimated to be about 0.93 nm and 1.2 μm, respectively, by AFM imaging (Fig. 6D). When GO and AChE were combined to form the AChE–GO composite (Fig. 6C), the overall structure appeared to be somewhat less disorderly and less thick than that with the GO-only structures. In this case, the globular shaped material seemed to be clearly imbedded in a relatively flowing structure. The appearance suggested that the AChE particles were embedded in a refined mesh of the GO polymer.
Quantitative analysis of GO Firstly, the RLS analysis was applied to study the interaction between small molecules and the bio-macromolecule (31). The RLS is a useful technique for the investigation of the nature of solutions, especially the structure and the size of the solutes (32–34). The RLS intensity versus emission wavelength plot (Fig. 7) of AChE/GO solutions was characterized by a growing peak at 375 nm, but there was no evidence of a GO peak. Peak growth suggested the formation of an AChE–GO complex. The formation of the growing band was described by a linear model involving the RLS band intensity and the concentration of GO, i.e. RLS intensity = 168.3cGO + 437.06. Thus, the concentration of GO can be established. The associated DL was 0.69 mg L–1, and the regression coefficient was 0.9777.
Figure 7. Resonance light-scattering spectra of the AChE samples in the presence of GO at different concentrations.
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Conclusions Initially, the optimum conditions for the enzymatic reaction were established i.e. volume (AChE) – 100.0 μL, concentration (ATChI) – 0.11 mmol L–1, concentration (DTNB) – 0.26 mmol L–1, temperature – 45 °C, and pH – 8.1. Under these conditions the estimated enzyme activity was 0.31 mmol min–1 μL–1. The enzymatic reactions were carried out with or without the inclusion of GO, and the enzyme activity and kinetic parameters were calculated and compared accordingly. The key finding was that the activity of AChE was inhibited by GO, and the likely reason for that observation was that the active site of the enzyme was occupied by GO. Spectroscopic techniques (UV–vis and fluorescence) were used to investigate the role of AChE in catalized reactions, and in this context, the role of some fluorescing amino acid residuals as well as changes in the protein secondary structure were discussed. This led to an investigation of the complex formation, AChE–GO, with the use of a titration method, and the formation of this complex was confirmed. Furthermore, the use of the SEM and RLS techniques confirmed that GO does inhibit the AChE activity by occupying the active site of AChE. In addition, it was also shown that the RLS technique could be successfully used for quantitative analysis of GO. Acknowledgements This work was supported by the National Natural Science Foundation of China (NSFC-21065007 and NSFC-21305061), the Natural Science Foundation of Jiangxi Province (20132BAB213011), the Jiangxi Provincial Department of Education (GJJ13026), the State Key Laboratory of Chemo/Biosensing and Chemometrics of Hunan University (SKLCBC-2013010) and the State Key Laboratory of Food Science and Technology of Nanchang University (SKLF-ZZA201302 and SKLF-ZZB201303).
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