Inhibition of immobilized acetylcholinesterase by aflatoxin B 1 in potentiometric biosensor

© 2016 K. V. Stepurska et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited


Introduction
Aflatoxins, the secondary metabolites of molds, are the life-threatening mycotoxins.An acute toxic effect of aflatoxins is associated with the fact that they are one of the most powerful hepatotropic poisons.Even a low level of these toxins in the diet may be detrimental for the human health.Aflatoxins are produced by some strains of microscopic fungi of the genus Aspergillus.They are able to grow and form toxins on various natural substrates such as raw materials for food products, fodder and other foodstuffs.
Under natural conditions, aflatoxins in large amounts occur most commonly in peanuts and corn; they can also accumulate in various nuts, oilseeds, wheat, barley, cocoa and coffee beans.Notably, aflatoxins practically are not destroyed in the course of ordinary cooking and food processing (the destruction point of aflatoxins is 268-269 °C).Currently, the aflatoxin family is represented by four natural aflatoxins B1, B2, G1, G2 and more than 10 compounds, which are the metabolites or derivatives of the main group (M1, M2, V2A, G2a, GM1, P1 , Q1, etc.) [1].ISSN 1993-6842 (on-line); ISSN 0233-7657 (print) Biopolymers and Cell. 2016. Vol. 32.N 4. P 271-278 doi: http://dx.doi.org/10.7124/bc.000929 The members of the blue (B) fluorescent group (aflatoxins B1 and B2) are characterized by the fusion of a cyclopentenone ring with a lactone ring of the coumarin moiety, whereas the green (G) fluorescent toxins (aflatoxins G1 and G2) contain a fusedring lactone [1].The capability of fluorescence determined almost all physical and chemical methods of quantitative and qualitative detection of aflatoxins, which include TLC [2], HPLC [3] and different combinations of liquid chromatography with mass spectrometry [4].
An alternative method of determining aflatoxin is an inhibitory analysis using the enzyme biosensors based on acetylcholinesterase (АChЕ) [5][6][7][8].The biosensor functioning is based on the following enzymatic reaction: In the course of enzymatic reaction (1), acetylcholinesterase decomposes acetylcholine to choline and acetic acid.Acetic acid dissociates, thus increasing the local concentration of protons in the working membrane.This, in turn, leads to changes in the solution pH close to the transducer surface, which is registered by a potentiometric transducer.Aflatoxins can inhibit the acetylcholinesterase activity and it is the basis for the development of AChE-biosensors for the aflatoxin determination.
The inhibitors may be reversible or irreversible.The reversible inhibitors weakly interact with their target enzyme and are easily removed, whereas the irreversible inhibitors form strong stable interactions and can be removed only chemically (if can be removed at all).The reversible inhibition reaches equilibrium depending on the inhibition constant, which characterizes the enzyme affinity for the inhibitor.The irreversible inhibition reaches maximum when the enzyme is completely bound with the inhibitor.The reversible inhibitors are further classified into competitive, noncompetitive, uncompetitive and mixed types according to the mechanism of their action.
The present work is devoted to a detailed study on biochemical properties of the inhibition of immobilized acetylcholinesterase by aflatoxin B1 within the potentiometric biosensor.To ascertain a type of inhibition is important for a better understanding of the mechanism of enzyme inhibition in biosensor [14].The updating of some inhibitory characteristics will also allow the optimization of the AChE-based biosensors for aflatoxin determination.

Materials
Acetylcholinesterase (AChE) from electric eel (EC 3.1.1.7),the activity of 426 U/mg, was immobilized on the surface of potentiometric transducer by the method of covalent crosslinking of enzyme in the vapour of 50 % aqueous solution of glutaraldehyde (GA) ("Sigma-Aldrich Chemie", Germany).Bovine serum albumin (BSA, fraction V) and glycerol of domestic production were used as additional substances for the enzyme immobilisation.

Potentiometric transducers
In the work, we used the portable measuring device and sensor chips fabricated at the V.Ye.Lashkaryov Institute of Semiconductor Physics (Kiev, Ukraine).The sensor chips contain the differential pair of pchannel transistors on the monocrystalline silicon substrate of total area 8x8 mm 2 .The sensor elements demonstrated the intrinsic pH-sensitivity of approximately 40 mV/рН and transconductance of 400-500 μA/V, thus providing pH-sensitivity of the transistor channel current of 15-20 μA/pH.The response of pH-FET sensor was measured by means of the current-to-voltage converter circuit with the sensors working in the current source mode with active load.The threshold voltage of the used pH-FETs was about -2.5 V.The measurements were performed with the initial channel current magnitude of approximately 500 μA, drain-to-source voltage of approximately 2 V, transistor bulk connected to the source [17].

Preparation and immobilization of bioselective elements
To prepare working enzyme membranes the following solution was used: 1 % acetylcholinesterase, 4 % BSA and 10 % glycerol in 20 mM phosphate buffer, pH 7. The mixture for reference membrane was prepared in the same way, but no enzyme was used, only BSA and glycerol of final concentrations 5 % and 10 % correspondingly were solved in 20 mM phosphate buffer, pH 7.After deposition of the prepared mixture on the working surface of potentiometric transducers, the latter were placed in saturated glutaraldehyde vapour for 20 min and then kept for 5 min in air at room temperature.Prior to the operation, the membranes were washed with buffer solution to remove the excess of unbound components.

Procedure of measurements
The measurements were carried out at room temperature in 5 mM phosphate buffer, pH 6.5, or in 2.5 mM "polymix" buffer of different pH, in an open cell with constant stirring.The substrate and inhibitor concentrations in the working cell were achieved by addition of aliquots of the corresponding stock solutions (Fig. 1).
The level of enzyme inhibition (I %) was evaluated by comparing the biosensor responses to the substrate concentration before (А 0 ) and after (А і ) inhibition according to the formula:

Results and Discussion
pH of the working buffer is a key parameter that is expected to affect the biosensor performance, since it is known to have a major impact on the enzyme stability and activity.In this study, 2.5 mM «polymix» working buffer was used to regulate pH value in the 4.5 to 9.5 range.Buffer concentration of 2.5 mM was chosen as it allowed getting sufficient biosensor response whereas at higher concentrations the signal decreased significantly.The biosensor response to 4 mM AChCl and the level of inhibition by 2 μg/ml AFB1 were measured changing pH by 0.5 unit at each measurement.The results are depicted in Fig. 2.
As can be seen, the optimal pH values for biosensor response operation were observed in the 6.5-8.5 range whereas the highest levels of inhibition were detected between pH 4.5 and 6.5.The best compromise was therefore achieved at pH 6.5.
Analyzing the type of inhibition of immobilized AChE by aflatoxin, the conclusion can be made about the nature of interaction.After inhibition, the biosensor response and, thus, the biomembrane activity can be completely restored by ordinary washing with the working buffer, which indicates weak aflatoxin-enzyme interaction, i.e. aflatoxin is a reversible inhibitor.
A graphical method that allows the determination of the type of reversible inhibition has been recently reported in the literature [13].In this method, a number of calibration curves are plotted, demonstrating the dependence of the degree of the immobilized enzyme inhibition in biosensor on different concentrations of the inhibitor.Each curve is plotted at a fixed value of substrate concentration, for instance, the substrate concentration equal to the K m value or the saturating concentration.For each curve, the degree of inhibition I 50 is determined.It is numerically identical to the inhibitor concentration, at which the degree of immobilized enzyme inhibition in biosensor is 50 %.Next, an analysis of the changes in the calibration curve and the inhibition degree I 50 allows the conclusion regarding the type of inhibition.If I 50 increases with increasing the substrate concentration, and the calibration curve shifts to the higher concentration range, it means that the inhibition is of competitive type.If I 50 decreases with increasing substrate concentration and the calibration curve shifts to the lower concentration range, it corresponds to the uncompetitive type of inhibition.In the case of the noncompetitive type of inhibition, I 50 and calibration curve do not change.Finally, in the case of inhibition of mixed type, I 50 increases with increasing substrate concentration likely to the competitive type of inhibition, but the calibration curve shift is significantly lower.For example, a 10-fold increase in the substrate concentration causes an increase of I 50 for the competitive type by 5.5 times whereas for the mixed type -by only 1.5 times.
Prior to the determination of the type of reversible inhibition, it was necessary to evaluate the apparent Michaelis constant K m app for immobilized AChE.Basically, the Michaelis constant for native enzymes is determined as half the maximum initial rate.With regard to the immobilized enzymes as the biosensor constituents, the estimation of this rate is almost impossible.It should be taken into account that it is inconsistent to equate the speed of biosensor response with the rate of biochemical reaction in the membrane, as the former is defined by certain electrochemical peculiarities of the registration method Fig. 2. Influence of pH on the AChE-based biosensor response (1) and inhibition level in the presence of 2 μg/ml aflatoxin B1 (2).Measurements were performed in 2.5 mM "polymix" buffer at room temperature.Fig. 3. Dependence of AChE-biosensor response on acetylcholine concentration.Measurements were performed in 5 mM phosphate buffer, pH 6.5, at room temperature.and diffusion properties.Considering the fact that the time of responses of the biosensor at all substrate concentrations is almost the same, we assume that it would be appropriate to apply directly the value of biosensor response for plotting the graphs as it is proportional to the initial rate of the enzymatic reaction with use of immobilized enzyme.Therefore, to determine the Michaelis constant for the enzyme immobilized on the surface of the potentiometric transducer, the curves of dependence of the biosensor response on the AChCl concentration were plotted.The data obtained are presented in both direct (Fig. 3) and inverse (Fig. 4) coordinates.
The graph (Fig. 5) shows that the maximum biosensor response is 77 μA, i.e. the maximum initial rate V max is proportional to this value.Determining the apparent Michaelis constant as half the maximum rate, we obtain K m app = 2.5 mM.Another method of the Michaelis constant determination suggests the presentation of the same data in inverted Lineweaver-Burk coordinates (Fig. 6).The obtained straight line cuts off on the X-and Y-axes the reciprocal values K m and V max , respectively.The equation of the straight line obtained (Fig. 6) is: y = 0.0334 x + 0.0116.From this equation, the reciprocal value 1/K m app = 0.3475.The apparent Michaelis constant determined by this method is K m app = 2.8 mM.Thus, the obtained apparent Michaelis constant of acetylcholinesterase immobilized on the surface of the potentiometric transducer was 2.3 mM or 2.8 mM, depending on the method of determination.For convenience, K m app = 2.5 mM was used further on.
For the next experiment on determining the type of inhibition of immobilized AChE by the "degree of inhibition" method, the substrate concentrations of 2.5 mM and 20 mM were fixed.They corresponded respectively to the apparent Michaelis constant K m app and 8K m app , which is saturating concentration.The calibration curves for aflatoxin B1 determination (dependence of degree of inhibition on the inhibitor concentration) were plotted for two fixed substrate concentrations (Fig. 5).
As seen, with an increase of the substrate concentration, I 50 also increases.Graphically, this corresponds to a rightward shift of the calibration curve.Since the shift is slight and the ratio of I 50 at substrate concentration 8K m app (I 50 = 21 μg/ml) to I 50 at substrate concentration K m app (I 50 = 10.5 μg/ml) is 2,  the conclusion can be made that the inhibition is of the mixed type.
It was interesting to confirm the obtained results using one of the traditional methods of identifying the type of inhibition, such as the Lineweaver-Burk, Dixon, Cornish-Bowden, Eadie-Hofstee methods.They are based on the construction of the Michaelis-Menten equation in different coordinate systems: 1/V versus 1/[S], v versus V/[S], 1/V versus [I] and [S]/V ([I]) respectively, where V -an initial rate of the enzymatic reaction, [S], [I] -concentrations of substrate and inhibitor respectively.We chose the Lineweaver-Burk method as the most commonly used.To invert the Michaelis-Menten equation into the Lineweaver-Burk plot it is necessary to determine the initial rates of the enzymatic reaction at different substrate concentrations in the presence of inhibitor and without it and to plot the 1/V vs 1/[S] graphs.The type of inhibition can then be identified by the analysis of these lines.For competitive inhibition, the lines intersection should be on the Y-axis (1/V), meaning that the maximum rate does not change, and the apparent Michaelis constant increases.For noncompetitive inhibition, the lines intersection should be on the X-axis (1/[S]), showing that in this case, in contrast, the apparent Michaelis constant remains unchanged, and the maximum rate decreases.For uncompetitive inhibition, both the maximum rate and apparent Michaelis constant decrease by the same times, graphically it looks like parallel straight lines.And finally, for mixed inhibition, the maximum speed decreases and the apparent Michaelis constant can either increase or decrease, therefore graphically the lines intersection should be not on X-and Y-axes.
During the experiment, the changes in the biosensor response to different substrate concentrations were investigated with no inhibitor as well as in the presence of various aflatoxin B1 concentrations (5 μg/ml and 10 μg/ml).The experimental results were presented in the double-inverted Lineweaver-Burk coordinates (Fig. 6).
From the equations of the regression lines (Fig. 6), the apparent Michaelis constants of immobilized AChE can be calculated in the absence of inhibitor (K m app = 2.8) and in the presence of 5 μg/ml inhibitor AFB1 (K m app = 2.1) and 10 μg/ml AFB1 (K m app = 1.8).The fact that the lines do not intersect on any of the axes and that at the increasing inhibitor concentration the values of both K m app and maximum biosensor response (proportional to V max ) decrease, indicates that aflatoxin B1 is a reversible acetylcholinesterase inhibitor of mixed type.

Conclusion
The detailed study of the mechanism of immobilized acetylcholinesterase inhibition by aflatoxin B1 within the potentiometric biosensor was presented in the work.The optimum pH of immobilized acetylcholinesterase was determined to be 6.5-8.5 for the substrate decomposition and 4.5-6.5 -for inhibition by aflatoxin B1.Using two methods, the "degree of inhibition" method and the Lineweaver-Burk plot, we analyzed the inhibitory effect of aflatoxin B1 on acetylcholinesterase, immobilized on the potentiometric transducer surface.Both methods showed that immobilized acetylcholinesterase inhibition by aflatoxin B1 is of mixed type.An increase in the substrate concentration was shown to result in increasing I 50 ; its value was 10.5 μg/ml at the substrate con-

Fig. 1 .
Fig. 1.Real response to 4 mM AChCl before and after inhibition by 4 µg/ml of АFВ1.Measurements were performed in 5 mM phosphate buffer, pH 6.5 at room temperature.

Fig. 4 .
Fig. 4. Dependence of the AChE-biosensor response on acetylcholine concentration in inverted Lineweaver-Burk coordinates.Measurements were performed in 5 mM phosphate buffer, pH 6.5, at room temperature.

Fig. 5 .
Fig. 5. Dependence of level of inhibition of immobilized AChE on aflatoxin B1 concentration in the presence of 2.5 mM (1) and 20 mM (2) AChCl.Measurements were performed in 5 mM phosphate buffer, pH 6.5, at room temperature.