Influence of macromolecular crowding on the oxidation of ABTS by hydrogen peroxide catalyzed by HRP

Influence of Macromolecular Crowding on the Oxidation of ABTS by Hydrogen Peroxide Catalyzed by HRP 
The interior of the living cell is highly concentrated and structured with molecules having different shapes and sizes. However, almost all experimental biochemical data have been obtained working in dilute solutions that do not reflect in vivo conditions.


INTRODUCTION
The cell cytosol is an aqueous medium that is crowded with macromolecules and solutes, which occupy up to 40% of its total volume [1].Therefore, biochemical reactions in vivo progress in solutions containing high concentrations of macromolecules.However, studies of biochemical processes in vitro have usually been performed in dilute solutions (typically concentrations of macromolecules less than 1 mg/mL).In the cellular environment, the rate of diffusion is reduced [2][3][4][5][6][7][8], and can even be anomalous at short time.In addition, the macromolecular crowding agent promotes processes such as protein folding, self-association and protein binding [9][10][11][12][13][14][15][16][17][18][19][20][21][22].Hence, the presence of large concentrations of inert solutes can affect the enzymatic activity.However, quite a few studies have explored the effects of crowding on enzyme catalysis, even in vitro.The first study on enzymatic reactions in macromolecular crowded media was carried out by Laurent in 1971 [23].He studied several reactions in polymer media as an initial attempt to describe how the environment affects the intracellular enzyme function, and in all cases studied, the presence of the macrolecules produced a moderate decrease in the apparent Michaelis-Menten (MM) constant, ‫ܭ‬ .Some years later, Minton and Wilf [9] studied the effect of macromolecular crowding on the various kinetic steps in the enzymatic processes of glyceraldehyde-3-phosphate dehydrogenase.They predicted that the rate of an enzymatic reaction will decrease when there is an increase in the concentration or size of the crowding agent.In other words, the excluded volume produces a decrease of both the MM constant, ‫ܭ‬ , and the catalytic constant, ݇ ௧ , when the enzymatic reaction follows the Michaelis-Menten mechanism.Nevertheless, most subsequent studies reported that a high concentration of neutral polymers only had a moderate influence on enzyme reactions.Briefly, a slight decrease in ‫ܭ‬ is frequently found, regardless of the properties of the crowding agent [9,[23][24][25][26][27][28][29][30].However, the effect of the crowding agent on ݇ ௧ is diverse: in some cases, ݇ ௧ increases [24,[28][29][30][31][32], whereas in other cases it decreases [9,[26][27]32].In a previous work, we studied the crowding effect of Dextrans of various molecular weights on the reaction rates of the hydrolysis of N-succinyl-L-phenyl-Ala-p-nitroanilide catalyzed by alpha-chymotrypsin [34].Our results pointed out that the volume occupied by the crowding agent, independent of its size, had an important role in the rate of this reaction.A ‫ݒ‬ ௫ decay and a ‫ܭ‬ increase were obtained when the Dextran concentration in the sample was increased.The rise of ‫ܭ‬ could be attributed to a slower diffusion of the protein due to the presence of crowding, whereas the decrease in ‫ݒ‬ ௫ could be explained by the effect of mixed inhibition by product, which is enhanced in crowded media.
Although considering the results revealed by the previous studies, the range of experimental conditions covered is not wide enough to fully understand the phenomenology of enzymatic kinetics in crowded media.In order to understand better the effect of macromolecular crowding brought on the enzyme kinetic, we have chosen a reaction catalyzed by Horseradish Peroxidase (HRP).HRP is a protein widely used in the field of biosensors due to its high specificity for hydrogen peroxide (H 2 O 2 ) and we consider that our results could be interesting in this field, too.Therefore, within this paper, we studied the way in which the kinetics of the oxidation of 2,2'-azino-bis(3ethylbenzothiazoline-6-sulfonate) (ABTS) by H 2 O 2 catalyzed by HRP is affected by the presence of crowding agents of several concentrations and sizes.Thus, we have investigated the effect of the excluded volume in this enzymatic reaction due to the presence of crowders.We chose this reaction as a model process, because it is a wellknown reaction [35] and there is only a minimal change in the excluded volume due to the small size of the substrates and products.Macromolecular crowding was mimicked using Dextrans from 5 to 150 kDa.Some advantages of using HRP protein in this research have been identified: on the one hand, the absence of known interactions with Dextrans (crowding agents); and on the other hand, the protein size (hydrodynamic radius, r h = 3.0 nm), which is intermediate between those of the selected crowding agents.Within this paper, we examine in what manner this known enzymatic reaction is affected by the presence of Dextrans of different sizes at distinct concentrations.In particular, we analyzed the effect of macromolecular crowding on the values of ‫ݒ‬ ௫ and ‫ܭ‬ parameters of this reaction.

RESULTS AND DISCUSSION
In order to carry out a study to understand the effect of macromolecular crowding on biological reaction, it is important that such a reaction to be accompanied by a minimal change in the excluded volume.Therefore, the substrates and the products must be tiny compared with the size of the protein and of the crowding agents.Hence, the effect of these molecules (i.e.substrates and products) on the excluded volume could be neglected.As a result, the effect of macromolecular crowding on the reaction can be interpreted mainly in terms of the presence of crowding agents.This represents one of the reasons for which we chose a well-known peroxidase-catalyzed reaction [36], namely the one electron oxidation of ABTS in the corresponding radical-cation, ABTS •+ , catalyzed by HRP, shown in equation 3 (3) Another reason is that this reaction can be easily monitored by UVspectroscopy.Since 70s, ABTS has been proposed to serve as a chromogen for H 2 O 2 assay using HRP, because it has a well-defined absorption maximum at 340 nm (ε 340 = 36,000 M -1 cm -1 ) [37].The radical-cation ABTS •+ has an absorption maximum at 414 nm (ε 414 = 36,000 M -1 cm -1 ); it also absorbs at 340 nm (ε 340 = 5400 M -1 cm -1 ).The ABTS method allows the easy quantification of the initial rates of the enzymatic reaction.Most of the commercially-available peroxidase assays are now using this method [38].As we investigate within this study the effect of macromolecular crowding on enzymatic reactivity, we need a well-behaved reaction of which kinetics can be interpreted with confidence.The oxidation of ABTS by H 2 O 2 is such a process.The values of Michaelis-Menten parameters, ‫ݒ‬ ௫ and ‫ܭ‬ reported in diluted solution and the values that we obtained using similar conditions are comparable [39].Regarding the enzymatic system chosen, we have investigated the effect brought by different concentrations and sizes of the crowding agent (Dextran).In all the cases, we observed a typical initial rise and a subsequent plateau in the absorbance/time plot of the released ABTS •+ .An example of the kinetic curves obtained is shown in Figure 1.
The initial velocity (v 0 ) was obtained by fitting the initial slope of these curves.The fitted values show a significant decrease of the initial velocity of the reaction with the increase of Dextran concentration.The dependence of v 0 on the substrate concentration for different Dextran concentrations and dimensions is illustrated in Figure 2.Each figure corresponds to a given Dextran concentration and shows the variation of v 0 with the concentration of ABTS.The Dextran concentration value given in mg/mL is proportional to the volume occupied by this crowding agent.Figure 2 reveals the dependence of v 0 -substrate concentration curves on the concentration of Dextran.For the same occupied volume, the v 0 -substrate concentration curves are similar irrespective of the obstacles dimension considered.Thus, the value of v 0 does not change with the size of Dextrans, but varies with their concentration, i.e. with the excluded volume.
Based on this similarity, for each Dextran concentration, the curves corresponding to the distinct Dextran sizes considered can be grouped into a single average v 0 -substrate concentration curve (Figure 3).
The absent dependence of the initial velocity on obstacle dimension is in accordance with the results of our previous work [34].In addition, these results are also in agreement with Minton et al. [2,[8][9][10][11][12], as these authors predicted that the excluded volume plays an important role in the enzymatic reactions that take place in macromolecular crowded media.We have found that, at least in two cases: the hydrolysis of N-succinyl-L-phenyl-Ala-p-nitroanilide catalyzed by alpha-chymotrypsin [34] and the oxidation of ABTS by H 2 O 2 catalyzed by HRP, the total volume excluded by the crowding agents brings a greater impact on the velocity of the reaction than the size of these agents.
In order to better understand in what way the excluded volume affects the hydrolysis reaction of HRP in crowded media, the values of ‫ܭ‬ and ‫ݒ‬ ௫ were calculated by fitting the curves in Figure 3 using equation 2. Table 1 shows the values obtained for these parameters as a function of the excluded volume.Our results indicate that both, the value of ‫ݒ‬ ௫ and ‫ܭ‬ decay as increasing the obstacle concentration in the sample.
Firstly we analyzed the ‫ܭ‬ behavior.We found (Table 1) that the values of ‫ܭ‬ for the oxidation of ABTS by H 2 O 2 catalyzed by HRP decreased with the rise of Dextran concentration, in other words, ‫ܭ‬ diminished with the obstacle excluded volume.This result contrast with that reported in our previous work [34].Within the study of hydrolysis of N-succinyl-L-phenyl-Ala-p-nitroanilide catalyzed by alphachymotrypsin in crowding media, an increase of ‫ܭ‬ with the concentration of Dextran was found.As was explained in detail by Pastor et al. [34] and previously reported by Gellerich et al. [24] and Wenner and Bloomfield [25], this behavior corresponded to a diffusion controlled enzymatic process for the case of alpha-chymotripsin.However, our actual results are in agreement with the major part of the results presented in other studies on crowding effects in enzymatic reactions [9,[24][25][26][27][28][29][30][31].Majority of these studies report a slight decrease of ‫ܭ‬ with respect to dilute solution.The decrease of ‫ܭ‬ with crowding could be ascribed to an increase in the ratio of activity coefficients between the native enzyme and the enzyme-substrate complex, or to an increase in chemical activity of the small molecules of substrate in highly non ideal crowded solution [27][28][29].The Michaelis-Menten constant depends on the activity coefficients of the free substrate in solution, so it can be expected to be sensitive to solution composition.In addition, substrate binding also requires the dehydration of both the active site and the substrate, and consequently may be dependent on water activity, which affects the ݇ ௧ value.Therefore, in cases in which the value of ‫ܭ‬ decays with the rise of obstacle concentration, the catalytic constant (݇ ௧ ) exhibits a greater role, as it can be affected by the environmental surroundings.
Secondly we analyzed the behavior of ‫ݒ‬ ௫ .We found (Table 1) a continuous diminishing of the values of this parameter while the concentration of Dextran increases.According to the enzymatic mechanism described in equation 1, ‫ݒ‬ ௫ is defined as ‫ݒ‬ ௫ ൌ ݇ ௧ ሾ‫ܧ‬ሿ ் , with ݇ ௧ ൌ ݇ ଶ .Therefore, its decrease could be interpreted in terms of crowding-induced variations of the catalytic rate constant or of the effective enzyme concentration.This result is in agreement with the major part of previous studies [9,[27][28][29]33] and also with the fact that in this work we found a decrease of ‫ܭ‬ values when the concentration of Dextran increases.A drop in ‫ݒ‬ ௫ is usually explained as a result of conformational changes of the enzyme active site that are produced by crowding-induced modifications of its surroundings [9,[27][28][29]31], which affects the ݇ ௧ value.However, we cannot estimate ݇ ௧ from ‫ݒ‬ ௫ , since it is known that the effective enzyme concentration increases with the excluded volume in crowded media [2,[8][9][10][11][12].With regard to our results, we can say that, for the investigated reaction catalyzed by HRP in in vitro crowded media, the catalytic constant (݇ ௧ ) exhibits a greater role and it might be affected by the changes in the environmental surroundings due to the presence of crowding agents.
In conclusion, we have studied the kinetics of the oxidation of ABTS by H 2 O 2 catalyzed by HRP as a model enzymatic reaction occurring in different in vitro crowded media, produced by Dextran of various concentrations and dimensions.Our results reveal that the volume occupied by the crowding agent has a significant impact on the rate of this reaction.We obtained a ‫ݒ‬ ௫ and ‫ܭ‬ decay along with the growth of obstacle concentration.Concerning this reaction, the data presented suggest an activation control of the enzymatic reaction in the studied system, meaning that the catalytic constant (݇ ௧ ) brings a significant contribution as it can be affected by the environmental surroundings.This contribution could be due to an increase in the ratio of activity coefficients between the nature enzyme and the enzyme-substrate complex due to presence of crowding agents, or an increase in chemical activity of water in highly crowded solution, or a crowding-induced conformational change in the enzyme active site, or could be the sum of all these factors.In our opinion, this must be taken into account when studying enzyme-catalyzed reactions that occur within the crowded physiological environment of the cell and also in biotechnology applications like biosensors development.

Oxidation of ABTS
All reactions were carried out at room temperature in phosphate buffer (0.1 M, pH = 7.4).The reaction mixture contained a fixed concentration of 10 nM of HRP and 10µM of H 2 O 2 , the concentration of ABTS varied between 0 and 23 µM, and the concentration of Dextran varied between 0 and 200 mg/mL.The reaction was initiated by the addition of HRP to a sample mixture of ABTS and H 2 O 2 and Dextran in phosphate buffer, using a syringe to favor mixing.The reaction progress was followed by monitoring the release of ABTS radical-cation (ABTS •+ ) (absorbance at 414 nm) with a UV spectrophotometer (UV-1603 Shimadzu).At the beginning of the reaction, the product absorbance increased linearly with time, and the velocity of the reaction was constant at short periods of time (Figure 1).Thus, the initial velocity, v 0 , was chosen as an experimental parameter, and it was calculated in all experiments as the slope of the linear fitting of the first 10s of the absorbance/time data.To reduce the standard error of v 0 , experiments were repeated from 3 to 5 times under identical conditions.This enzymatic reaction can be considered a single enzyme-substrate reaction and can be studied using the Michaelis-Menten equation following the irreversible reaction scheme that was first proposed by Henri [36] (1) where k 1 , k -1 , and k 2 are rate constants.The difficulty of following an enzymatic reaction was largely solved when Michaelis and Menten showed that, under certain conditions, e.g. an excess of substrate, enzyme activity could be studied by measuring the initial rate of product formation.Since then, enzyme kinetic parameters have usually been determined using an expression for the velocity of product formation that is known as the Michaelis-Menten equation where v max is the maximum velocity defined as ‫ݒ‬ ௫ ൌ ݇ ௧ ሾ‫ܧ‬ሿ ் and, for the classical enzymatic mechanism depicted in equation 1, ‫ܭ‬ ൌ ሺ݇ ିଵ ݇ ଶ ሻ ݇ ଵ ⁄ is the Michaelis-Menten constant and ݇ ௧ ൌ ݇ ଶ .

FIGURE 2 .
FIGURE 2. Michaelis-Menten plot that relates the reaction rate v 0 to the substrate

FIGURE 3 .
FIGURE 3. Michaelis-Menten plot that relates the reaction rate v 0 to the substrate

Table 1 .
Kinetic constants of the oxidation of ABTS by peroxide hydroxide catalyzed by HRP for different concentrations of Dextran (in mg/mL).The value of the fitting parameter r 2 is also given.Example of kinetic curves for different concentrations of ABTS (6, 10, 13, 16, 20 and 23 µM, respectively) in samples with 25 mg/mL of 50 kDa Dextran, 10 µM of H 2 O 2 and 10 nM of HRP.