KINETICS OF QUERCETIN NITRATION BY HORSERADISH PEROXIDASE

In this study we investigated the kinetics of the nitration of quercetin by horseradish peroxidase. Quercetin nitration reaction was followed by recording the spectral changes over the time at 380 nm. The reaction rate increases with increasing of the quercetin concentration and follows the Michaelis-Menten type kinetics. Kinetic parameters of the studied enzymatic reaction were determined. Acta Medica Medianae 2013;52(2):5-9.


Introduction
Flavonoids are the major class of plant polyphenolics which comprise several thousand compounds sharing a common phenylchromane skeleton. This basic structure allows a variety of substitution patterns leading to different flavonoid subclasses, such as flavonols, flavones, flavanones, flavanols, anthocyanins, dihydroflavonols, isoflavones and chalcones (1). Flavonoids are extensively distributed in the medicinal plants, vegetables, fruit juices, and a variety of beverages (tea, coffee, wines, and fruit drinks) (2). Biological effects of flavonoids are linked to their potential cytotoxicity and their capacity to interact with enzymes through protein complexation. Furthermore, flavonoids act as scavengers of free radicals (3). It has been reported that consuming food which contains flavonols is related to the decrease in risks to arteriosclerosis (4). They also have antimicrobial properties (3) and might have some antidepressant activity (5). Havsteen (6) published a detailed review on the biochemistry and medical significance of the flavonoids.
Flavonoids, and particularly quercetin derivatives, have received more attention as dietary constituents during the last few years (2). Quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one, Figure 1) is one of the major polyphenols in many plant foods (1). He has shown to be an effective inhibitor of xanthine oxidase and lipoxygenase, the enzymes involved in processes such as inflammation, atherosclerosis, cancer and ageing (7,8).
Enzymatic methods have become increasingly important in chemical synthesis in the past three decades. Crude and purified enzymes have been used on both laboratory and industrial scale to synthesize value-added compounds which may be difficult to produce using traditional chemical methods. The field of enzymatic synthesis is currently dominated by the use of hydrolases such as lipases, proteases, and glycosidases, mainly due to their ease of use, high stability and activity under synthetic conditions, and their independence from expensive cofactors. The use of oxidative enzymes in synthesis, however, has been more challenging because these enzymes are often less stable and typically require cofactors. Nonetheless, interest in developing processes using oxidative enzymes is increasing (9). The oxidation of tyrosinase on quercetin is well described (10). Nitrate is secreted into the oral cavity as a salivary component. Bacteria in the cavity can reduce nitrate to nitrite. On the other hand, vegetables and fruits contain quercetin glycosides. When foods that contain quercetin glycosides are ingested, the glycosides are hydrolyzed to quercetin, and the quercetin can be oxidized by peroxidase-dependent reactions in the oral cavity (4, 11). Budde et al. (9) have shown that soybean peroxidase is an effective catalyst for the nitration of phenols in the presence of various organic co-solvents at concentrations up to 40% (v/v). The mild reaction conditions (ambient temperatures and neutral pH) make this approach the most appropriate for complex compounds (both natural and synthetic) with limited aqueous solubility and with labile functional groups. The aim of this work was to investigate the nitration of quercetin by horseradish peroxidase. Kinetic parameters of the studied enzymatic reaction were determined.

Spectrophotometric assays
Kinetic assays were carried out by measuring the appearance of the product in the reaction medium on VARIAN Cary-100 Spectrophotometer and controlled by using a VARIAN Cary-100 UV-Winlab software. Firstly, the reaction mixture was scanned at wavelengths of 200-800 nm, and then the quercetin nitration reaction was followed by recording of spectral changes over the time at 380 nm.
Enzymatic reaction was performed in such a way that phosphate buffer and water were always first added in a tube. Then, sodium nitrite solution and quercetin solution were added. Afterwards, the solution of horseradish peroxidase was added and finally the reaction began by adding solution of hydrogen peroxide.
The reaction medium contained 2500 μl of 50 mM phosphate buffer pH 7, about 2000 μl of distilled water, 500 μl of 50 mM sodium nitrite solution, 30, 80 or 120 μl of 10 mM solution of quercetin, 25 μl of peroxidase solution (0.34 mg of peroxidase dissolved in 50 ml of cold phosphate buffer), and 100 μl of 10 mM solution of hydrogen peroxide.

Determination of kinetic parameters of enzymatic reaction
Kinetic parameters (Michaelis constant, K m , and maximal rate, V max ) were determined from the graphic of linear Michaelis-Menten equation. Catalytic constant, k +2 , represents the number of moles of the product formed per time unit with one mole of pure enzyme, which is saturated with substrate. The efficiency of the enzyme activity was calculated as the ratio k +2 /K m .

Results and discussion
The progress of the studied enzymatic reaction at quercetin concentrations of 0.06 mM, 0.16 mM and 0.24 mM are given in Figures 2-4, respectively. In all studied reactions a decrease of absorbance at 380 nm in the function of reaction time was observed, which indicates a decrease in the concentration of quercetin in the mixture due to the occurrence of the nitration reaction products.
The slopes of the graphics obtained (Figures 2-4) are directly proportional to the rates of enzymatic reaction at different quercetin concentrations. On the basis of the quercetin nitration rate dependence upon its concentration ( Figure 5), it can be concluded that reaction rate increases with increasing of the substrate concentration and follows Michaelis-Menten type kinetics.
From the Lineweaver-Burk plot of enzymatic nitration at different quercetin concentrations ( Figure 6) the kinetic constants K m (0.0833 mM) and V max (0.02629 ΔA/mM) were obtained. The ratio of the maximum reaction rate and Michaelis's constant (V max /K m ) was 0.3156 mM ΔA 380 /min mM. The value of the catalytic constant, k +2 , is calculated as the ratio of the maximum rate and enzyme concentration, amounting 0.0011 ΔA 380 /min nM. The efficiency of the enzyme activity was calculated on the basis of the values of k +2 and K m , amounting 0.01356 min/mM nM.
The subject of our further study will be the identification of the products of enzymatic modification as well as testing of their pharmacological activities.

Conclusions
The kinetics of nitration of quercetin by horseradish peroxidase was studied. Based on the obtained results, the following conclusions can be drawn: -under the conditions described in this paper the enzymatic nitration of quercetin was carried out; reducing the concentration of quercetin in the mixture indicates the formation of products; -quercetin nitration was followed by recording of spectral changes over the time at 380 nm; -the reaction rate increases with increasing of the quercetin concentration and follows Michaelis-Menten type kinetics; -kinetic constants K m and V max amounted to 0.0833 mM and 0.02629 ΔA/mM, respectively; -efficiency of the enzyme activity amounted to 0.01356 min/mM nM; -to the best of our knowledge, this is the first report on the nitration of quercetin by horseradish peroxidase.