Purification and characterization of glutathione S-transferase from blueberry fruits (Vaccinium arctostaphylos L.) and investigated of some pesticide inhibition effects on enzyme activity

摘要

Pesticides cause pollution by remaining in water, soil, fruits and vegetables for a long time and also reach human through the food chain. It was thought that some pesticides used in agriculture could adversely affect the antioxidant enzyme system and the minimum inhibition values were studied. glutathione s-transferase (GST), an important antioxidant enzyme, catalyzes the conjugation of glutathione with toxic metabolites. It was purified from the blueberry fruits. The purification of the enzyme was performed separately by affinity and gel filtration chromatography. The purity of the enzyme was determined by SDS-PAGE electrophoresis. Characterization studies were done for the enzyme. For this purpose, optimal pH, temperature, Km and Vmax values for GSH and CDNB were also determined for the enzyme as 7.2 in K-phosphate buffer, 50 °C, 1.0 M, 7.0 in K-phosphate buffer, 1.57 mM; 0.17 mM and 0.048 EU/mL, 0.0159 EU/mL, respectively. Additionally, inhibitory effects of some pesticides; dichlorvos, acetamiprid, cyhalothrin, haloxyfop-p-Methyl, 2,4 dichlorophenoxy acetic acid, cypermethrin, imidacloprid, fenoxaprop-p-ethyl, glyphosate isopropylamine salt were examined the enzyme activity in vitro by performing Lineweaver–Burk graphs and plotting activity % IC50 and Ki values were calculated for each of pesticides. All of the pesticides inhibited the GST enzyme at millimolar level. Pesticide showing the best inhibitory effect was found as dichlorvos. The Ki value which is the inhibition constant of this pesticide was 0.0175 ± 0.005.