PURIFICATION AND INVESTIGATION OF SOME KINETIC PROPERTIES OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE FROM PARSLEY (PETROSELINUM HORTENSELEAVES

Abstract

In this study, glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps: preparation of homogenate, ammonium sulfate fractionation, and DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 8.79% and had a specific activity of 2.146 U (mg protein)(-1). The overall purification was about 58-fold. Temperature of +4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method, at 340 nm. In order to control the purification of enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 77.6 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a molecular weight of 79.3 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found to be 6.0, 8.0, and 60 degrees C, respectively. Moreover, KM and Vmax values for NADP+ and G6-P at optimum pH and 25 degrees C were determined by means of Lineweaver-Burk graphs. Additionally, effects of streptomycin sulfate and tetracycline antibiotics were investigated for the enzyme activity of glucose-6-phosphate dehydrogenase in vitro.

Glucose‐6‐phosphate dehydrogenase partitioning in two‐phase aqueous mixed (nonionic/cationic) micellar systems

“…Although it is usually asymptomatic, hemolytic anemia can be developed in the affected individual after exposure to oxidative stresses, or with some infections such as hepatitis (Chim et al, 2001;Krzelj et al, 2001). As a result, several biochemical and medical studies involving G6PD have been carried out (Altikat et al, 2001;Au et al, 2000;Coban et al, 2002;Ishibashi et al, 1999) . This enzyme is also of great interest as an analytical reagent, being used in various quantitative assays, including the measurement of creatin-kinase activity for diagnosis of heart and skeletal-muscle diseases, the measurement of hexose concentrations, and as a marker for enzyme immunoassays (Lojudice et al, 2001).…”