A rapid method for the purification of glucose-6-phosphate dehydrogenase from bovine lens

Abstract

This paper describes a simple and rapid method for the purification of glucose-6-phosphate dehydrogenase from bovine lens, together with analysis of the kinetic behaviour and some properties of the enzyme. The purification consisted of two steps, 2′,5′-ADP-Sepharose 4B affinity chromatography and DEAE Sepharose Fast Flow ion exchange chromatography in procedure which took two working days. The enzyme was obtained with a yield of 13.7% and had a specific activity of 2.64 U/mg protein. The overall purification was about 19,700-fold. The molecular weight of the enzyme was found to be 62±3 kDa by Sephadex G-200 gel filtration chromatography. A protein band corresponding to a molecular weight of 69.2±3.2 kDa was obtained on SDS polyacrylamide slab gel electrophoresis. On chromatofocusing, lens glucose-6-phosphate dehydrogenase gave a single peak at p I 5.14. The activation energy of the reaction catalyzed by the enzyme was calculated from Arrhenius plot as Ea=5.88 kcal/mol. The pH versus velocity curve had two peaks at pH 7.7 and 9.6. By the double-reciprocal plots and the product inhibition studies, it was shown that the enzyme follows ‘Ordered Bi Bi’ sequential kinetics. From the graphical and statistical analyses, Km NADP + , Km G-6-P, Ki NADPH, Ki 6-PGA were estimated to be 0.008±0.002, 0.035±0.013, 0.173±0.007 and 1.771±0.160 mM, respectively. The observed kinetic behaviour of glucose-6-phosphate dehydrogenase from bovine lens was in accordance with the enzyme from other sources.