Abstract
Alcohol dehydrogenase (EC 1. 1. 1. 1) in skipjack liver was extracted with sodium phosphate buffer solution and purified by ammonium sulfate fractionation and chromatographies on Toyopearl HW-55F, Butyl-Toyopearl 650M, and Blue-Toyopearl 650 ML. At the final step, it was separated into two fractions (ADH-1 and ADH-2). By this method, a final specific activity (ADH-1) of 590 units/mg and purification of 118.0-fold was attained. With respect to ADH-2, a final specificactivity of 1063 units/mg and purification of 212.6-fold was achieved. The apparent molecular weights were estimated to be about 140, 000 (ADH-1) and 130, 000 (ADH-2) by gel filtration on Toyopearl HW-55F. ADH-1 gave a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis which also revealed that this enzyme was composed of identical subunits with a molecular weight of 33, 000. ADH-2 showed a single band with MW of 66, 000. In relation to ADH-2, the optimum temperature was about 40°C. ADH-2 was stable at 30°C for 30min but completely inactivated at 40°C for 30min. The optimum pH was about 10 and ADH-2 was stable at pH 7-9 but was unstable when the pH was lower than 7.0. ADH-2 was activated by Co2+ and Mn2+, but was inhibited by Hg2+, Zn2+, and Cu2+.
