Purification and characterization of alcohol dehydrogenase from liver of skipjack Katsuwonus pelamis

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 650ML. 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 specific activity 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-I 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 degrees C. ADH-2 was stable at 30 degrees C for 30 min but completely inactivated at 40 degrees C for 30 min. 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+.