PURIFICATION AND CHARACTERIZATION OF ALCOHOL-DEHYDROGENASE FROM LIVEROF SKIPJACK KATSUWONUS-PELAMIS

Alcohol dehydrogenase (EC 1.1.1.1) in skipjack liver was extracted wit h sodium phosphate buffer solution and purified by ammonium sulfate fr actionation and chromatographies on Toyopearl HW-55F, Butyl-Toyopearl 650M, and Blue-Toyopearl 650ML. At the final step, it was separated in to two fractions (ADH-1 and ADH-2). By this method, a final specific a ctivity (ADH-1) of 590 units/mg and purification of 118.0-fold was att ained. 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 b and on sodium dodecyl sulfate-polyacrylamide gel electrophoresis which also revealed that this enzyme was composed of identical subunits wit h 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 deg rees C. ADH-2 was stable at 30 degrees C for 30 min but completely ina ctivated at 40 degrees C for 30 min. The optimum pH was about 10 and A DH-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+.