Purification and biochemical characterization of polyphenol oxidases from embryogenic and nonembryogenic cotton (Gossypium hirsutum L.) cells.

Polyphenol oxidases (PPOs) were isolated from cell suspensions of two cultivars of cotton (Gossypium hirsutum L.), and their biochemical characteristics were studied. PPO from Coker 312, an embryogenic cultivar, showed a highest affinity to catechol 20 mM, and PPO from R405-2000, a nonembryogenic cultivar, showed a highest affinity to 4-methylcatechol 20 mM. The optimal pH for PPO activity was 7.0 and 6.0 for Coker 312 and R405-2000, respectively. The enzyme had an optimal temperature of 25 degrees C and was relatively stable at 20-30 degrees C. Reducing sodium metabisulfite, ascorbic acid, dithiothreitol, SnCl(2), and FeCl(3) markedly inhibited PPO activity, whereas its activity was highly enhanced by Mg(2+), Ca(2+), and Mn(2+) and was moderately inhibited by Ba(2+), Cu(2+), and Zn(2+). The analysis revealed a single band on the sodium dodecyl sulfate polyacrylamide gel electrophoresis which corresponded to a molecular weight of 55 kDa for Coker 312 and 42 kDa for R405-2000.