Purification and Properties of Phenoloxidase from Spinach Leaves

A phenoloxidase from spinach leaves was extensively purified to homogeneity. The molecular weight of the purified enzyme was 150,000 by gel filtration, and 40,000 by SDS-polyacrylamide gel electrophoresis. The enzyme contained 2 atoms of copper per 150,000 of molecular weight. The optimum pH of the enzyme action is near 6.5. The enzyme lacks cresolase activity. Km and k0 were measured for some 4-substituted catechols and oxygen. Log k0 values for catechols substituted in position 4 by -N02, -CHO, -CH3, -COOH, or -CH2CH2COOH were confirmed to fit Hammett relationships (ρ-Values for a and am are - 3.652 and — 4.007, respectively), but not for log Km values. Log(k0/Km) values for the same substrates were confirmed to have this relationship, but protocatechuic and hydrocaffeic acids deviated from this linearity.