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 -NO2,-CHO,-CH3,-COOH,or -CH2CH2COOH were confirmed to fit Hammett relationships(p-Values for σp and σm are -3.652 and -4.007,respectively),but not for long Km values.Long (K0 /Km) values for the same substrates were confirmed to have this relationship,but protocatechuic and hydrocaffeic acids deviated from this Iinearity.