Purification of Dehydroascorbate Reductase from Spinach and Its Characterization as a Thiol Enzyme

Dehydroascorbate (DHA) reductase (EC 1.8.5.1) was purified from spinach leaves to nearly a homogeneous state, judging from polyacrylamide gel electrophoresis. The enzyme was found to be a monomer with a molecular weight of 23,000 with apparent Km values for GSH and DHA of 2.5 and 0.07 nu, respectively. Kinetic studies indicated that the reaction proceeds by an ordered or random mechanism involving the formation of a ternary complex. The enzyme was inhibited by thiol reagents and DHA could partly protect the enzyme thiol group. Incubation of the inactivated enzyme with GSH led to slight recovery of the activity. NADH, NADPH, cysteine or reduced thioredoxin could not replace GSH as an electron donor. Thus, the thiol group(s) participates in the reaction of DHA reductase.