Purification of Ascorbate Peroxidase in Spinach Chloroplasts; Its Inactivation in Ascorbate-Depleted Medium and Reactivation by Monodehydroascorbate Radical

Ascorbate specific peroxidase in chloroplasts was purified from spinach leaves. Spinach chloroplast peroxidase was a monomer with a molecular weight of about 30,000 and showed an absorption spectrum similar to a hemoprotein. The enzyme lost its activity within a minute in the absence of ascorbate under aerobic conditions. In addition to ascorbate, 20% sorbitol was necessary to stabilize the enzyme. The inactivation of the enzyme in the ascorbate-depleted medium was protected by other electron donors, pyrogallol, guaiacol and pyrocatechol, whose oxidation rates were very low compared with that of ascorbate. The inactivated enzyme recovered its activity with monodehydroascorbate radicals generated by the ascorbate-ascorbate oxidase system. A mechanism of inactivation and reactivation of ascorbate peroxidase is proposed.