Purification and characterization of ascorbate peroxidase in roots of Japanese radish

Abstract

Ascorbate peroxidase (APX) was purified to homogeneity from roots of Japanese radish ( Raphanus sativus L.). The root APX was monomeric with a molecular mass of 28 kD and was stabilized by ascorbate. The enzyme utilized mainly ascorbate as a substrate, within a narrow optimum around pH 6.0, but could not use guaiacol, 3,3′-diaminobenzidine, pyrocatechol or d-iso-ascorbate. The purified APX was labile in the absence of ascorbate. Spectral analysis and inhibitor studies revealed the presence of a heme moiety and the participation of an SH group for enzymic activity. Antibodies raised against root APX reacted to extracts of spinach leaf, maize seedling and Brassica root, but not to guaiacol peroxidase from Japanese radish roots. The amino acid sequence of the N-terminal region of the root APX exhibited homology to the cytosolic forms of APX from pea, maize, and a deduced sequence from the Arabidopsis genome, but not to tea chloroplastic APX. These results suggest that the Japanese radish root APX is a novel cytosolic enzyme.