PURIFICATION AND CHARACTERIZATION OF ASCORBATE PEROXIDASE IN CHLORELLA-VULGARIS

Chlorella vulgaris contained only one isoform of ascorbate peroxidase (AsAP) as the hydrogen peroxide (H2O2)-scavenging system except for ca talase at a specific activity of 3.3 +/- 0.2 units/mg protein. The act ivity of glutathione peroxidase was not detected in the extracts from cells grown in the absence and presence of sodium selenite. We detecte d the activity of monodehydroascorbate reductase involved in the regen eration of ascorbate, but we failed to detect the dehydroascorbate red uctase activity. AsAP has been purified to electrophoretic homogeneity from Chlorella cells. The enzyme was a monomer with a molecular mass of 32 kDa using gel filtration and SDS-polyacrylamide gel electrophore sis. The enzyme showed higher specificity with ascorbate than with pyr ogallol. The Km values of the enzyme for ascorbate and H2O2 were 111 /- 8.9 and 20 +/- 2.5 mu M, respectively. When the enzyme was diluted with the ascorbate-deleted medium, the half inactivation time was appr oximately 15 min. The absorption spectra of the purified enzyme and th e inhibition by cyanide and azide showed that it is a hemoprotein. The enzyme was markedly inhibited by 0.2 mM p-chloromercuribenzoate. The enzyme cross-reacted by immunoblotting with the monoclonal antibody ra ised against Euglena cytosolic AsAP. The amino acid sequences in the N -terminal region of Chlorella AsAP showed no significant similarity to any other AsAPs from higher plants and algae. (C) Societe francaise d e biochimie et biologie moleculaire/Elsevier, Paris.