Peroxidase Isoenzymes from Horseradish Roots

The reactivity of histidine in horseradish peroxidase isoenzymes Al and C was determined by titrating with diazonium-lH-tetrazole. No exposed histidine residue was detected in the native isoenzymes. Upon removal of the heme prosthetic group, however, all three histidine residues became titratable. Studies with p-chloromercuribenzoate and 14C-iodoacetamide indicated that the 6 half-cystine residues were present as three disulfide linkages. No sulfhydryl groups were detected, even in the presence of 8 M urea. Gel filtration studies showed that reduction and carboxymethylation of the three disulfide bonds yielded a single molecular weight species similar in size to the apoperoxidase. Upon electrophoresis the reduced and carboxymethylated derivatives migrated as a single protein band. These findings show that the three disulfide linkages were intrachain (connecting different parts of a single polypeptide chain) and not interchain (connecting separate polypeptide chains). Tryptic peptide map experiments showed that peroxidase isoenzymes can be segregated into at least three distinct groups; an anionic group, consisting of perioxidase Al and A2 ; a second anionic group consisting only of peroxidase A3 ; and a cationic group consisting of peroxidase B and C. There appear to be many points of difference in primary structure among the three groups. The differences between the isoenzymes within a single group, i.e. between peroxidase Al and A2 or between peroxidase B and C, were not established.