The purpose of this research was to characterize the antibody-mediated inhibition of horseradish peroxidase activity. Inhibition of enzyme activity was determined when alternate substrates were used. Typical biphasic patterns of inhibition were observed with each of five electron donors and three hydroperoxides. In no case was inhibition complete (100%). Inhibition assays with varied substrate concentrations revealed a dependence of anticatalytic activity upon the hydroperoxide concentration. Increased hydroperoxide concentration elevated inhibition levels. Inhibition is not dependent upon the electron donor concentration.

The dependence of inhibition levels on hydroperoxide concentration suggests that antibody may make the horseradish peroxidase basic isoenzyme more susceptible to peroxide inactivation. Kinetic data show increased enzyme affinity for hydrogen peroxide in the presence of antibody. In addition, acidic peroxidases that are less susceptible to peroxide are poorly inhibited by antibasic horseradish peroxidase.

Spectrophotometric experiments with an electron donor analogue indicated that antibody does not inhibit horseradish peroxidase activity by preventing electron donor binding. Inhibitory antibody binding sites could not be determined because fragments of the enzyme capable of binding inhibitory antibody could not be produced.

Antisera prepared against acidic horseradish peroxidase isoenzymes were used in immunological procedures (precipitation, anticatalytic assays) to assess the homology of horseradish, radish and turnip peroxidases. Acidic horseradish peroxidases with similar physicochemical properties are distinguishable by immunological methods. Interspecies relationships are stronger among acidic peroxidases than relationships between acidic and basic horseradish isoenzymes. These results support an evolutionary conservation of acidic isoenzymes and suggest that each isoenzyme group catalyzes specialized plant functions.