Effect of γ-radiation on the charge and size properties of horseradish peroxidase

Horseradish peroxidase (EC. 1.11.1.7) has been irradiated in dilute aqueous solution under argon with60 Co γ-rays. A G value of ∼0.09 has been found, indicating that peroxidase is relatively resistant to ionizing radiation. Isoelectric focusing of the irradiated peroxidase revealed: (1) a prevailing dose-dependent shift to components with lower isoelectric points and (2) multiple new enzymatically active components. By thin-layer gel chromatography, formation of aggregates partially retaining enzyme activity has been detected. Radiation-induced aggregation depended strongly on enzyme concentration; in diluted solutions much fewer aggregates were formed. Aggregation proceeded stepwise, the degree of polymerization increasing with dose. Noncovalent bonds and disulfide links appear to be involved in aggregation. Fragments smaller than the monomer have not been detected. The charge properties of the aggregates and remaining monomers, isolated from irradiated peroxidase by gel chromatography, were characterize...