Radiation Damage to Crystalline Ribonuclease: Importance of Free Radicals in the Formation of Denatured and Aggregated Products

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Abstract
The production of denatured and aggregated products from crystalline RNase was studied following irradiation by 60Co Y-rays in vacuo, in O2 and H2S, and in the presence of the paramagnetic ion, Cu++. The yields of these products were determined by gel filtration of the irradiated enzyme on G-75 Sephadex. Aggregation occurred through relatively slow free radical reactions which were eliminated or altered by free radical scavengers such as O2 and H2S. Conversely, the yield of denatured products was enhanced from 1.2/100 eV (in vacuo) to 2.4/100 eV by these gases. The yield of both aggregated and denatured molecules decreased in the presence of Cu++ ions, suggesting that these ions reduce the effective radiation dose. Detailed reaction schemes were proposed to account for the rates of formation of the products, and the yields deduced from these schemes were in reasonable agreement with the yields for inactivation of the enzyme under the same conditions. Two peaks, which eluted in the region of the aggregates, were identified as dimers and trimers of RNase. The trimer was observed even at low radiation doses.