Radiation Damage to Crystalline Ribonuclease: Identification of Polypeptide Chain Breakage in the Denatured and Aggregated Products

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Abstract
Gamma-irradiated crystalline ribonuclease was chroma-tographically separated into 3 distinct components classified as denatured, aggregated, and native, on the basis of the physical alterations which occurred in the conformation of these molecules. Chromatography on G-75Sephadex failed to reveal the presence of any low-molecular-weight products in the irradiated enzyme. However, after reduction of the disulfide bridges in mercaptoethanol and chromatography on G-75 Sephadex, low-molecular-weight products indicative of polypeptide chain breakage were detected from both the denatured and the aggregated products. The presence of fragments of the reduced protein was confirmed by velocity sedimentation measurements. Fragments were not observed from the "native" component, which contained partially damaged molecules. These results indicated that polypeptide chain breakage occurred on exposure of RNase to ionizing radiation but that it was masked by the presence of disulfide bonds. Evidence was obtained which suggested that free amide groups were produced as a result of the breakage of the polypeptide chain. A high yield of fragments was observed following irradiation in H2S and in vacuo. The inability of H2S to prevent the main-chain breakage suggested that this cleavage occurred at an early step in the radiation process. The rupture of the polypeptide chain was an important factor, but not the exclusive cause of the inactivation of crystalline ribonuclease by ionizing radiation.

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