Structural Investigation of Radiation-induced Aggregates of Ribonuclease

Summary

Following irradiation of bovine pancreatic ribonuclease in aqueous solution with ⁶⁰Co γ-rays protein aggregates are formed. The nature of the bonds linking these radiation-induced aggregates together has been investigated by chromatographic and electrophoretic methods. Thin-layer gel filtration and polyacrylamide gel electrophoresis, both in the presence of sodium dodecyl sulphate, demonstrated the existence of covalent crosslinks between the aggregates. However, noncovalent crosslinking also plays a role in the radiolysis of ribonuclease.

Thin-layer gel filtration with and without 6M urea and 2 per cent β-mercaptoethanol added to the gel, revealed that only part of the covalent bonds between the aggregates consisted of disulphide linkages. By separation of the reduced aggregates by thin-layer gel filtration and electrophoresis, both with SDS, this finding was substantiated. Densitometric measurements indicated for example that the percentage of covalently linked dimers held together by disulphide bridges amounted to about 40–45 per cent, whereas the remaining 55–60 per cent of the dimers must be linked by other covalent bonds.

The existence of covalent crosslinks other than disulphide bonds was also confirmed by isoelectric focusing. By this method definite differences were established between the proteolytic hydrolysates of the reduced aggregates and the reduced monomer of γ-irradiated ribonuclease.

• bovine pancreatic ribonuclease
• radiation-induced aggregates