Radiation-produced aggregation and inactivation in egg white lysozyme.

Chromatographically homogeneous egg white lysozyme has been subjected under reduced pressure to 0.67-m.e.v. y-rays. At 37% destruction of enzymic activity, three inactive aggregates and one partially active fraction have been isolated by salt precipitation and chromatographic procedures. The aggregates, upon reduction with 2-mercaptoethanol and reaction with iodoacetic acid, give derivatives with molecular weights of 12,000 to 15,750 in comparison with one of 14,500 for reduced carboxymethylated lysozyme. One urea-insoluble aggregate becomes soluble upon reduction and, after air oxidation, in dilute solution gives active enzyme in 15% yield. Mixed disulfides of cystine and this or other inactive fractions also give significant (10 to 20%) yields of active enzyme upon incubation with cysteine. Disulfide analyses showed close to the expected number of disulfide bonds in two of the mixed disulfide derivatives if one assumes no fragmentation but simply aggregation of lysozyme monomer molecules. Amino acid and disulfide content of the active component from reactivation of one mixed disulfide derivative agreed well with that found for the active component from reactivation of the mixed disulfide of native lysozyme. Thus a significant portion (15 to 20%) of radiation inactivation in solid lysozyme can be explained by the rupture of disulfide bonds followed by formation of “incorrect” intermolecular disulfide bonds.