Radiation-produced Aggregation and Inactivation in Egg White Lysozyme

C.O Stevens,H.E Sauberlich,Glenn R Bergstrom

doi:10.1016/s0021-9258(18)96075-8

C.O Stevens, H.E Sauberlich + Show 1 more

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https://doi.org/10.1016/s0021-9258(18)96075-8

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Abstract

Abstract Chromatographically homogeneous egg white lysozyme has been subjected under reduced pressure to 0.67-m.e.v. γ-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.

Highlights

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
The radiation dose level (26.0 mrads) at which 37% of enzymic activity is destroyed in dry lysozyme was chosen for large scale fractionation procedures
It would be difficult to ascertain whether enzymic activity loss occurred through a primary structural change resulting from a single, supposedly lethal, event (27) or a secondary change resulting from a second or third ionization within or near the enzyme molecule

Summary

SUMMARY

Homogeneous egg white lysozyme has been subjected under reduced pressure to 0.67-m.e.v. We have not been able to detect production of sulfhydryl groups in egg white lysozyme (mucopeptide N-acetylmuramylhydrolase, EC 3.2.1.17) after radiation doses of 26 mrads when 37% of enzymjc activity had been destroyed (5), nor have we, or other workers, been able to detect significant destruction of constituent amino acids during the irradiation of lysozyme (6) or RNase (4) in the solid state at doses at which loss of enzymic activity occurs None of these measurements on whole irradiated samples would be capable of reflecting such changes as disulfide interchange in the inactive portion of the sample. With the same enzyme after y-irradiation under reduced pressure, Friedberg and Hayden (4) could detect only small amounts of

PROCEDURE

RESULTS

Gel retention volume measurements on Sephadex G-100 in