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. 1977 Jun;74(6):2362–2366. doi: 10.1073/pnas.74.6.2362

Formation of the four isomers of hen egg white lysozyme containing three native disulfide bonds and one open disulfide bond

A Seetharama Acharya 1,2, Hiroshi Taniuchi 1,2
PMCID: PMC432171  PMID: 267931

Abstract

Reduced partially carboxymethylated hen egg white lysozyme (mucopeptide N-acetylmuramoylhydrolase; EC 3.2.1.17) (approximately 0.8 mol of [1-14C]carboxymethyl groups) was air oxidized at pH 8.0 and 37° in the presence of 1.5 mM 2-mercaptoethanol for 36 hr. Gel filtration of this product gave the lower (native) and higher hydrodynamic volume forms, both containing radioactivity (approximately 35 and 65%, respectively). Ion exchange chromatography of the lower hydrodynamic volume forms yielded renatured lysozyme, two major radioactive samples (LHC and LHD) eluting at the positions of monocarboxymethylated lysozyme, and two minor radioactive samples eluting at the positions of dicarboxymethylated lysozyme. Sample LHC (approximately 23% of the radioactivity) was essentially homogeneous with respect to electrophoretic mobility, exhibited approximately 39% of the enzymic activity of lysozyme, and contained 0.95 mol of [14C]carboxymethyl groups. Sample LHD (approximately 8% of the radioactivity) was also enzymically active and contained approximately 0.5 mol of [14C]carboxymethyl groups; this low value is apparently due to contamination of noncarboxymethylated species. The radioactive tryptic peptides from samples LHC and LHD were characterized. The results indicated that all eight isomers, containing three presumably native disulfide bonds and one free and one carboxymethylated sulfhydryl group, are formed on air oxidation of reduced partially carboxymethylated lysozyme. Since in each of these isomers the formation of one of the four native disulfide bonds is permanently blocked, it would follow that no one of the four disulfide bonds of native lysozyme is obligatory in the formation of the other three native disulfide bonds.

Keywords: protein folding, renaturation of reduced proteins, native hydrodynamic volume, intermediates, carboxymethylated lysozyme

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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