The Sulfhydryl Groups of Papaya Lysozyme and Their Relation to Biological Activity

Papaya lysozyme, by virtue of its ability to react with a variety of sulfhydryl reagents, has been shown to have four -SH groups per molecule.These groups are available for reaction in the native protein but react much more rapidly when denaturants are present.By stepwise titration of the --SH groups with monovalent mercurials, active derivatives of the enzyme can be prepared with up to three -SH groups blocked.When more than three groups are blocked activity falls off sharply.Evidence from mercaptide exchange experiments and from Hg*f papaya lysozyme derivatives indicate that one sulfhydryl group is essential for activity.Titration of the enzyme -SH groups was associated with increased lability to heat and decreased solubility.A simplification of the method for isolation ot papaya lysozyme is described, as well as a method for isolation of Nacetyl-o-glucosamine oligosaccharides.Tri-N-acetyl-o-plucosamine was found to be a convenient substrate for study of papaya lysozyme activity.In the original description of papaya lysozyme by Smith et al. (I) and in later report's by Howard and Glazer (2, 3) the en-z>-me was shown to contain 8 hnlf~cystine residues per mole.The failure of iodoacetic acid or p-mercuribenzoate to inhibit lysis of Sarcina lutea led the original authors to conclude that sulfhydryl groups were not essential for activity and since that time it has been tacitly assumed that the enzyme has four intramolecular disulfide bonds per molecule.Recently, Bare1 et al. ( 4) have shoxvn that all disulfide bonds of papaya lysozyme can be reduced in the presence of 8 M urea, and upon removal of urea, reformation of these bonds occurs associated with regeneration of full biological activity.