Selective Chemical Cleavage of the Peptide Bond at N'-Formylkynurenine in Ozone-Oxidized Hen Egg-White Lysozyme1

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Abstract
Conditions for the selective cleavage of the kynurenyl bond in oxidized hen egg-white lysozyme [EC 3.2.1.17] in aqueous hydrazine solution were established at around pH 5.8. The cleavage reaction was best performed at room temperature or 30°C by direct reaction of oxidized lysozyme with 2 M hydrazine containing 8 M urea or 5 M guanidine hydrochloride at the optimum pH for 4 days or by reaction in the same medium for 24 h after anhydrous hydrazine treatment. The reduced and S-carboxymethylated (Rcm) derivative of an inactive oxidized lysozyme containing one N'-formylkynurenine residue (1-NFK-lysozyme) was subjected to reaction with hydrazine under the established conditions, and was split into two polypeptides. These polypeptides were isolated by gel-filtration on Sephadex G-50. Amino acid compositions and terminal residues of these polypeptides were analyzed, and the formation of 4-acylamino-6- (2′-aminophenyl)-2,3,4,5-tetrahydropyridaz-3-one upon hydrazinolysis of the N'-formylkynurenyl bond was tested. These analyses showed that one of the two isolated polypeptides was the N-peptide corresponding to residues 1 to 62 and the other was the C-peptide consisting of residues 63 to 129 in Rcm-1-NFK-lysozyme. It was deduced that N'-formylkynurenine in 1-NFK-lysozyme is present at position 62 in place of tryptophan in the native protein.