Preparation and properties of a lysozyme derivative in which two domains are cross-linked intramolecularly between trp62 and asp101

A lysozyme derivative in which two domains were cross-linked intramolecularly was newly prepared by means of a two-step reaction. First,the β-carboxyl group of Asp1O1 in lysozyme was selectively modified with 2-(2-pyridyldithio)ethylamine in the presence of l-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride. After reduction of the pyridyldithio moiety of Asp101 modified lysozyme at pH4.5 with dithiothreitol, the derivative was allowed to cross-link intramolecularly by reaction with 1,3-dichloroace-tone at pH 7. Intramolecularly cross-linked lysozyme thus formed was purified by gel chromatography followed by ion-exchange chromatography. Based on the results of¹H-NMR and peptide analyses, it was concluded that Asp101 was cross-linked to Trp62 with a -CH₂COCH₂SCH₂CH₂NH- bridge in this derivative. The derivative showed minor but distinct activity against Micrococcus lysodeikticus and glycol chitin. Its melting temperature for thermal denaturation was higher by 7.3°C than that of native lysozyme at pH3.