Preparation and Properties of a Lysozyme Derivative in Which Two Domains Are Cross-Linked Intramolecularly between Trp62 and

A lysozyme derivative in which two domains were cross-linked intramolecularly was newly prepared by means of a two-step reaction. First, the /?-carboxyl group of AsplOl 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 AsplOl modified lysozyme at pH 4.5 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 of H-NMR and peptide analyses, it was concluded that AsplOl 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 tempera-ture for thermal denaturation was higher by 7.3C than that of native lysozyme at pH 3. The stabilization of a protein is one of the ultimate goals in protein engineering. To date, the introduction of intra-molecular cross-linking into proteins by use of gene engi