An intramolecular cross-linkage of lysozyme. Formation of cross-links between lysine-1 and histidine-15 with bis(bromoacetamide) derivatives by a two-stage reaction procedure and properties of the resulting derivatives.
Hen egg white lysozyme was treated at pH 5.5 with four bifunctional reagents, bis(bromoacetamide) derivatives [BrCH2CONH(CH2)nNHCOCH2Br, 1-n, n = 0, 2, 4, and 6], to alkylate His-15 monofunctionally. The excess bifunctional reagent was then removed, and the pH was raised to 9.0 to allow the other end of the reagent molecule to react. The shortest reagent (1-0) gave no intramolecularly cross-linked lysozyme derivative but only histidine-15-modified lysozyme monomer and intermolecularly cross-linked lysozyme dimer. However, the reagents with longer arms (1-2, 1-4, and 1-6) gave lysozyme derivatives cross-linked intramolecularly between the nitrogen at epsilon 2 of His-15 and the epsilon-amino group of Lys-1 without formation of any other intramolecularly cross-linked lysozyme derivative. These results are consistent with our previous proposal that lysozyme has a small hydrophobic pocket that binds small molecules in the direction from His-15 to Lys-1 [Yamada, H., Uozumi, F., Ishikawa, A., & Imoto, T. (1984) J. Biochem. (Tokyo) 95, 503-510]. The thermal stabilities of three cross-linked lysozymes thus obtained were investigated in 0.1 M acetate buffer containing 3 M guanidine hydrochloride at pH 5.5. All derivatives were stabilized but to different degrees. The derivative cross-linked with 1-4 was most stabilized (2.3 kcal/mol), but the derivatives cross-linked with the reagents both shorter (1-2) and longer (1-6) than 1-4 were less stabilized (both 1.6 kcal/mol).(ABSTRACT TRUNCATED AT 250 WORDS)
D000081:Acetamides / Q000494:pharmacology*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002645:Chickens; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D003959:Diamines / Q000494:pharmacology*; D004529:Egg White; D006639:Histidine; D007700:Kinetics; D008239:Lysine; D009113:Muramidase / Q000378:metabolism*; D011108:Polymers / Q000494:pharmacology; D013816:Thermodynamics
D000081:Acetamides; D000596:Amino Acids; D003432:Cross-Linking Reagents; D003959:Diamines; D011108:Polymers; C016272:N-bromoacetamide; D006639:Histidine; D009113:Muramidase; D008239:Lysine