Hen egg white lysozyme was treated at pH 5.5 with four bifunctional reagents, bis(bromoacetamide) derivatives [BrCH2CONH(CH2).,NHCOCH2Br, 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 ε2 of His-15 and the ε-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). The decreased entropies of the denatured polypeptide chains due to the present cross-links were theoretically predicted to be -9.1 to -9.2 eu, which corresponded to stabilization energies of about 3.0 kcal/mol in all cases around the melting temperatures of the cross-linked derivatives. The lower than predicted stabilizations observed and the magnitude of the stabilizations suggest that the introduction of the cross-link causes a strain in the native state of lysozyme depending on the length of the cross-link and this strain effect of the destabilization of the denatured state mentioned above. Therefore, it is concluded that maximum stabilization can be achieved only when the proper length of a cross-link is introduced into a protein. The activities of the cross-linked lysozymes against glycol chitin at pH 5.5 increased first on increasing temperature, reached the maxima, then decreased, and vanished at 100 °C. Below 60 °C, all activities increased similarly to that of native lysozyme, but above 60 °C, the activities of the derivatives were signficantly higher than that of native lysozyme. The orders of the optimum temperatures and the maximum activities were exactly the same as the order of the thermal stabilities. The mechanism of the decrease of the activity at high temperatures was discussed.
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