Enhanced Reactivity of Lysozyme with Formaldehyde during Cold Denaturation

The reactivity of lysozyme with formaldehyde at low temperatures (0 to −20 °C) was compared with lysozyme reactivity at 37 °C. Lysozyme reaction with formaldehyde, in the presence and absence of sodium cyanoborohydride, was also compared. The progress of reactions was evaluated by measurement of protein primary amino groups, lysozyme enzymatic activity, and occurrence of cross-linkage as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results obtained indicated that the chemical modification of lysozyme by formaldehyde was significantly enhanced at cold freezing temperatures (−6 and −10 °C) when compared with 37 °C. Cold-temperature protein modification with formaldehyde gave rise to inactive enzyme which was partially cross-linked. Reductive methylation at 37 °C did not appreciably affect enzyme activity or cause protein cross-linkage. Taken together, these results gave a strong indication that the observed enhanced reactivity of lysozyme with formaldehyde at low temperature was likely due to cold denaturation of the protein.