An improved method for preparing lysozyme with chemically ¹³C-Enriched methionine residues using 2-aminothiophenol as a reagent of thiolysis

Jones et al. have reported that the ε-carbons of methionine residues in myoglobin can be enriched with stable isotope (¹³C) in two steps, i.e., methylation of methionine residues with ¹³CH₃I in the protein and thiolysis using dithiothreitol. Using their method, we failed to prepare active lysozyme in which the ε-carbons of methionine residues are enriched with ¹³C, because many side reactions took place under the thiolysis condition (pH 10.5, 37°C). When we employed 2-aminothiophenol as a reagent for thiolysis, the reduction proceeded under a weakly acidic condition to afford fully active lysozyme, in which the ε-carbons of two methionine residues were enriched with ¹³C, in a 30% yield. Analysis of the ¹³C-edited NOESY spectra of ¹³C-enriched methionine lysozyme in the absence and presence of a substrate analogue indicated the occurrence of conformational change around Met 105 in lysozyme.