An Improved Method for Preparing Lysozyme with Chemically 13C-Enriched Methionine Residues Using 2-Aminothiophenol as a Reagent of Thiolysis.

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  • Abe Yoshito
    Graduate School of Pharmaceutical Sciences, Kyushu University 62
  • Ueda Tadashi
    Graduate School of Pharmaceutical Sciences, Kyushu University 62
  • Imoto Taiji
    Graduate School of Pharmaceutical Sciences, Kyushu University 62

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説明

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 [Jones, W. C., Rothgeb, T. M., and Gurd, F. R. N. (1976) J. Biol. Chem. 251, 7452-7460]. 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.

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