1. An improved method for preparing lysozyme with chemically 13C-enriched methionine residues using 2-aminothiophenol as a reagent of thiolysis.
- Author
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Abe Y, Ueda T, and Imoto T
- Subjects
- Binding Sites, Carbon Isotopes, Methionine metabolism, Methylation, Muramidase chemistry, Muramidase metabolism, Nuclear Magnetic Resonance, Biomolecular, Protein Conformation, Sulfhydryl Compounds chemistry, Sulfhydryl Compounds metabolism, Aniline Compounds chemistry, Isotope Labeling methods, Methionine chemistry, Muramidase chemical synthesis
- Abstract
Jones et al. have reported that the epsilon-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 epsilon-carbons of methionine residues are enriched with 13C, because many side reactions took place under the thiolysis condition (pH 10.5, 37 degrees 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 epsilon-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.
- Published
- 1997
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