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 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.
Abe
Y
Y
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka.
Ueda
T
T
Imoto
T
T
D000814:Aniline Compounds / Q000737:chemistry*; D001665:Binding Sites; D002247:Carbon Isotopes; D007553:Isotope Labeling / Q000379:methods*; D008715:Methionine / Q000737:chemistry* / Q000378:metabolism; D008745:Methylation; D009113:Muramidase / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism
D000814:Aniline Compounds; D002247:Carbon Isotopes; D013438:Sulfhydryl Compounds; C005464:2-aminothiophenol; D008715:Methionine; D009113:Muramidase