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

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Abstract

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.

Original languageEnglish
Pages (from-to)1153-1159
Number of pages7
JournalJournal of Biochemistry
Volume122
Issue number6
DOIs
Publication statusPublished - Jan 1 1997

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Muramidase
Methionine
Carbon
Methylation
Myoglobin
Dithiothreitol
Isotopes
2-aminothiophenol
Substrates
Proteins

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

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title = "An improved method for preparing lysozyme with chemically 13C-Enriched methionine residues using 2-aminothiophenol as a reagent of thiolysis",
abstract = "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.",
author = "Yoshito Abe and Tadashi Ueda and Taiji Imoto",
year = "1997",
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doi = "10.1093/oxfordjournals.jbchem.a021875",
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TY - JOUR

T1 - An improved method for preparing lysozyme with chemically 13C-Enriched methionine residues using 2-aminothiophenol as a reagent of thiolysis

AU - Abe, Yoshito

AU - Ueda, Tadashi

AU - Imoto, Taiji

PY - 1997/1/1

Y1 - 1997/1/1

N2 - 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.

AB - 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.

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U2 - 10.1093/oxfordjournals.jbchem.a021875

DO - 10.1093/oxfordjournals.jbchem.a021875

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JO - Journal of Biochemistry

JF - Journal of Biochemistry

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