Reduction of disulfide bonds in proteins by 2-aminothiophenol under weakly acidic conditions

Research output: Contribution to journalArticle

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Abstract

We developed a method for reducing disulfide bonds in proteins under weakly acidic conditions by use of 2-aminothiophenol. The disulfide bonds in hen egg-white lysozyme, ribonuclease A, and soybean trypsin inhibitor were quantitatively reduced by 2-aminothiophenol in phosphate buffer, pH 6, containing 8 M Gdn HCI, 1 mM EDTA, and 20% ethanol, for 60 min at 40°C. On analysis of the RP-HPLC patterns of tryptic peptides, which were derived from reduced and S-alkylated lysozyme and ribonuclease A at pH 6, it was confirmed that no side reaction occurred. Moreover, the reduction under weakly acidic conditions was demonstrated to be applicable for the location of such a labile residue as O-acetylated tyrosine.

Original languageEnglish
Pages (from-to)52-67
Number of pages16
JournalJournal of Biochemistry
Volume115
Issue number1
DOIs
Publication statusPublished - 1994

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Pancreatic Ribonuclease
Muramidase
Disulfides
Egg White
Trypsin Inhibitors
Human computer interaction
Soybeans
Edetic Acid
Tyrosine
Buffers
Proteins
Ethanol
Phosphates
High Pressure Liquid Chromatography
Peptides
2-aminothiophenol
hen egg lysozyme

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

Reduction of disulfide bonds in proteins by 2-aminothiophenol under weakly acidic conditions. / Abe, Yoshito; Ueda, Tadashi; Imoto, Taiji.

In: Journal of Biochemistry, Vol. 115, No. 1, 1994, p. 52-67.

Research output: Contribution to journalArticle

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AB - We developed a method for reducing disulfide bonds in proteins under weakly acidic conditions by use of 2-aminothiophenol. The disulfide bonds in hen egg-white lysozyme, ribonuclease A, and soybean trypsin inhibitor were quantitatively reduced by 2-aminothiophenol in phosphate buffer, pH 6, containing 8 M Gdn HCI, 1 mM EDTA, and 20% ethanol, for 60 min at 40°C. On analysis of the RP-HPLC patterns of tryptic peptides, which were derived from reduced and S-alkylated lysozyme and ribonuclease A at pH 6, it was confirmed that no side reaction occurred. Moreover, the reduction under weakly acidic conditions was demonstrated to be applicable for the location of such a labile residue as O-acetylated tyrosine.

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