Preparation of a two-disulfide bonded enzymically active derivative from hen egg lysozyme.

A. S. Acharya, H. Taniuchi

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

A method has been developed for preparation of an enzymically active two-disulfide bonded derivative from hen egg lysozyme. Lysozyme (0.15 mM) is incubated with 2 mM dithiothreitol at pH 7.8, 23 degrees for 40 min. The products are reacted with [1-14C]iodoacetic acid and then purified by gel filtration and ion-exchange chromatography. An enzymically active derivative containing 4 mol of [1-14C] carboxymethyl groups and no free sulfhydryl groups is obtained in approximately 18% yield. Examinations of hydrodynamic volume, tryptophan fluorescence, CD and tryptic peptides containing [1-14C] carboxymethyl cysteine indicate that this derivative contains two presumably native disulfide bonds and two open disulfide bonds between Cys 6 and Cys 127 and between Cys 76 and Cys 94. The rest of the species in the incubation mixture are intact lysozyme. Thus, the species containing two presumably native disulfide bonds and four free sulfhydryl groups at Cys 6, Cys 76, Cys 94 and Cys 127 appears to be only the intermediate accumulating during reduction of lysozyme with dithiothreitol.

Original languageEnglish (US)
Pages (from-to)503-509
Number of pages7
JournalInternational Journal of Peptide and Protein Research
Volume15
Issue number5
StatePublished - May 1980
Externally publishedYes

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Disulfides
Muramidase
Derivatives
Dithiothreitol
Iodoacetic Acid
Ion Exchange Chromatography
Hydrodynamics
Chromatography
Tryptophan
Gel Chromatography
Cysteine
Ion exchange
Fluorescence
Gels
Peptides
hen egg lysozyme

ASJC Scopus subject areas

  • Biochemistry

Cite this

Preparation of a two-disulfide bonded enzymically active derivative from hen egg lysozyme. / Acharya, A. S.; Taniuchi, H.

In: International Journal of Peptide and Protein Research, Vol. 15, No. 5, 05.1980, p. 503-509.

Research output: Contribution to journalArticle

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N2 - A method has been developed for preparation of an enzymically active two-disulfide bonded derivative from hen egg lysozyme. Lysozyme (0.15 mM) is incubated with 2 mM dithiothreitol at pH 7.8, 23 degrees for 40 min. The products are reacted with [1-14C]iodoacetic acid and then purified by gel filtration and ion-exchange chromatography. An enzymically active derivative containing 4 mol of [1-14C] carboxymethyl groups and no free sulfhydryl groups is obtained in approximately 18% yield. Examinations of hydrodynamic volume, tryptophan fluorescence, CD and tryptic peptides containing [1-14C] carboxymethyl cysteine indicate that this derivative contains two presumably native disulfide bonds and two open disulfide bonds between Cys 6 and Cys 127 and between Cys 76 and Cys 94. The rest of the species in the incubation mixture are intact lysozyme. Thus, the species containing two presumably native disulfide bonds and four free sulfhydryl groups at Cys 6, Cys 76, Cys 94 and Cys 127 appears to be only the intermediate accumulating during reduction of lysozyme with dithiothreitol.

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