Evaluation of irreversible protein thermal inactivation caused by breakage of disulphide bonds using methanethiosulphonate

Junichiro Futami, Ai Miyamoto, Atsushi Hagimoto, Shigeyuki Suzuki, Midori Futami, Hiroko Tada

Research output: Contribution to journalArticle

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Abstract

Many extracellular globular proteins have evolved to possess disulphide bonds in their native conformations, which AIDS in thermodynamic stabilisation. However, disulphide bond breakage by heating leads to irreversible protein denaturation through disulphide-thiol exchange reactions. In this study, we demonstrate that methanethiosulphonate (MTS) specifically suppresses the heat-induced disulphide-thiol exchange reaction, thus improving the heat-resistance of proteins. In the presence of MTS, small globular proteins that contain disulphides can spontaneously refold from heat-denatured states, maintaining wild-type disulphide pairing. Because the disulphide-thiol exchange reaction is triggered by the generation of catalytic amounts of perthiol or thiol, rapid and specific perthiol/thiol protection by MTS reagents prevents irreversible denaturation. Combining MTS reagents with another additive that suppresses chemical modifications, glycinamide, further enhanced protein stabilisation. In the presence of these additives, reliable remnant activities were observed even after autoclaving. However, immunoglobulin G and biotin-binding protein, which are both composed of tetrameric quaternary structures, failed to refold from heat-denatured states, presumably due to chaperon requirements. Elucidation of the chemical modifications involved in irreversible thermoinactivation is useful for the development of preservation buffers with optimum constitutions for specific proteins. In addition, the impact of disulphide bond breakage on the thermoinactivation of proteins can be evaluated using MTS reagents.

Original languageEnglish
Article number12471
JournalScientific Reports
Volume7
Issue number1
DOIs
Publication statusPublished - Dec 1 2017

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Disulfides
Sulfhydryl Compounds
Proteins
Denaturation
Chemical modification
Stabilization
Hot Temperature
methanethiosulfonate
Heat resistance
Conformations
Buffers
Immunoglobulin G
Thermodynamics
Heating

ASJC Scopus subject areas

  • General

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Evaluation of irreversible protein thermal inactivation caused by breakage of disulphide bonds using methanethiosulphonate. / Futami, Junichiro; Miyamoto, Ai; Hagimoto, Atsushi; Suzuki, Shigeyuki; Futami, Midori; Tada, Hiroko.

In: Scientific Reports, Vol. 7, No. 1, 12471, 01.12.2017.

Research output: Contribution to journalArticle

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