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.
Highlights
Globular proteins usually exist in equilibrium between folded and unfolded states
Bond breakage and disulphide-thiol exchange reactions are accelerated under alkaline conditions[13]; these chemical modifications can be suppressed under acidic conditions
Glycinamide is a superior additive for the prevention of irreversible denaturation[1], through interaction with the molecular surfaces of aromatic groups, as demonstrated with hen egg lysozyme (HEL)[19]
Summary
Globular proteins usually exist in equilibrium between folded and unfolded states. Under physiological conditions, this equilibrium greatly favours the folded state. The reversible refolding ability from a heat-induced unfolded state is a factor in the thermal stability of a globular protein. Bond breakage and disulphide-thiol exchange reactions are accelerated under alkaline conditions[13]; these chemical modifications can be suppressed under acidic conditions. The suppression of disulphide-thiol exchange reactions in heat-denatured proteins has been achieved through the addition of copper(II) ions[14,15]. We investigated methanethiosulphonate (MTS) as an effective additive to suppress irreversible denaturation by heat-induced disulphide-thiol exchange reactions. Because catalytic amounts of perthiol, generated by β-elimination of disulphide bonds, accelerate the disulphide-thiol exchange reaction, protection of perthiol/thiol by MTS molecules prevented irreversible denaturation. Analysis of irreversible denaturation using MTS reagents can help evaluate the contribution of disulphide bonds in the thermal stability of proteins, as well as their protein function
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