Author(s): Volkin DB, Klibanov AM
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Abstract To ascertain the upper limit of protein thermostability and to evaluate the introduction of additional -S-S- bridges for the enhancement of protein thermostability by site-directed mutagenesis, the stability of cystine residues at 100 degrees C has been investigated in a number of unrelated proteins. All proteins studied (more than a dozen) undergo heat-induced beta-elimination of cystine residues in the pH range from 4 to 8. The time courses of this process closely follow first-order kinetics indicating that the stability of a cystine residue is not significantly affected by its position in the polypeptide chain. Furthermore, the first-order rate constants of beta-elimination of disulfide bonds at 100 degrees C are remarkably similar for different proteins--0.8 +/- 0.3 h-1 and 0.06 +/- 0.02 h-1 at pH 8.0 and 6.0, respectively. Thus, this process is relatively independent of both the primary structure and the elements of higher structures remaining in proteins in 100 degrees C. beta-Elimination of disulfides produces free thiols that cause yet another deleterious reaction in proteins--heat-induced disulfide interchange. This reshuffling of -S-S-bonds, which is much faster than beta-elimination, has also been quantitatively characterized.
This article was published in J Biol Chem
and referenced in International Journal of Waste Resources