Amino acid disulfide interchange

To ascertain the upper limit of protein thermostability and to evaluate the effect of additional disulfide bridges on the enhancement of protein thermostability, additional cysteine residues were introduced into several unrelated proteins by site-directed mutagenesis and deactivation behavior tested at 100°C (Volkin, 1987). All the proteins investigated underwent heat-induced beta-elimination of cystine residues in the pH 4—8 range with first-order kinetics and similar deactivation constants kj that just depended on pH 0.8 0.3 h-1 at pH 8.0 and 0.06 0.02 h 1 at pH 6.0. These results indicate that beta-elimination is independent of both primary amino acid sequence and the presence of secondary structure elements. Elimination of disulfides produces free thiols that cause yet another deleterious reaction in proteins, heat-induced disulfide interchange, which can be much faster than beta-elimination. 

There is another phenomenon, regarded as a deteriorative change in the protein of soy milk, caused also by the evaporation of water. This is a film formation on the surface of soy milk, which occurs when heated soy milk is kept open to the air. This phenomenon is observed not only in heated soy milk but also in heated cow s milk. Film formation of soy milk occurs only when the soy milk is heated above 60°C and there is evaporation of water from the surface of the soy milk. The mechanism of protein insolubilization is basically the same as that of soy milk powder produced from heated soy milk (10. When water is removed from the surface of heated soy milk by evaporation, the molecular concentration of protein near the surface increases locally and the exposed reactive groups of the denatured molecules come close enough to interact intermolecularly both by hydrophobic interactions and through the sulfhydryl/disulfide interchange reaction to form a polymerization (film) on the surface. The upper side of the film contains more hydrophobic amino acids because of orientation of the hydrophobic portions of the unfolded molecules to the atmosphere rather than into the aqueous solution. 

Fio. 8. (A) Hypothetical transition state in the electrophilic catalysis of thiol-di-suliide interchange. (B) General acid-base catalysis of thiol-disulfide interchange by a hypothetical amino group. 

The method of extracting protein material from wool by splitting the peptide bonds while leaving the disulfide bonds intact has attracted some attention. Such a procedure might retain the original conformation of the amino acid chains if disulfide interchange during hydrolytic extraction could be avoided. 

Common to all of these processes, is the concept of protein denaturation, the definition of which has come under scrutiny (Stanley and Yada, 1992). The term denaturation has been defined in terms of the effect on the protein structure, as simply a major change from the original native structure, without alteration of the amino acid sequence, i.e., without severance of any of the primary chemical bonds which join one amino acid to another (Tanford, 1968). One criticism of this definition lies in its exclusion of oxidation-reduction or interchange reactions of covalent disulfide bonds, which may be crucial in the denaturation process for some proteins. Denaturation has also been defined with respect tp its effect on protein functionality, as any nonproteolytic modification of the unique structure of a native protein giving rise to definite changes in chemical, physical or biological properties (Neurath and colleagues, as cited by Colvin, 1964). 

Since reductive S-alkylation of disulfide bonds introduces unnatural amino acid side chains into the protein and, therefore, cannot serve as a useful model for nutritional and toxicological studies, we initiated systematic studies of effects of thiols on the inhibitory process. Such thiols are expected to interact with inhibitor disulfide bonds via sulfhydryl-disulfide interchange and oxidation reactions (Friedman, 1973). 

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