In the foregoing examples, protein disulfide groups appear to participate in interchange reactions under conditions of denaturation. No doubt most disulfide groups in native proteins are either hindered or otherwise unreactive (33) , so that some disruption of protein structure is necessary to make them available for reaction. Without this restriction, disulfide exchange reactions could take place indiscriminately in physiological systems, and chaos would ensue in the living cell. In the case of simpler peptides and amino acids, however, the disulfide groups are readily available, and interchange reactions often take place with amazing ease, even with peptides which contain no sulfhydryl groups as initiators.

This fact was first reported by Sanger (34) after observations that an acid hydrolyzate of insulin may contain many more cystine peptides than can be accounted for by any unique structure for the protein. In a subsequent study, Ryle and Sanger (35) demonstrated that disulfide interchange in peptide solutions at 37 °C can take place either in neutral or in alkaline medium or else in strongly acid medium (7 to 12N HC1) with little or no exchange observed in moderately acid solutions. The former process is accelerated by an increase in pH or by the addition of mercaptans and inhibited by sulfhy-dryl-blocking reagents, whereas disulfide interchange in strongly acid solution is inhibited by added mercaptans. Cognizant of these facts. Sanger and his associates (36) were able to devise conditions for the hydrolysis of insulin so as to avoid disulfide interchange and the resulting artifacts among the peptides produced.

Additional examples of disulfide exchange reactions both in acidic and in alkaline media have been described by Schoberl and Grafje (37), who also found that illumination with ultraviolet light promotes disulfide interchange. Although the latter observation suggests the possibility of disulfide interchange by a free radical mechanism, because of the pH dependence of the photochemically induced exchange, the authors consider it probable that the illumination in some way promotes the formation of mercaptide ions rather than initiating a free radical process.

Ressler (38) has observed that on standing at room temperature in sodium bicarbonate solution the cystine-containing peptide hormone oxytocin undergoes loss of biological activity accompanied by aggregation and decreased solubility. This behavior is ascribed to a disulfide interchange reaction which produces intermolecular disulfide linkages; the latter can be cleaved, and biological activity can be partially restored, by treatment of the inactivated hormone with cysteine, glutathione, or hydrogen sulfide. Since the inactivation takes place under the mild alkaline conditions ordinarily used for the preparation of dinitrophenyl derivatives of proteins and peptides, the need for caution is noted in the interpretation of results involving dinitrophenyl derivatives of disulfide-contain-ing peptides.

Reaction Mechanisms

The sulfhydryl-initiated disulfide interchange illustrated in Fig. 1 undoubtedly is analogous to the intermediate step in the reduction of simple disulfides by mercaptans, a reaction which has been studied extensively (39) and which is known to proceed by nuclec-philic attack of a mercaptide anion (RS-) on the disulfide bond. Unless they are strongly influenced by other factors, such as changes in electrostatic repulsive forces between the charged protein molecules (1), disulfide interchange reactions of this type would be expected to proceed at a more rapid rate as the pH, and thus the mercaptide ion concentration, is increased. In the protein experiments discussed above in which comparisons have been made, an increase in pH has been found to enhance phenomena ascribed to the disulfide interchange reaction.

In the case of peptides which contain no sulfhydryl groups, the disulfide interchange reaction in neutral or alkaline solution appears to proceed by a similar mechanism, the initiating sulfhydryl group being produced by the hydrolysis of a disulfide bond (55). In strongly acid medium, on the other hand, disulfide exchange takes place by a different mechanism, demonstrated recently by Benesch and Benesch (40) to involve electrophilic attack of a sulfenium cation (RS+) on the disulfide bond. Under the action of strong hydrochloric acid, the following reactions occur:

The known reaction of mercaptans with sulfenyl chlorides (41) appears to be partially reversible, so that small amounts of sulfenyl chloride are formed from the disulfide (reaction 1). The sulfenyl chloride then reacts with a disulfide, in a manner also described by Moore and Porter (42), to form the mixed disulfide and to regenerate a new sulfenyl chloride which carries on the process (reaction 2). Marked enhancement of disulfide exchange is observed upon the addition of small amounts of sulfenyl chlorides or sulfenic acids (RSOH), which in strong acid can give rise to sulfenium ions, as well as of hydrogen peroxide, which reacts with disulfides to produce sulfenium ions. That the acid-catalyzed exchange is inhibited by the addition of mercaptans is readily understandable, since the presence of these substances reverses reaction 1 and thus depletes the amount of sulfenyl chloride, the concentration of which is already rate-limiting.

Physiological Processes

The foregoing examples of disulfide interchange reactions are in vitro processes which take place when a protein or peptide is subjected to chemical or physical manipulation. Whether similar interchange reactions take place in vivo is a question of considerable interest, and there are now strong indications that sulfhydryl-initiated disulfide interchange may play a role in certain important physiological processes.

One such phenomenon is the clotting of blood fibrinogen (43). It is well established by the work of Robbins (44) and of Lorand (45) that the fibrin clot formed from the action of thrombin on purified fibrinogen (fibrin-s) is different from the physiological clot obtained from the coagulation of blood or of recalcinated plasma (fibrin-i). Fibrin-s is soluble in 5M urea or in weak acid or alkali, whereas fibrin-i is insoluble in these reagents and also possesses much greater mechanical strength. The formation of the insoluble type of fibrin requires the presence both of calcium ions and of a nondialyzable, heat-labile substance present in blood plasma, known variously as Laki-Lorand (L-L) factor, fibrin-stabilizing factor (FSF), or urea-insolubility factor. The properties of fibrin-i indicate that this clot possesses stable cross linkages not present in fibrin-s, whereas the fact that fibrin-i dissolves readily in a mixture of urea and thioglycolic acid (46) suggests that these additional cross linkages are disulfide bonds.