The observation that small amounts of sulfhydryl-blocking reagents prevent the formation of urea-insoluble fibrin clots led Loewy and Edsall (47) to propose that fibrin-/ is cross-linked by intermolecular disulfide bonds produced through a sulfhydryl-initiated interchange of the disulfide groups of fibrin-s. With the subsequent isolation of the fibrin stabilizing factor in purified form (31, 48-50), and the demonstration that this substance contains from one (49) to two (50) equivalents of titratable sulfhydryl per 100,000 grams of protein, sulfhydryl which is essential for FSF function, this factor would appear to possess the necessary qualifications to be an initiator of disulfide interchange.

During the clotting process, sulfhydryl groups are produced whose reactivity appears to be considerably increased over that of the original FSF sulfhydryl (49). Inactivation of FSF by pretreatment with silver ions requires ten times the silver concentration necessary to prevent fibrin-i formation when the silver is present during the clotting process, whereas iodoacetamide will inhibit fibrin-i formation but will not inactivate FSF on pretreatment. These observations of Loewy's are reminiscent of Kolthoffs (11, 12) studies on aggregated plasma albumin after guanidine hydrochloride treatment, and they are consistent with the involvement of a sulfhydryl-disulfide interchange mechanism in the formation of fibrin-i.

Whether the sulfhydryl groups of FSF are the only such groups which can initiate the conversion of fibrin-s to fibrin-i is not certain. Earlier findings that simple mercaptans (47) or plasma albumin (51) could promote this transformation have been attributed by Lorand and Jacobsen (52) to a regeneration of inactivated FSF tenaciously bound to the protein employed, since, with their fibrinogen preparations, neither cysteine nor plasma albumin showed any FSF activity. However, Loewy (49) has prepared fibrinogen which by immunological criteria appears to be free of bound FSF, and preliminary experiments with this material indicate that simple mercaptans do show FSF activity when present in rather high concentration. Thus, further study is required before agreement can be reached as to the specificity of the FSF as the reaction initiator. In any case, the concept that FSF, augmented in some way by calcium ions, effects cross linking of fibrin molecules through a sulfhydryl-disulfide interchange reaction provides a reasonable explanation for the formation of the physiological fibrin-i clot.

A second biological phenomenon which may involve sulfhydryl-disulfide interchange is that of cell division. It has long been known (53) that during mitosis the acid-soluble sulfhydryl (glutathione) content of the sea urchin egg exhibits a cyclical variation; it decreases after fertilization, reaches a minimum at about the time of spindle formation, and returns to its original level prior to cleavage. After observations that the protein sulfhydryl content of the sea-urchin egg likewise varies, but with an inverse relationship to the variation of the glutathione level, and also that the isolated mitotic apparatus is soluble in sodium thioglycc-late, Mazia (54) proposed that disulfide bonds are important structural features of the mitotic apparatus and that the formation of this entity may involve the polymerization of small protein molecules through a glutathione-initi-ated disulfide interchange reaction. The construction of the mitotic apparatus is considered to involve both gelation of protein through intermolecular disulfide bond formation and orientation of the gel structure through secondary bond production; the orientation but not the gelation process is prevented by the mitotic inhibitor, colchicine (55).

The participation of sulfhydryl groups in mitosis is further indicated by the reversible blockage of the mitotic cycle of sea-urchin eggs by the addition of small amounts of mercaptoethanol (56), whereas the ability of mercaptoethanol to induce twin formation, when present during a particular stage of the cleavage cycle of Dendraster eggs, suggests that the simple mercaptan competes with protein sulfhydryl groups in the formation of disulfide bonds involved in interblastomere linkages (57). Although many details of the complex physiological process of mitosis remain to be elucidated, the intriguing suggestion that sulfhydryl-disulfide interchange plays an important role affords a promising approach for further investigation.

Eldjarn and Pihl (58) have reported that cystamine and cysteamine administered to a mouse rapidly become incorporated into the blood proteins, apparently by a sulfhydryl-disulfide exchange. This finding suggests that disulfide interchange reactions with body constituents may be intimately concerned with the protective action of these sulfur compounds against ionizing radiation.

Whether sulfhydryl-disulfide interchange plays a role in other biological phenomena remains to be investigated, although certain additional suggestions along these lines have been put forward. In view of the previously mentioned inactivation and reactivation of oxytocin, presumably by a disulfide exchange mechanism, Ressler (38) has suggested the possibility that disulfide-containing peptide hormones, such as oxytocin and vasopressin, may exist physiologically in an inactive form, with activation taking place at appropriate sites under the influence of sulfhydryl compounds such as glutathione. After observations of what appears to be a long-range intramolecular sulfhydryldisulfide interchange in bovine plasma albumin mediated through the bound water lattice, Klotz (23) pointed out that such a mechanism could furnish a means of electron transport in oxidation-reduction reactions involving sulfhydryl enzymes, especially in systems which appear to transfer energy over a distance. Other processes in which the possibility of disulfide interchange should be considered include the production of keratin and the formation of such physiological protein gels as the mitachondrial framework or the lens of the eye. Disulfide linkages are relatively abundant in most protein molecules, whereas sulfhydryl and disulfide are potentially among the most reactive of the protein functional groupings, although, as was pointed out above, their reactivity ordinarily is more or less restricted by the characteristic structure of the protein molecule. It is not unreasonable to consider that the physiological initiation and control of many important processes in living organisms may depend on factors which establish and regulate conditions under which interaction between protein sulfhydryl and disulfide groups can take place.