As an outgrowth of studies of the effect of disease on the heat coagulability of serum proteins, it was observed that the single sulfhydryl group of human or bovine plasma albumin exerts a marked influence on the nature of the coagula obtained when solutions of such proteins are heated (118). Clots formed by the thermal denaturation of unmodified bovine plasma albumin at neutral pH are opaque, friable, and synerizing, whereas those formed from albumin which has had its sulfhydryl group destroyed or blocked by treatment with an appropriate "sulfhydryl reagent" are transparent, firm, and non-synerizing (Fig. 33). Moreover, in the absence of the sulfhydryl group, solid gels are obtained with solutions of much lower albumin concentration than that required for gel formation when the protein sulfhydryl group is present. In analogy to established concepts of fibrin clot structure, it appeared that the clear non-synerizing coagula consist of a regular three-dimensional network of protein chains with many interstitial spaces for water to be bound, whereas the opaque synerizing clots possess additional side-by-side association to give heterogeneous clumps which scatter light and have relatively few interstitial spaces for binding water. It was concluded that during thermal denaturation the albumin sulfhydryl group in some way must promote this lateral association of protein chains. But how a single group can so markedly influence the aggregation behavior of a large molecule containing nearly 600 amino acid residues presented a challenging question.

Insight into the nature of this sulfhydryl-induced protein aggregation was obtained from studies of the urea denaturation of plasma albumin. The previously known ability of albumin and certain other proteins to form clear firm gels when exposed to concentrated urea or guanidine hydrochloride was found to depend on the protein sulfhydryl group (67). Gelation in urea is favored by increased pH, inhibited by oxygen, and eliminated by blockage of the sulfhydryl. The capacity for gelation is restored to sulfhydryl-free albumin by the addition of trace amounts of either sulf-hydryl-containing proteins or simple mercaptans. Since the observed phenomena appeared to involve a stoichiometry quite different from that usually encountered in protein reactions, it was proposed that, under conditions of protein denaturation, a sulfhydryl group reacts with a disulfide group to form an intermolecular disulfide linkage, at the same time generating a new sulfhydryl group capable of reiterating the process (Fig. 34). By such a chain reaction, a single sulfhydryl group can bring about the interchange of a large number of protein disulfide groups, leading to a three-dimensional gel network in the case of urea denaturation and to sideby-side aggregation in the case of thermal denaturation where no bound urea molecules are present to hold the protein chains apart.

Fig. 34.-Sulfhydryl-disulfide interchange in proteins.

Quantitative evidence confirming the foregoing concept was obtained from measurement of viscosity changes in albumin solutions too dilute to form solid gels in urea (28). On exposure to concentrated urea, dilute solutions of bovine plasma albumin show a large immediate increase in viscosity, followed by a further gradual but prolonged viscosity rise (Fig. 35). The secondary viscosity change, but not the first, is abolished by treatment of the protein with sulfhydryl reagents, and it can be restored to iodoacet-amide-treated albumin by the addition of trace amounts of simple mer-captans. Thus this secondary viscosity change in dilute solution reflects the same process which leads to gelation in more concentrated solution, namely, a sulfhydryl-initiated disulfide interchange.

In the case of thermal denaturation, measurements of viscosity and sedimentation of dilute albumin solutions, as well as the effect of traces of mercaptans in the thermal coagulation of iodoacetamide-treated albumin, furnish support for the concept that lateral association of protein chains by sulfhydryl-initiated disulfide interchange takes place when albumin solutions are heated (29).

Fig. 35.-Effect of 1 equivalent of silver nitrate on the viscosity change of an albumin-urea solution.

Since the publication of the above considerations, the occurrence of disulfide interchange during protein reactions has been confirmed by a large number of investigators. In addition to aggregation phenomena accompanying protein denaturation, the concept has been utilized to explain such varied phenomena as the long-range elasticity of wool, the rheolog-ical properties of kneaded dough, and artifacts arising in the hydrolysis of proteins and peptides. Evidence has been put forward that certain important physiological processes, such as the clotting of blood, the formation of the mitotic apparatus in cell division, and the physiological action of the peptide hormone, vasopressin, may involve sulfhydryl-disulfide interchange reactions. A survey of the reported investigations concerned with sulfhydryl-disulfide interchange in proteins and peptides has been published recendy (115).