A number of observations confirm the occurrence of disulfide interchange during thermal denaturation of proteins. The effect of oxidizing and reducing agents on the viscosity and turbidity of heated /?-lactoglobulin solutions led Zittle and Delia Monica (13) to conclude that the protein sulfhydryl groups promote lateral association of peptide chains, with resulting opacity, although the effect is less pronounced than in the previously mentioned case of bovine plasma albumin (1, 6). From the influence of sulfhydryl reagents on the sedimentation behavior of thermally denatured bovine plasma albumin and on its solubility in 83-percent acetic acid, Steinrauf and Dandliker (14) have shown that during the first 30 seconds at 100°C and pH 5.6, intramolecular rearrangement of hydrogen bonds takes place, after which a rapid intermolecular disulfide interchange commences, leading to polymerization. Somewhat similar conclusions were reached by Warner and Levy (15) working at the lower temperature of 65.7°C. Kinetic investigations, involving sedimentation studies of the intermediate products formed in this mild thermal denaturation of bovine plasma albumin in the presence and absence of sulfhydryl reagents, indicate that the initial reaction is an intramolecular sulfhydryl-disulfide interchange and that aggregation takes place subsequently, in part by an exchange mechanism of an intermolecular type. Apparently, precipitation of a protein with alcohol is an especially favorable condition for disulfide interchange. In addition to the previously mentioned experiments with ovalbumin (9), Straessle (16) has reported that treatment of human plasma mercaptalbumin with cold aqueous ethanol of more than 60 percent alcohol content by volume can cause partial conversion of the protein to a dimer, a reaction which does not occur with iodoacetamide-treated albumin under similar conditions. This sulfhydryl-initiated cross-linking reaction provides an explanation for the earlier observation of Cohn, Hughes, and Weare (17) that aggregation of protein takes place during the methanol extraction of lipids from albumin. Recently, Rouser (18) has reported that nonprotein sulfhydryl compounds in plasma exert a marked influence on the alcohol precipitation of the protein. On the addition of ethanol, plasma from normal individuals gives a coarse granular precipitate, whereas plasma low in cysteine, obtained from chronic lymphatic leukemia patients, as well as solutions of commercial human plasma albumin, give a fine, difficult-to-filter precipitate which is slowly transformed to the coarse granular type upon addition of small amounts of cysteine.

Fig. 1. Sulfhydryl-disulfide interchange in proteins.

The sulfhydryl-initiated transformation of intramolecular disulfide bonds to intermolecular linkages with accompanying gelation has been clearly demonstrated by Benesch and Benesch (19). who used a model protein, thiolated gelatin. This substance is prepared by treatment of gelatin, which itself contains no sulfhydryl or disulfide groups, with N-acetylhomocysteine thiolactone. Oxidation of the thiolated protein with ferricyanide in dilute solution gives a protein with intramolecular disulfide linkages, as indicated by the lack of change in its viscosity and sedimentation characteristics on oxidation. Treatment of a solution of the oxidized protein with a trace of mercaptoethylamine causes an immediate transformation into a firm, heat-stable gel similar to that produced by oxidation of the thiolated gelatin in concentrated solution where intermolecular disulfide bonds are formed directly.

In a practical application of sulfhydryl-disulfide interchange, Arnold (20) has cross linked a monomolecular layer of fibrinogen to afford a semipermeable protein membrane for use in model cell membrane studies. A thin layer of fibrinogen in saline is floated carefully on an aqueous surface, and the addition of cysteine to the water effects two-dimensional cross linking of the protein to form a stable film.

A somewhat different protein phenomenon considered to involve sulfhydryl-disulfide interchange is the long-range elasticity of wool. When a wool fiber is stretched in water by an amount greater than about 30 percent, irreversible structural alterations take place which have been postulated to result from the rupture of disulfide linkages (21). Since the sulfhydryl content of wool was found to be unaltered by the stretching process, Burley (22) has suggested that stretching brings a sulfhydryl group into contact with a disulfide group whereupon interchange takes place to form a new disulfide linkage in a manner which relieves the mechanical strain. In support of this exchange mechanism, Burley observed that wool fibers with their sulfhydryl groups blocked by previous reaction with iodoacetamide or N-ethylmalei-mide stretch at a much slower rate and to a lesser extent than do untreated, sulfhydryl-containing fibers under the same conditions. Moreover, untreated fibers which stretch readily in water are resistant to stretching in 0.1 N hydrochloric acid, an environment unfavorable for the sulfhydryl-disulfide reaction.

Klotz and his coworkers (23) have studied the interaction between the sulfhydryl group of bovine plasma albumin and the disulfide-containing dyestuff 2, 2'-(2-hydroxy-6-sulfonaph-thyl-l-azo)-diphenyl disulfide. In this case, only the first stage of the sulfhydryl-disulfide chain reaction appears to take place. To explain the stoichiometry observed, the authors propose a novel type of long-range intramolecular sulfhydryl-disulfide interchange in the albumin molecule, which involves electron transport by way of the hydration lattice of the protein.

Two examples of reversible protein aggregation in the absence of denaturing agents have been ascribed to sulfhydryl-disulfide interchange. These are the association of soluble feather keratin, which takes place as the protein concentration is increased (24). and the dimerization of bovine plasma albumin, which occurs when the pH is lowered to 3.4 or below (25). These phenomena differ from those previously described not only in that they are manifest in the absence of conditions which disrupt the protein structure but also in that the aggregation appears to be freely reversible by such relatively mild manipulations as dilution or raising of the pH to neutrality. Moreover, in the case of bovine plasma albumin, the aggregation is observed in a pH region where sulfhydryl-initiated disulfide interchange ordinarily does not occur. Although these examples may represent rather special cases of disulfide exchange, it would seem advisable to consider the alternative possibility that some other type of sulfhydryl-dependent aggregation may be involved.

It is now established that sulfhydryl groups can contribute to the association of protein units in ways other than through disulfide bonds. In addition to the well-known dimerization of albumin by reaction of sulfhydryl groups with divalent mercury (26), it appears that the sulfhydryl group itself can interact with some other protein group or groups to form a stable but reversible linkage. Participation of sulfhydryl in a thiazoline structure, first proposed by Linderstr0m-Lang and Jacobsen (27), has been demonstrated by Calvin (28) to exist under certain conditions in the peptide glutathione. Madsen and Cori (29) have observed a reversible, sulfhy-dryl-dependent aggregation of the enzyme phosphorylase. Deutsch and Morton (30) have reported an association of human serum macroglobulin units which appears to involve sulfur-containing groups, and Lorand (31) has described a somewhat similar aggregation for the fibrin stabilizing factor of blood plasma. Finally, the existence of a stable but reversible intramolecular "sulfhydryl bond" has been proposed (32) to account for several rather unusual properties of bovine plasma albumin. Thus, the multiple possibilities for the role of sulfhydryl groups in linking protein units together should be borne in mind when one is considering sulfhydryl-dependent aggregation phenomena in protein systems.