Reduction and Reoxidation of a Critical Disulfide Bond in the Rabbit Antibody Molecule*

Treatment of 6.5-S rabbit antibody with pepsin removes a large inactive fragment. The residual portion of the molecule, which is still bivalent, has a molecular weight of 106,000 and a sedimentation coefficient of 4.6 to 5.0 S (1, 2). Reduction of a single, highly labile disulfide bond cleaves this fragment approximately in half (2, 3). The resulting 3.5-S fragments are univalent and inhibit specific precipitation (1). Upon reoxidation 5-S protein is formed once again; and if specifically purified antibody was the starting material, most of the original precipitating activity is restored (4). *%ntibody of mixed specificity can be prepared by reducing and reoxidizing a mixture of antibodies specific for two different antigens (5, 6). The univalent fragments formed by the action of pepsin and a reducing agent are similar in many properties to those produced by proteolysis with papain (1, 4, 7). The two univalent fragments of an individual molecule appear to be similar. Evidence for this is the fact that they migrate as a single 3.5-S peak in the ultracentrifuge (7) and are eluted together from carboxymethyl cellulose (8). In addition, fingerprinting studies by Gitlin and Mcrlcr (9) suggest that the number of peptides liberated from y-globulin is approximately half the number of peptide bonds broken, which is consistent with the possibility of symmetry within the molecule. Fahey and Askonas (10) have shown that the electrophoretic mobilities on starch gel of the active fragments of individual molecules of mouse y-globulin are similar. Five additional disulfide bonds can be cleaved in univalent fragments, formed by treatment with pepsin and reducing agent, without change in sedimentation coefficient or appreciable loss of specific activity (11, 12). When such fragments are allowed to recombine only 5-S protein is formed (11, 12). The absence of products of larger molecular size indicates that only the sulfhydryl groups liberated from the disulfide bond linking the univalent fragments are available for recombination among fragments. Retention of specific activity and molecular size after reduction was subsequently reported by Grossberg, Stelos, and Pressman (13), who investigated the univalent fragments produced by treatment with papain. In a preliminary communication (14), it was reported that the critical disulfide bond which links the univalent fragments after pepsin treatment can also be reduced readily in the untreated