Separation of univalent fragments from the bivalent rabbit antibody molecule by reduction of disulfide bonds

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Nonprecipitating active fragments of rabbit antiovalbumin γ-globulin, having approximately the same sedimentation coefficient (3.5 S) and inhibitory effect on the homologous precipitin reaction as the univalent fragments produced by papain, can be prepared by treatment with pepsin in the presence of a reagent capable of breaking disulfide bonds. The reaction can also be carried out by successive treatments with pepsin and one of several such reagents. Cysteine, 2-mercaptoethylamine, thioglycolate, and cyanide were effective. In the absence of enzyme, but under otherwise identical conditions, these compounds had no apparent effect on the antibody. The combining sites on fragments of an antihapten antibody produced by this method were found, by hapten-binding measurements, to be essentially intact, although the capacity to form specific precipitates was lost. This fact and the close similarity to the univalent fragments formed by the action of papain indicate that the active split products produced by pepsin under these conditions are univalent. Peptic digestion causes a decrease in sedimentation coefficient to about 5 S, but the capacity to form specific precipitates is largely retained, indicating that the 5 S fragment is bivalent. Further treatment with a disulfide-splitting reagent results in the formation of fragments with the properties of univalent antibody and s ≅ 3.5. The results indicate that the proteolytic enzyme splits off an inactive portion of the molecule; and that subsequent to this reaction the bivalent residue can be divided into two univalent fragments by breaking the disulfide bonds which hold them together. Because compounds which react with disulfide groups have always been used with papain, as activators of the enzyme, it appears that a similar mechanism may obtain in the digestion of rabbit antibody by that enzyme. Since Porter has shown that the two active fragments (3.5 S) released by papain are nearly identical in molecular weight and amino acid composition, the possibility is suggested that the biosynthesis of rabbit antibody may include linkage of these two nearly identical univalent subunits through one or more disulfide bonds.