THE SUBUNITS IN RABBIT ANTI-FORSSMAN IGM ANTIBODY

Abstract

Radioactively labeled reduced and alkylated subunits of IgM [immunoglobulin M] rabbit anti-Forssman antibody were obtainedby 2 distinct methods. Antibody was eluted from formalin treated sheep erythrocytes at pH 10.6, followed by filtration through Sephadex G-200 and centrifugation in 10-30% sucrose density gradients. The antibody was labeled with 125I at a level of 9-120 atoms of I per mole, and reduced with .002 [image] dithiothreitol and alkylated with .003 [image] iodoacetamide. In the other method, IgM antibody was precipitated with a water-insoluble preparation of purified Forssman hapten. Antibody in the complexes was reduced and at the same time released by treatment with .006 [image] dithiothreitol, followed by 14c-iodoacetamide. Reduced and alkylated subunits were thus obtained trace labeled either with 125I in tyrosine or with 14C at substituted sulhydryl residues. Their behavior in all subsequent tests was similar. On gel filtration the subunits travelled between IgG [immunoglobulin G] and albumin; in sucrose density gradients they sedimented between albumin and a marker (pepsin digested IgG) M.W. 100,000. Gel filtration in .04 [image] sodium dodecylsulfate or 6 [image] guanidine showed that free L and H chains were present. At the low concentrations examined (<100 [mu]g/ml) the subunits consisted of one H and one L chain. The subunits bound much more weakly than the original IgM to sheep erythrocytes. On passage through columns of boiled sheep erythrocyte stroma on a supporting matrix exactly half the subunits were specifically retarded, implying that only half had antibody activity. Reasons are given why antibody active and inactive subunits are probably derived from the same molecules. The structure of IgM antibody is discussed in relation to its special capacity to bind to surfaces with repeating antigenic structures.