Study of properties of structural subunits of IgM immunoglobulin obtained by reduction with 2-mercaptoethanol or by oxidative sulphitolysis.

Abstract

IgM was isolated from pig serum by isoelectric precipitation and gel filtration. Different methods of breaking down the disulphide bonds and of isolating subunits of the IgM molecule—oxidative sulphitolysis and reduction by 0.1m 2-mercaptoethanol in the absence of a disaggregating agent, oxidative sulphitolysis in the presence of 6m urea and reduction by 0.3m 2-mercaptoethanol in medium containing 6m and 8m urea—were compared. Degraded material was separated by gel filtration on Sephadex G-100 or G-200 in 0.05m formic acid with 6m or 8m urea. Oxidative sulphitolysis or reduction by 0.1m 2-mercaptoethanol without a disaggregating agent did not yield pure H andl chains. Oxidative sulphitolysis was the more effective. Oxidative sulphitolysis in 6m urea medium severely damaged the material. Reduction of IgM by 0.3m 2-mercaptoethanol in 6m or 8m urea also altered its immunochemical properties. The possible presence of light chains in the heavy chain fraction cannot likewise be excluded in this case. The results are in agreement with experiments showing that the molecular weight of the IgM heavy chain is greater than that of the IgG heavy chain.