Formation of non-covalently linked IgM polymers: Dependence on the protein sulfhydryl groups and zinc ions

Abstract

Splitting of native polymeric IgM to tree non-covalently linked half sub-units and free J-chains was obtained by reduction with 0.2M mercaptoethanol. Removal of the reducing agent by dialysis for 24 hr or several days in the presence of 10 mm of cheluting agents gave no polymers, while dialysis in the presence of 20 μM zinc ions gave up to 97% of polymers. The polymers showed a sedimentation rate and protein profile in the ultracentrifugc and J-chain content similar to that of native IgM.

The polymers made by dialysis for 24 hr were non-covalently linked since they were split by iodoacetamide orN-ethyl maleimide to free non-covalently linked half sub-units and free J-chains and by sodium dodecyl sulphate mostly to free chains. Of the polymeric molecules prepared by dialysis for several days, some of the molecules were found to be stabilized by disulphide bridges.

It was concluded that in order to obtain IgM polymersin vitro from reduced IgM, the sub-units and J-chains must have their sulfhydryl groups intact and zinc ions must be present. It seems likely that the formation of covalently linked IgM polymers alsoin vivo is preceded by the formation of non-covalently linked polymers.