Copper-catalysed reoxidation of human monoclonal IgM

Human monoclonal IgM was dissociated into subunits by reduction with 0.01 M dithiothreitol and then separated from the latter with minimal loss of free sulphydryl groups by gel filtration. Incubation in pH 8.0 buffered saline at 25 degrees C for 1 or 2 h resulted in oxidation of approximately 10% of the sulphydryl groups and no significant polymer formation. Incubation for 20 h resulted in oxidation of approximately 60% of the sulphydryl groups and appearance of 15--55% polymers sedimenting at 17S. Oxidation of the sulphydryl groups during the 20 h incubation period was decreased, and reassociation of the subunits was prevented by the addition of 10(-3) M ethylenediamine tetraacetic acid to the buffered saline. In contrast, 90% of the sulphydryl groups were oxidized and 75% of the subunits were reassociated after only 30 min when incubated in 10(-5) M cupric ion. The reassociated IgM sedimented as a major and a minor component at 15S and 19S, respectively. Urea-SDS polyacrylamide gel electrophoresis further demonstrated that extensive interchain bonding also occurred. Neither the yield of polymers nor the relative quantities of the reassociated components were altered by increasing the incubation time to 20 h.