Copper-Catalysed Reoxidation of Human Monoclonal IgM

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Abstract
Human monoclonal Ig[immunoglobulin]M was dissociated into subunits by reduction with 0.01 M dithiothreitol and separated from the latter with minimal loss of free sulphydryl groups by gel filtration. Incubation in pH 8.0 buffered saline at 25.degree. C for 1 or 2 h resulted in oxidation of approximately 10% of the sulfhydryl groups and no significant polymer formation. Incubation for 20 h resulted in oxidation of approximately 60% of the sulfhydryl groups and appearance of 15-55% polymers sedimenting at 17S. Oxidation of the sulfhydryl groups during the 20 h incubation period was decreased, and reassociation of the subunits was prevented by the addition of 10-3 M EDTA to the buffered saline. Of the sulfhydryl groups, 90% 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 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.