ISOLATION OF MONOMERIC SUBUNIT OF IMMUNOGLOBULIN M WITH ITS INTERCHAIN DISULFIDE BONDS INTACT

Immunoglobulin M (IgM) consists of five monomeric subunits, IgMs, each apparently comprising two μ chains and light chains. The distribution of disulfide bonds between μ chains within IgMs and those joining the μ chains of one IgMs to another is not known with certainty. Our goal was to obtain IgMs with its original inter-μ-chain and μ-light-chain disulfide bonds intact, and to determine whether one or two disulfide bonds join each IgMs to another subunit. The amount of IgMs released from IgM by reduction could be controlled by adjusting the concentration of mercaptoethylamine. In our hands, reduction of IgM with 0.015 M mercaptoethylamine resulted in a mixture containing about 50% IgMs. The subunit, isolated by filtration of the mixture through Bio-Gel P-200, had a sedimentation coefficient of 6.17 S at a concentration of 6.21 mg/ml, electrophoretic mohility similar to its parent IgM, maintained the specific antigenic sites on the μ chain, and comprised both μ and light chains. Regardless of the time of reduction from 5 to 40 min with 0.015 M mercaptoethylamine, IgMs contained about two carboxymethylcysteine residues per molecule. In addition, IgMs isolated after 30-min reduction failed to dissociate in propionic acid into μ and light chain until it subsequently was reduced with 2-mercaptoethanol. In conclusion, the data indicated the IgMs has maintained its original interchain disulfide bond integrity, and each IgMs is bound to another by a single disulfide bond. © 1968, American Chemical Society. All rights reserved.