Primary and secondary tryptic cleavages of human IgM at high temperature.

Previously it was shown that the tryptic digestion of human IgM at 65 C yield Fc'mu fragments in addition to Fabmu and (Fc)5mu fragments. This Fc'mu was found to be derived from the Cmu4 domain of the mu-chain. Additional studies were done on the effects of tryptic digestion time and temperature on the proteolytic process of IgM, IgM digestion by 2% trypsin at 65 C produced (Fc)8mu and Fc'mu in approximately a 3:2 ratio. While an appreciable amount of intact (Fc)5mu was present along with Fc'mu in the IgM digest, no residual "(Fc)5mu fragment" with its C-terminal segment missing was found. If the temperature of digestion is held constant at either 56 C or 60 C and the digestion time is varied from 20 to 90 min, there is also progressive cleavage of IgM with a concomitant increase in the yield of Fc'mu. It appears that the tryptic digestion of IgM is a stepwise process and that the primary cleavage of IgM occurs at Arg-325(2) of all ten mu-chains and the (Fc)5mu is subsequently degraded to Fc'mu fragments by secondary cleavage.