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Primary and secondary tryptic cleavages of human IgM at high temperature
Abstract:
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.
Insights
Tryptic digestion of human immunoglobulin M (IgM) yields Fc'mu fragments. This process involves stepwise cleavage, primarily at Arg-325, degrading the (Fc)5mu fragment into Fc'mu.
Area of Science:
- Immunology
- Protein Chemistry
- Biochemistry
Background:
- Human immunoglobulin M (IgM) is a crucial antibody involved in the immune response.
- Previous studies indicated tryptic digestion of IgM yields Fc'mu fragments from the Cmu4 domain.
Purpose of the Study:
- To investigate the effects of tryptic digestion time and temperature on IgM proteolysis.
- To elucidate the stepwise mechanism of IgM digestion and Fc'mu fragment generation.
Main Methods:
- Tryptic digestion of human IgM at varying temperatures (56-65°C) and times (20-90 min).
- Analysis of resulting fragments, including Fabmu, (Fc)5mu, and Fc'mu.
Main Results:
- IgM digestion at 65°C yielded Fc'mu fragments and Fabmu fragments.
- Digestion at 65°C produced (Fc)8mu and Fc'mu fragments in a 3:2 ratio.
- Progressive cleavage of IgM and increased Fc'mu yield were observed with varied digestion times at 56°C or 60°C.
Conclusions:
- Tryptic digestion of IgM is a stepwise proteolytic process.
- Primary cleavage occurs at Arg-325 on the mu-chains.
- (Fc)5mu fragments are subsequently degraded into Fc'mu fragments.