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Mercaptan-induced fragmentation of a subunit-like proteolytic fragment of immunoglobulin M
Abstract:
Limited papain hydrolysis of immunoglobulin M (IgM) produces a subunit-like proteolytic fragment designated IgM(p) (Inman & Hazen, 1968). In the presence of mercaptans, IgM(p) partially dissociated into Fc(mu)-like and Fab(mu) fragments. Treatment of residual IgM (that remaining after a papain digestion) with 2mm-mercaptoethylamine resulted in fragmentation of the same type that occurs in a routine limited digestion of IgM with papain, although exogenous enzyme was not added to the mixture. When IgM was hydrolysed with (14)C-labelled papain, a small quantity of the enzyme was found to be associated with the residual IgM and IgM(p) fractions. IgM and IgM 7S subunit (IgM(s)) that had been exposed to papain in the absence of activating mercaptan and separated from the enzyme by gel filtration also fragmented when subsequently treated with 2mm-mercaptoethylamine. The fragments resembled those produced during a typical limited papain digestion of IgM. It was concluded that mercaptoethylamine induced fragmentation of IgM(p) by activating adsorbed papain.
Insights
Mercaptoethylamine activates adsorbed papain to fragment immunoglobulin M (IgM) and its subunits. This explains how IgM(p) fragments form without adding more enzyme during papain digestion.
Area of Science:
- Biochemistry
- Immunology
Background:
- Limited papain hydrolysis of immunoglobulin M (IgM) yields IgM(p) fragments.
- IgM(p) can dissociate into Fc(mu)-like and Fab(mu) fragments in the presence of mercaptans.
Purpose of the Study:
- To investigate the mechanism of IgM fragmentation by papain and mercaptoethylamine.
- To determine if adsorbed papain plays a role in IgM fragmentation.
Main Methods:
- Limited papain hydrolysis of IgM.
- Treatment with mercaptoethylamine.
- Radioactive labeling of papain with (14)C.
- Gel filtration chromatography.
Main Results:
- Residual IgM and IgM(p) fractions showed fragmentation similar to routine papain digestion when treated with mercaptoethylamine.
- (14)C-labeled papain was found associated with residual IgM and IgM(p) fractions.
- IgM and IgM 7S subunits (IgM(s)) exposed to papain, then separated from the enzyme, fragmented upon subsequent treatment with mercaptoethylamine.
Conclusions:
- Mercaptoethylamine induces fragmentation of IgM(p) by activating adsorbed papain.
- Adsorbed papain is responsible for the observed fragmentation of IgM and its subunits.
- This mechanism explains fragmentation patterns observed in IgM digestion studies.