Pronase and proteinase K digestion of human immunoglobulin M.
Human 19S IgM was digested with pronase and proteinase K. Proteolysis was relatively fast, producing Fab2 mu-like fragments (approx. mol. wt 114,000) and Fab mu-like fragments (approx. mol. wt 46,500) as major products. Immunochemical analysis indicated that the fragments produced by either enzyme are very similar and that they are produced by cleavage at the C mu 2-C mu 3 and C mu 1-C mu 2 domain junctions respectively. An intermediate species of mol. wt 74,300, immunologically identical to F(ab)2 mu, was also identified. This is thought to represent an F(ab)2 mu fragment with one Fab mu fragment removed. Fc mu-related fragments were not identified in the digestion mixture with either enzyme. Covalently linked and non-covalently linked 7S human IgM (IgMs and IgMr respectively) were digested with pronase and proteinase K. IgMs was degraded very rapidly by either enzyme, producing relatively stable F(ab)2 mu- and Fab mu-like fragments. These fragments were similar in mol. wt and immunochemical properties to those produced from 19S IgM. IgMr was also degraded rapidly by either enzyme, in this case producing Fab mu-like fragments with no detectable F(ab)2 mu-like fragments. The kinetics of digestion and nature of the products suggest that cleavage of 19S IgM by pronase or proteinase K proceeds via an initial attack at the C mu 2-C mu 3 junction, followed by further degradation at the Cmu 1-C mu 2 junction. The results obtained using 7S IgM show that the intersubunit disulphide bonds, and the associated pentameric structure, are responsible for the relative resistance of 19S IgM to proteolysis. The inter-heavy-chain disulphide bonds, in particular the bond at cys 337, are responsible for the limited susceptibility of F(ab)2 mu-like fragments to proteolysis.
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K; D010450:Endopeptidases; D006801:Humans; D005779:Immunodiffusion; D007122:Immunoelectrophoresis; D007140:Immunoglobulin Fab Fragments / Q000032:analysis; D007075:Immunoglobulin M; D010446:Peptide Fragments / Q000032:analysis; D011402:Pronase; D014357:Trypsin
D007140:Immunoglobulin Fab Fragments; D007075:Immunoglobulin M; D010446:Peptide Fragments; D010450:Endopeptidases; D014357:Trypsin; D019286:Endopeptidase K; D011402:Pronase