A comparison of the actions of trypsin and pepsin on porcine immunoglobulin M and their effects on biological activity

The action of trypsin at 55°C and pH 8.3 on pig IgM anti-Salmonella has been compared with the action of pepsin at 37°C and pH 4.6. Both processes cause the gradual removal of Fab arms and C_μ2 domains to produce eventually an (Fc)₅ fragment. However, during tryptic digestion Fab arms are preferentially removed from the same subunit, whereas peptic digestion causes random removal from any subunit. At intermediate stages of digestion both processes produce partially fragmented molecules which consist of an (Fc)₅ portion still attached to limited numbers of Fab arms. Both processes cause a gradual decrease in the ability of molecules to agglutinate Salmonella, but complement fixation by the complexes declines much more rapidly. A stage is reached where molecules having four Fab arms can still agglutinate but there is no complement fixation. However, the remaining arms on the tryptic molecules are distributed in pairs on the same subunit, whereas those on the peptic molecules are distributed randomly. Hence the number of remaining Fab arms, rather than their distribution, appears to be the critical factor which influences biological activity. A possible explanation for this is discussed.