Enzymatic hydrolysis of tetanus toxin by intrinsic and extrinsic proteases. Characterization of the fragments by monoclonal antibodies.

Enzymatic fragments of tetanus toxin were characterized by immunoblotting using a set of previously characterized antibodies (Ahnert-Hilger et al. (1983) and a set of novel antibodies. The selected antibodies recognized the light chain, fragment C (beta 1), and the complementary piece (beta 2) of the heavy chain when blotted on nitrocellulose. All toxin preparations contained intrinsic esteroprotease activity which became manifest in the presence of urea. The main split product was a fragment (MW 100 000) reacting with anti-fragment C and anti-beta 2 antibodies. Toxicity does not depend on this protease activity. Some crude preparations of tetanus toxin contain another split product (MW 47 000) which resembles fragment C. The main product of papain hydrolysis is fragment C, which appears as a double band under nonreducing conditions but is homogeneous when reduced. Chymotryptic digestion hydrolyses the heavy chain well but leaves the light chain largely intact. Tetanus toxin is very resistant against trypsin as compared with other proteases, although this enzyme splits numerous different links. Our data show the usefulness of immunoblotting with monoclonal antibodies in analytical work with tetanus toxin, and the relevance of intrinsic proteases.