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

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Abstract

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 antibodis recognized the light chain, fragment C (Β 1), and the complementary piece (Β 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-Β 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 fragmentC, 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.

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Abbreviations

T:

Tetanus toxin

H:

heavy chain of tetanus toxin

L:

light chain of tetanus toxin

L,Β 1Β2 :

recognized by the respective antibodies, x strong reaction, (x) faint reaction

NC:

nitrocellulose

PAGE:

polyacrylamide gel electrophoresis

SDS:

sodium dodecyl sulphate

PMSF:

phenyl methylsulphonyl fluoride

TBS:

tris buffered saline (50 mM tris-HCl pH 7.4, 150 mM NaCl)

DCC:

diphenylcarbamyl chloride

TPCK:

L-1-Tosylamide-2-phenylethyl chloromethyl ketone

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Some of the results were presented at the Seventh International Conference on Tetanus (Goretzki and Habermann 1984)

This communication contains parts of the M.D. thesis of K. Goretzki

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Goretzki, K., Habermann, E. Enzymatic hydrolysis of tetanus toxin by intrinsic and extrinsic proteases. Characterization of the fragments by monoclonal antibodies. Med Microbiol Immunol 174, 139–150 (1985). https://doi.org/10.1007/BF02298124

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  • DOI: https://doi.org/10.1007/BF02298124

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