Chains and fragments of tetanus toxin. Separation, reassociation and pharmacological properties

Abstract

Tetanus toxin, as obtained from bacterial culture filtrates, consists of two chains. Since their roles in poisoning are unknown, we have made a detailed study of their preparation, reassociation and pharmacological activity. 1. Two-chain tetanus toxin (pI 6.0) was subjected to isoelectric focussing under reducing conditions in 2M urea. Both light (pI 4.8) and heavy (pI 7.2) chains separated as nearly homogeneous proteins of low toxicities. Upon removal of urea and reoxidation, partial homodimerization by formation of disulfide bonds took place in the purified fractions. The toxin was reconstituted nearly quantitatively by covalent heterodimerization of the complementary chains, as shown by SDS/gel electrophoresis, toxicity studies, inhibition of evoked [3H]noradrenaline release and binding to rat brain membranes. 2. Accordingly, fragment B (pI 5.6) resulting from papain hydrolysis, was separated into a light chain and the N-terminal moiety of the heavy chain, called fragment beta 2 (pI 7.1 and 6.8, two maxima). Removal of urea and reoxidation led to reconstitution of fragment B. Covalent linkage did not occur between the two parts of the heavy chain, or between the light chain and the C-terminal part of the heavy chain. 3. The heavy chain alone inhibited K+-evoked [3H]noradrenaline release from a rat brain homogenate. However, the concentration-response ratio was flat and 10-100-fold higher concentrations were required than with native or reconstituted two-chain toxin. The light chain was inactive. Purified heavy chain but not light chain decreased the [3H]noradrenaline content, whereas the two-chain toxin increased it. Binding to rat brain membranes was assessed by competition with 125I-labelled two-chain toxin. In hypotonic buffer, the heavy chain, the papain fragment C and native and reconstituted two-chain toxin had comparable affinities to membranes. In isotonic buffer the heavy chain displayed an about 1000-fold lower affinity than native or reconstituted two-chain toxin. The light chain did not bind to membranes in either test. Our data indicate that (a) the light chain and the N-terminal part of the heavy chain are held together not only by one disulfide bond but also by hydrogen bonds and ionic forces to yield a two-chain toxin or fragment B and (b) both chains contribute to the actions of the toxin in vivo and in vitro, and to its binding.