STRUCTURE-FUNCTION RELATIONSHIP OF TETANUS NEUROTOXIN MOLECULE: ANALYSES WITH COMPLEMENTARY POLYPEPTIDE FRAGMENTS AND A SUBFRAGMENT

ABSTRACT Two complementary polypeptide fragments (fragment ≪K, mol. wt. 53,000; fragment β, mol. wt. 107,000) were isolated and purified from the mildly trypsin-treated toxin (mol. wt. 160,000) prepared from bacterial extracts. These purified fragments were antlgenically active and native enough to be reconstituted to the whole toxin molecule. A subfragment (fragment β-1, mol. wt. 50,000) was obtained by further trypsin digestion of fragment β and purified. Immunochemical analyses employing the above three fragments and horse antitoxin sera revealed that tetanus toxin possesses four distinct kinds of antigenic determinants carried respectively by fragments α and β-1 and fragment “β-2” (designated for the other portion of fragment than fragment β-1), and one “conformational” determinant present on the whole toxin but not present or exposed in the isolated fragments α and β. While approximately one-fourth of the amount of the antibodies precipitable by the whole toxin could not be precipitated by fragments α and β, almost all of the neutralizing activities of the antitoxin were lost by precipitation with fragments α and β. The loss of the major part of the neutralizing activities was due to precipitation with fragment β, about half of which was with fragment β-l. Anti-fragment α antibody occupied only a small part of the antitoxin. All three fragments were immunogenic; fragment β elicited the highest and fragment CK the lowest amounts of antitoxin in rabbits. Mild papain treatment of the toxin, prepared from bacterial extracts, yielded two antlgenically active components; one antlgenically identical with fragment β-1 and the other, a complex of fragments α and β-2 (fragment α.β-2). Fragments α, β and β-1 bound specifically to synaptosomes prepared from rabbit spinal cord and to ganglioside-cerebroside complex. The submolecular structure of tetanus toxin is discussed.