Isolation and purification of two antigenically active, "complimentary" polypeptide fragments of tetanus neurotoxin.
Tetanus neurotoxin (molecular weight approximately 160,000) was purified from bacterial extracts (intracellular toxin) and mildly trypsinized and from culture filtrates (extracellular toxin). Both purified preparations could be dissociated reversibly into two polypeptide chains, with molecular weights of 53,000 (fragment alpha) and 107,000 (fragment beta), by treatment with 100 mM dithiothreitol (DTT) and 4 M urea with concomitant loss of toxicity. Upon removal of DDT and urea from the dissociated toxin preparation by dialysis, these fragments reassociated, forming the whole toxin. The two fragments were isolated and purified from the dissociated toxin by gel filtration on an Ultrogel AcA 44 column equilibrated with buffer containing 2 M urea and 1 mM DTT. The preparation of fragment alpha was nontoxic whereas that of fragment beta was slightly toxic. Immunodiffusion analyses, using horse antitoxin, showed that the antigenicities of fragment alpha and fragment beta were distinct from each other but were partially identical with that of undissociated toxin. The abilities of these fragments to precipitate antitoxin were lost on heating at 60 C for 5 min. The molecular substructure of tetanus neurotoxin is discussed on the basis of these findings.
D000942:Antigens, Bacterial / Q000302:isolation & purification*; D002850:Chromatography, Gel; D003017:Clostridium tetani / Q000276:immunology*; D004229:Dithiothreitol; D004591:Electrophoresis, Polyacrylamide Gel; D005428:Flocculation Tests; D006358:Hot Temperature; D010455:Peptides / Q000276:immunology* / Q000302:isolation & purification; D013744:Tetanus Toxin / Q000302:isolation & purification* / Q000378:metabolism; D014508:Urea
D000942:Antigens, Bacterial; D010455:Peptides; D013744:Tetanus Toxin; D014508:Urea; D004229:Dithiothreitol