Enzymatic fragmentation of tetanus toxin. Identification and characterization of an atoxic, immunogenic fragment

Purified filtrate tetanus toxin was subjected to limited digestion with papain and the resulting fragments were separated by gel exclusion chromatography and characterized. One atoxic fragment was shown to react with antiserum against tetanus toxoid and was capable of inducing antibodies in rabbits that neutralized native tetanus toxin, The fragment had an estimated molecular weight of 56,000 by SDS polyacrylamide gel electrophoresis and 62,000 by sedimentation equilibrium. In the presence of a reducing agent, the fragment yielded two components with approximatec molecular weights of 23,000 and 32,000. Thus, it appears that the atoxic, immunogenic fragment is composed of two peptides joined by at least one disulfide bond. The fragment was examined by circular dichroism and data analysis indicated the presence of considerable beta-structure, but little, if any, alpha-helicity. This is significantly different from the estimates for filtrate toxin. 29% alpha-helicity and 23% beta-structure. Above 250 nm, the circular dichroic spectrum of the fragment was also distinct from that of intact toxin.