Chemical purification of the Ascoli antigen from infected mice has been accomplished by methanol precipitation in the cold. Maximum recovery of active substance occurred at 60% methanol concentration.
Paper electrophoresis and ultracentrifugation showed that much of the crude Ascoli was made up of particulate material which gave unsatisfactory results in electrophoresis and was not amenable to analysis by ultracentrifugation. Determinations for nonglucose amine showed that alcohol precipitation concentrated this component.
Paper chromatography showed that crude normal and crude infected Ascoli contained glycine, proline, aspartic acid, leucine, glutamic acid, histidine, alanine and rnethionine. The alcohol-purified Ascoli contained glycine, glutarnic acid, and alanine only.
Centrifugation of the crude infected Ascoli and the purified Ascoli at 60,000 rpm gave a gelatinous sediment which contained a negligible amount of protein but which was active both as a sensitizer and inhibitor in the hemagglutination test and which protected mice against challenge with fully virulent Pasteurella tularensis. Chymotrypsin treatment of this gelatinous material decreased nitrogen from 2.17 mg/ml to 0.2 mg/ml without decreasing the serological activity. Further tests for the characterization of this antigen are planned.