100 PROTEIN POISONS 



hereafter described as being obtained from the poison from 

 agar-grown cultures. This demonstrates that the poison is 

 an integral part of the cellular substance, and it is evident 

 that the bacterial cell must synthetically produce this 

 protein body during its growth from the chemical con- 

 stituents of the medium. 



When the protein is split up by dilute alkali in absolute 

 alcohol according to the method described, the poison is 

 in solution in the alkaline alcohol. The preparation is 

 filtered and the filtrate neutralized with hydrochloric acid, 

 avoiding an excess of acid. This throws down the greater 

 part of both base and acid as sodium chloride, which is 

 removed by filtration. In this way a solution of the poison 

 in absolute alcohol is obtained. This is evaported in 

 vacua at 40, redissolved in absolute alcohol to remove 

 traces of sodium chloride, and again evaporated in vacua 

 at 40 or less. Evaporation may be done in an open dish, 

 but the toxicity of the substances is somewhat decreased 

 when this is done. The poisonous part of the protein 

 molecule when obtained in this way and powdered, when 

 there is no water present, forms a dark brown scale which 

 pulverizes into a lighter brown powder. 



It should be clearly understood that we regard this 

 method of extracting the poisonous group from the protein 

 molecule as by no means ideal. We know that it is crude 

 and that much of the poison is destroyed in the process. 

 In disrupting a protein by our method with dilute alkali 

 in absolute alcohol, ammonia is given off and the odor of 

 this gas is apparent even at the end of the third extraction. 

 An effort was made to discover how much nitrogen was 

 converted into ammonia in the process. A device was 

 arranged for conducting the ammonia into standard acid, 

 and four 10-gram samples of Witte's peptone were extracted 

 with 2 per cent, sodium hydrate in absolute alcohol, one 

 for three hours in a current of air, the others in a current 

 of hydrogen for two and one-half, eight and one-half, and 

 nineteen and one-half hours respectively. At the end of 

 each operation the excess of acid was titrated with deci- 



