BACTERIAL CELLULAR SUBSTANCE 69 



added, and the mixture poured into three volumes of 

 alcohol. An aqueous solution of the precipitate thus 

 obtained did not reduce Fehling's solution, but after boiling 

 with hydrochloric acid it reduced both Fehling's and 

 Nylander's solutions, and also gave the furfurol test, thus 

 showing the presence of a carbohydrate. The original 

 powder burned readily, puffing up and glowing as does 

 nucleic acid, then fusing and leaving a dark ash. Two 

 determinations of phosphorus gave the following: 



Weight of Weight of 



sample. pyrophosphate. Weight of P. Per cent, of P. 



. 4723 gram . 0524 gram . 01450 gram 3 . 09 



0.6469 gram 0.0685 gram 0.01895 gram 2.93 



In our attempts to extract the poisonous groups from 

 bacterial proteins this carbohydrate gave us great trouble. 

 It is readily soluble in water, whether acid or alkaline, and 

 more or less soluble in alcohol, the degree of solubility 

 depending upon the strength of the alcohol. In absolute 

 alcohol it is insoluble, but it cannot be precipitated com- 

 pletely from aqueous solution by the addition of alcohol. 

 Concentrated solutions and residues obtained by evapora- 

 tion in vacuo are sticky and unsatisfactory in all attempts 

 at purification. As we ascertained after much experimenta- 

 tion, the poisonous group in the protein molecule is freely 

 soluble in absolute alcohol, and finally when we disrupted 

 the protein molecule with a dilute solution of alkali in 

 absolute alcohol we secured a complete separation of the 

 poisonous group from both carbohydrates. Therefore the 

 best material in which the bacterial or other protein carbo- 

 hydrates can be studied is the non-poisonous portion after 

 complete removal of the poisonous group by heating the 

 protein repeatedly with a 2 per cent, solution of sodium 

 hydroxide in absolute alcohol. This method will be dis- 

 cussed in detail later, but it needs to be stated here that 

 when this is done the poisonous group, free from any trace 

 of either carbohydrate, goes into solution in the alkaline 

 alcohol, while the non-poisonous part, or the haptophor, as 



