BACTERIAL CELLULAR SUBSTANCE 81 



Hydrolysis of the Cellular Protein of Bacillus Coli Com- 

 munis. Three hundred and fifty grams of this cellular 

 protein was thoroughly mixed with 1100 c.c. of hydrochloric 

 acid, of specific gravity 1.19, and allowed to stand over- 

 night. The next morning it was found to be a frothy mix- 

 ture of deep purple color (Liebermann's reaction). It was 

 placed in a 2-liter flask connected with a long reflux con- 

 denser, and boiled on the sand-bath for six hours, when it 

 no longer gave the biuret reaction. It was then filtered 

 through heavy paper, giving a clear brown filtrate and 

 leaving a heavy deposit of humus on the paper. The latter 

 was repeatedly extracted with dilute hydrochloric acid, 

 the extracts concentrated, and added to the filtrate The 

 humus, air-dried, weighed 77 grams, making 22 per cent, 

 of the cellular protein. 



The filtrate was concentrated in vacuo to half its volume, 

 placed in a freezing mixture, saturated with hydrochloric 

 acid gas and left in the ice-box for two days. By this time 

 the hydrochloride of glutamic acid had crystallized out. 

 An equal volume of ice-cold alcohol, previously saturated 

 with hydrochloric acid gas, was added, and the glutamic 

 acid collected, dried over solid sodium hydrate, and 

 weighed. It yielded 10.5 grams of 3 per cent, of the cellular 

 protein. 



The filtrate from the glutamic acid was esterified with 

 hydrochloric acid gas, followed by gentle heat. The alcohol 

 was distilled off in vacuo, an equal volume added, again 

 saturated with the gaseous acid, heated, and removed by 

 distillation, this being repeated several times. The fluid 

 was left in the ice-box for forty-eight hours, when crystals 

 were deposited. These were purified by reprecipitating 

 them from alcoholic solution with hydrochloric acid gas. 

 The melting-point was 144 and the crystals were identified 

 as the ethyl ester of glycocoll. The yield of glycocoll was 

 1 gram. 



The filtrate from the glycocoll containing the hydro- 

 chlorides of the esters was treated as already stated and 

 fractioned with the following yields: 

 6 



