36 THE GENERAL CHARACTERS OF THE PROTEINS 



Method. 



The method adopted is the Osborne- Harris modification of the 

 original Hausmann method, with a further modification suggested by 

 Giimbel, viz., the employment of a vacuum at a temperature of 40 C., 

 for the distillation of the ammonia after treatment with magnesia. 



About i gram of protein is boiled with 20 per cent, hydro- 

 chloric acid until the solution no longer gives the biuret reaction, 

 usually from seven to ten hours. It is then evaporated at 40 under 

 diminished pressure to 2-3 c.c. and the bulk of the hydrochloric acid 

 is thereby removed. The residual solution is then transferred to a 

 flask with about 350 c.c. of water, and a cream of magnesia, which 

 has been freed from every trace of ammonia by prolonged boiling 

 is then added until in slight, but distinct, excess. After distilling 

 and determining the ammonia by warming to 40 in vacuo, and 

 passing the distillate into a known quantity of a standard acid solu- 

 tion, the solution in the flask is filtered through a nitrogen-free filter 

 paper, and the residue thus collected washed thoroughly with water 

 and the nitrogen determined in it together with the paper by 

 Kjehldahl's method (" humin " nitrogen). The filtered solution is 

 next concentrated to 100 c.c., cooled to 20 C., 5 grams of sulphuric 

 acid added, and then 30 c.c. of a solution containing 20 grams of phos- 

 photungstic acid and 5 grams of sulphuric acid per 100 c.c. After 

 twenty-four hours the precipitate is filtered off and washed with a solu- 

 tion containing 2*5 grams of phosphotungstic acid and 5 grams 

 of sulphuric acid per 100 c.c. The washing is effected by rinsing the 

 precipitate from the filter into a beaker and returning to the paper 

 three successive times, each portion of the wash solution being allowed 

 to run out completely before the next is applied. About 200 c.c. of 

 washings are thus obtained. The nitrogen contained in the precipi- 

 tate (" basic " nitrogen) is then determined by transferring it to a 

 Jena glass flask of about 600 c.c. capacity and digesting with 35 c.c. 

 concentrated sulphuric acid for seven or eight hours. During diges- 

 tion potassium permanganate crystals are added three or four times. 

 In a few cases, when the phosphotungstic acid precipitate is small, 

 less sulphuric acid is used, enough being taken in each case to 

 prevent too violent bumping. The remaining nitrogen, belonging 

 chiefly to monoamino acids, is found by subtracting the sum of the 

 nitrogen found in the preceding operations from the total nitrogen 

 contained in the protein under examination. 



Skraup has recently shown that two - thirds of the amide- 

 nitrogen is evolved in a very short time, even with the use of 

 dilute acids for hydrolysis. 



SECTION XIV. THE SULPHUR, PHOSPHORUS AND HALOGEN 

 CONTENT OF PROTEINS. 



The Sulphur Content. 



Far more characteristic of the individual protein than the per- 

 centage of nitrogen is that of the sulphur. Although the latter element 

 is not contained in large quantity, yet the variations in its amount 

 are considerable, and its percentage may be regarded as one of the 



