43 6 H'iston and its Prcparation [April 



avoided. Filter off the alcohol and extract the precipitated material 

 directly with 0.8 per cent. hydrochloric acid Solution. A clear, 

 bluish white, readily filterable, extract will result. Extraction with 

 hydrochloric acid Solution should be continued for several days. 

 In our experiments it was found necessary to repeat the extrac- 

 tion with hydrochloric acid Solution several times to obtain all the 

 histon, Preliminary extraction of the glands with alcohol removes 

 none of the histon (as was detemiined by study of the extract) and, 

 by precipitating all the proteins in the glands, the alcohol prevents 

 the presence of protein impurities in the acid extract. The histon 

 can then be precipitated from the acid extract by whatever method 

 is desired, as already outlined. 



3. Proteins left after removal of histon from thymus extract. 

 Some experiments were conducted to detemiine if possible the 

 merits of the contentions of Lilien feld, Huiskamp and Bang as to 

 the nature of the histon complex in the cell. These views were 

 outlined in detail on page 428, The procedure was as f ollows : 

 Thymus glands were minced and extracted with water for 48 hours 

 in a refrigerator. The extract was decanted, filtered, precipitated 

 with a few drops of conc. hydrochloric acid Solution and the super- 

 natant fluid treated with hydrochloric acid until a strength of 0.8 

 per cent. was present; after standing for several days, the acid 

 Solution was filtered off and the precipitate extracted with 0.8 per 

 cent. hydrochloric acid Solution. This process was repeated until 

 the hydrochloric acid extract failed to give an ammonia-precipitate 

 or a biuret reaction. The histon-f ree residue was then washed with 

 alcohol until it was free from acid, treated with 0.3 per cent. potas- 

 sium hydroxid Solution, in which it dissolved slowly but com- 

 pletely, and the Solution filtered, placed in a parchment bag and 

 dialysed free from hydroxyl ions. No precipitate resulted, the Solu- 

 tion remaining clear. (Toluene was used as a preservative during 

 this process.) To the neutral Solution 10 per cent. calcium chlorid 

 Solution was now added (2 c.c. of 10 per cent. calcium chlorid 

 Solution to each 100 c.c. of liquid). A copious precipitate resulted, 

 the filtrate from this mass giving a good biuret test. Addition of 

 more calcium chlorid to the filtrate failed to produce further 

 precipitation. 



