HEXON BASES. 427 



The method which Kossel and Kutscher i have employed for the isola- 

 tion and separation of the hexon hases in a very condensed form is as fol- 

 lows : For every gram of proteid 3 c.c. of concentrated HjSOi and 6 c.c. 

 of water are taken and the mixture is hoiled for 14 hours under an inverted 

 condenser. The liquid is then made up to a definite volume and the total 

 nitrogen in a definite portion can be determined. Hot, saturated baryta 

 is then added till only a slight acidity remains, after which the BaSOi is 

 drained and thoroughly washed. The filtrate and wash-water are com- 

 bined and the total nitrogen in a portion, is again determined. The 

 diflference represents the nitrogen in the humin substances, which are 

 dragged down with the BaSOj. 



In another portion the ammonia is determined by distillation with mag- 

 nesia. The ammonia is expelled from the remaining total fluid by evap- 

 oration with magnesia. The combined ammonia-free fluids are then ren- 

 dered strongly alkaline with baryta and the resultant precipitate is 

 thoroughly washed. The filtrate and wash-water are combined and acid- 

 ulated with HjSOi. The precipitate which forms is likewise thoroughly 

 washed and the liquids are united, made up to a definite volume and the 

 total nitrogen in a portion again determined = Humin nitrogen II. 



To separate the bases present arginin and histidin are precipitated to- 

 gether, while the lysin remains in solution. For this purpose the acid 

 liquid is diluted, heated on the water-bath and silver sulphate is added 

 till a drop of the liquid added to an excess of baryta yield a yellow pre- 

 cipitate. When this point is reached the liquid is cooled to 40° and sat- 

 urated with baryta. The precipitate of arginin- and histidin-silver is 

 removed at once and washed thoroughly with baryta. 



To separate the arginin and histidin, the precipitate is suspended in 

 dilute sulphuric acid and decomposed with hydrogen sulphid. The sul- 

 phid precipitate is removed and thoroughly washed. The combined fil- 

 trates are evaporated to expel the gas, then made up to a definite volume 

 and the total nitrogen in a portion is determined. The solution is then 

 neutralized with baryta, after which barium nitrate is added till all the 

 sulphuric acid is precipitated. The precipitate is removed and well washed. 

 The filtrates are united, concentrated to about 300 c.c, after which silver 

 nitrate is added until a drop of the liquid when added to an excess of 

 baryta gives a yellow precipitate. The solution is carefully neutralized 

 with baryta, after which the reagent is added from a burette in small por- 

 tions at a time till all the histidin has been precipitated. This point is 

 reached when a portion of the liquid slowly added to weak ammoniacal 

 silver nitrate yields no precipitate. If a precipitate forms and is soluble 

 in excess of ammonia, histidin is present. 



The histidin-silver precipitate is filtered off and washed well. The fil- 

 trates contain arginin. To separate the histidin the precipitate is suspended 

 in water, acidulated with HjSOi, and decomposed with hydrogen sulphid. 

 The combined filtrate and wash-water are then freed from sulphuric acid 

 by means of baryta. The latter is removed and the final filtrate is evap- 

 orated to dryness. The residue is taken up with silver nitrate solution to 

 which a drop of nitric acid was added and the solution is filtered. The 

 filtrate on evaporation yields the crystalline histidin dichlorid, which is dried 

 in a vacuum at 40° and weighed. 



The above arginin filtrate is saturated with powdered baryta and the 

 resultant precipitate is well washed, after which it is suspended in acidu- 

 lated water and decomposed with hydrogen sulphid. The combined fil- 

 trates are evaporated and then made up to a definite volume. The nitro- 

 gen in a portion is determined and the total amount of arginin is calcu- 



^ZeUg. physiol. Chem., 31, 166-178 ; 25, 178. 



