ADVANCED PHYSIOLOGICAL CHEMISTRY 453 



Digestion is allowed to proceed until the bromine water reaction is maximal. 

 The digest is then boiled, cooled and filtered, and H 2 S0 4 added to the filtrate, so 

 as to bring the amount of H 2 S0 4 in the latter to 5-6 per cent. If any precipitate 

 is hereby formed it should be filtered off. The clear filtrate is then mixed with an 

 excess of an acid solution of mercuric sulphate (10 per cent, mercuric sulphate 

 dissolved in 10 per cent. H 2 S0 4 and filtered). This reagent may precipitate 

 besides tryptophane some tyrosin and cystin. 



From tyrosin the precipitate is freed by washing it with 5-6 per cent, H 2 S0 4 , 

 the mercury compound of tyrosin being very soluble in this. From cystin (which 

 is scanty in a digest of casein) the tryptophane is separated by reprecipitation. 

 For this purpose the washed mercury precipitate is suspended in water and 

 decomposed with H 2 S gas. To complete this reaction the suspension must be 

 saturated with the gas, then warmed and saturated again. The HgS precipitate 

 is filtered off, the filtrate warmed to rid it of H 2 S, then acidified to 5-6 per cent. 

 H 2 S0 4 , and the mercuric sulphate reagent added to it until a small permanent 

 precipitate is produced. This is mainly cystin, and is filtered off. The trypto- 

 phane in the filtrate is then completely precipitated by mercuric sulphate, and 

 the resulting precipitate treated exactly like the first one. 



In this way a solution of tryptophane in 5-6 per cent. H 2 S0 4 is obtained. The 

 H 2 S0 4 is now precipitated by adding Ba(OH) 2 water in the heat and filtering. 

 Great care should be taken that the filtrate contains no excess either of H 2 S0 4 or 

 of Ba(OH). 2 . The watery solution of tryptophane is then mixed with half its 

 bulk of alcohol and evaporated on a water bath. During evaporation small 

 quantities of alcohol are added from time to time to prevent the browning which 

 occurs if watery solutions of tryptophane are heated alone. Evaporation pro- 

 ceeds till crystallisation commences, when the basin is removed and allowed to 

 stand. The crystals (glistening plates) are collected on a filter, and, to purify 

 them, may be recrystallised. 



A solution of the crystals gives the bromine and the glyoxylic 

 reactions very distinctly, and if the crystals be heated in a dry test- 

 tube indol and skatol (see p. 238) are evolved. 



Hopkins and Cole have shown the constitution of tryptophane to be 

 skatol-amido-acetic acid, its formula, therefore, is : 



CH 3 



C 6 H 4 <>C - CH(NH Q )COOH. 

 NH 



By allowing anaerobic bacteria to grow in a nutritive solution 

 containing tryptophane, the amido group of the side chain is 

 split off as ammonia and skatol-acetic acid is formed. If aerobic 

 bacteria are now introduced they oxidise the ^skatol-acetic acid, 

 first of all into skatol-carbonic acid, then into skatol, then into indol 

 (see p. 238). 



