PHYSIOLOGICAL CHEMISTRY 395 



orotatory (a) D in aqueous solution= - 10'8. The leucin which is obtained by 

 boiling protein with baryta, or that obtained synthetically (by the action of 

 ammonia on a-bromocaproic acid) is optically inactive, and the dextrorotatory form 

 may be obtained from this by allowing penicillium glancum (a fungus) to grow on 

 a solution of it. The fungus destroys the levo-rotatory part, but leaves the 

 dextro-rotatory, untouched. Moulds, yeasts and ferments act much more ener- 

 getically on naturally occurring than on synthetic isomers. 



Tryptophane. 1 If bromine water be cautiously added to a tryptic digest of 

 several days' standing a deep violet-red colour will result, and if the mixture be 

 shaken with amyl alcohol, this latter will take up the colour. The glyoxylic 

 reaction (see p. 303) v;ill also be very distinct in the digest even after the Biuret 

 reaction has disappeared (i.e. after the protein molecule has been quite destroyed). 

 Both these reactions are due to tryptophane, which is closely related in its 

 chemical structure to certain of the aromatic substances that are produced by 

 the bacterial digestion of protein. 



Separation of Tryptophane. A large amount (500 gr.) of commercial 

 casein (plasmon or protene) is mixed with liq. pancreaticus (200 c.c. Benger) and 

 0'8 % Na^COa, and placed in an incubator for about a week. The ferment should 

 be added, half at the beginning and the remainder three or four days later. Anti- 

 septics should be added. 



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

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

 as to bring the amount of H 2 S0 4 in the latter to 5-6 %. 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 % mercuric sulphate dissolved 

 in 10 % H 2 SO 4 ) and filtered. This reagent may precipitate, besides tryptophaue, 

 some tyrosin and cystin. 



From tyrosin the precipitate is freed by washing it with 5-6 % 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 decom- 

 posed 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 

 oft, the filtrate warmed to rid it of H 2 S, then acidified to 5-6 % H 2 S0 4 , and the 

 mercuric sulphate reagent added to it until a small permanent precipitate is pro- 

 duced. This is mainly cystin, and is filtered off. The tryptophane 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 % H 2 SO 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 proceeds till 



*A single digestion mixture may be employed for the separation of leucin, 

 tyrosin and tryptophane, but in such a case both fibrin and casein ought to be 

 added, since casein is the only common protein which yields any large amount of 

 .tryptophane. It also contains a considerable amount of tyrosin. 



