310 PRACTICAL PHYSIOLOGY 



adding powdered barium carbonate ; filter off the barium sulphate, 

 evaporate the filtrate to small bulk, and add a drop or two of very weak 

 ferric chloride solution. A violet colour results. 



2. Separation of Leucine. The tyrosine-free filtrate is evaporated 

 till a skin of leucine forms on the surface. It is then mixed while still 

 warm with several times its bulk of alcohol, whereby a precipitate 

 (previously known as antipeptone) separates out, which after cooling 

 can be removed by filtration. This precipitate consists of a mixture of 

 several bodies, including lysine, histidine, and arginine. The filtrate is 

 evaporated on the waterbath until all the alcohol has been driven off. 

 It is then boiled with lead carbonate and filtered. The lead is removed 

 from the filtrate by means of H 2 S, the PbS separated by filtration, and 

 the final filtrate accurately neutralised with weak NaOH. By now 

 concentrating by evaporation on the waterbath and cooling leucine 

 will separate out. 



Reactions of Leucine. (1) It is much more soluble in water than is 

 tyrosine ; it is soluble also in alcohol. 



(2) When heated in a piece of dry glass tubing, a sublimate forms on 

 the cool parts of the tube. 



(3) Like other amino acids, it gives off ammonia gas when heated in a 

 test tube with a piece of solid caustic potash and a few drops of water. 

 If the melt be cooled, dissolved in water, and then acidified with 

 sulphuric acid, it gives a smell of valerianic acid on heating. 



(4) Scherer's Test. Heat some leucine with a drop of nitric acid on a 

 piece of platinum foil, add to the dry residue some caustic potash, when 

 a yellow stain results. Heat still further, and the stain rises up into a 

 globule which runs off the platinum. 



(5) Examine a solution of leucine with the polariscope (p. 212). It is 

 Icevo-rotatory (a) D in aqueous solution = - 10-8. The leucine which 

 is obtained by boiling protein with baryta, or that obtained 

 synthetically is optically inactive, and the dextro-rotatory form may be 

 obtained from this by allowing penicillium glaucum (a fungus) to grow 

 on a solution of it. The f ungus destroys the laevo-rotatory part, but 

 leaves the dextro-rotatory untouched. Moulds, yeasts and ferments 

 act much more energetically on naturally occurring than on synthetic 

 isomers. 



Tryptophan. 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. 196) will 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 tryptophan, 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 Tryptophan. A large amount (500 gr.) of commercial 

 casein is mixed with liquor pancreaticus (200 c.c. Benger) and 0-8 per 

 cent. Na 2 CO 3 , 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. Antiseptics should be added. 



1 A single digestion mixture may be employed for the separation of leucine, 

 tyrosine and tryptophan, 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 tryptophan. It also contains a considerable amount of tyrosine. 



