394 PRACTICAL PHYSIOLOGY 



Test the reaction towards litmus and the intensity of the tryptophane reaction 

 on each succeeding day. When the tryptophane reaction becomes very intense (in 

 about five days) proceed to isolate leucin and tyrosin in the following manner : 



The digest is rendered faintly acid with acetic acid, boiled and filtered hot. A 

 sample of the filtrate is removed and tested for proteose. A negative result is 

 usually obtained. 



1. Separation Of Tyrosin. The remainder is evaporated on the water-bath 

 to a thin syrup. This is allowed to stand on ice or in a cold place for several 

 days. White flocculi of tyrosin separate out. These are filtered through fine 

 muslin, and removed to a beaker by means of a jet of cold distilled water and 

 washed several times with distilled water by decantation. They are then dis- 

 solved by boiling with water made alkaline by the addition of a few drops of 

 ammonia, and the resulting solution is quickly filtered hot. The filtrate is heated 

 till all the ammonia is expelled ; it is then cooled, when the tyrosin separates out 

 as a white precipitate. This is collected on a filter paper, washed, and dried. 

 The following reactions may be applied to the resulting powder : 



(1) Tyrosin is insoluble in cold water, slightly soluble in hot water, and very 

 soluble in dilute alkali. 



(2) A solution in hot water gives a red colour on the addition of Millon's 

 reagent. This is because tyrosin contains an aromatic radicle (p. 390). 



(3) Piria's Test. Place some of the powder in a dried test tube, add about 2 c.c. 

 concentrated sulphuric acid, and place the test tube in a boiling water-bath for 

 half an hour. Now cool and dilute with water, transfer to an evaporating basin, 

 and remove the sulphuric acid by 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. This reaction is 

 due to the formation of tyrosin-sulphuric acid. 



2. Separation Of Leucin. The tyrosin-free filtrate is evaporated till a skin 

 of leucin 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 water-bath 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 water- bath and cooling leucin will separate out. 



Reactions of Leucin. (1) It is much more soluble in water than is tyrosin ; 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 valerian ic acid on heating. 



(4) Scherer's Test. Heat some leucin 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 leucin with the polariscope (p. 282). It is levo- 



