ADVANCED CHEMICAL PHYSIOLOGY 309 



fine point at both ends, is filled with egg white, the ends closed in the 

 flame, and the tubes then heated in a boiling waterbath so that a 

 column of coagulated albumin is obtained. It is then cut into seg- 

 ments of equal length, and two of these are placed in a test tube or 

 Petri dish which contains the pepsin solution, acidified with 0-2 per 

 cent, hydrochloric acid. Two similar tubes are placed in another test 

 tube or Petri dish with another pepsin solution of different strength. 

 Both are placed in the incubator for several (ten) hours. The length 

 of dissolved protein column is then measured in both cases, and the 

 desired result is obtained by squaring this distance. 



Thus if in one test tube the length were 2, and in the other 3, the 

 strength of the two pepsin solutions has the ratio of 4 to 9. 



This method is only accurate when weak pepsin solutions are used. 

 If more than 4 mm. of protein are digested, the estimation must be 

 repeated with diluted solutions. 



Isolation of Amino Acids. Mince up a pig's pancreas thoroughly, 

 and shake it in a flask with 500 c.c. of water containing 3 c.c. of a 

 saturated solution of sodium carbonate, and 3 c.c. of chloroform. Add 

 also about 200 gms. of blood fibrin, which has previously been soaked 

 in 1 per cent, sodium carbonate solution. Place the flask in an incubator 

 at body temperature, and after three days test the reaction of the digest 

 towards litmus. If acid, add more sodium carbonate till distinctly 

 alkaline. Also remove about 10 c.c. and filter into a test tube. To 

 this sample carefully add a few drops of bromine water. A violet 

 colour results, the intensity of which should be carefully noted. This 

 colour reaction is due to tryptophan, an aromatic amino acid which is 

 liberated by the actipn of trypsin. 



Test the reaction towards litmus and the intensity of the tryptophan 

 reaction on each succeeding day. When the tryptophan reaction 

 becomes very intense (in about five days) proceed to isolate leucine 

 and tyrosine 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 Tyrosine. 'The remainder is evaporated on the 

 waterbath to a thin syrup. This is allowed to stand on ice or in a cold 

 place for several days. White flocculi of tyrosine 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 dissolved 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 tyrosine 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 ) Tyrosine 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 tyrosine contains an aromatic radicle 

 (p. 196). 



(3) Pirid'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 waterbath for half an hour. Now cool and dilute with water, 

 transfer to an evaporating basin, and remove the sulphuric acid by 



