PROTEINS 87 



7. Folin and Denis's Test. 1 To 1-2 c.c. of the solution to be tested add an 

 equal volume of a special reagent (containing 10 per cent sodium tungstate, 2 

 per cent phosphomolybdic acid and 10 per cent phosphoric acid) and 3-10 c.c. 

 of a saturated solution of sodium carbonate. A blue color indicates tyrosine. 

 It is said to detect i part in one million. 



Abderhalden 2 claims the reagent also reacts with tryptophane, 

 oxytryptophane and /-oxyproline. 



EXPERIMENTS ON LEUCINE 



Make the following tests upon the leucine crystals already prepared 

 or upon some pure leucine furnished by the instructor. 



i, 2 and 3. Repeat these experiments according to the directions given 

 under Tyrosine (pages 86 and 87). 



PREPARATION OF CYSTINE S 



From 50 to 500 grams of wool or hah* is pushed into a (Jena) flask and con- 

 centrated hydrochloric acid (200 c.c. to each 100 grams of wool) is added. In 

 order to get a part of the acid quickly to the bottom of the flask a part of the acid 

 may be put in first, then the wool, and finally the remaining acid. A condenser 

 consisting only of a glass tube 2 to 3 ft. long is inserted and the mixture is boiled 

 until the biuret reaction is entirely negative. The wool dissolves in a few min- 

 utes and if much cystine is desired more wool and acid can then be introduced. 

 After three to five hours' boiling with moderate quantities of wool the biuret 

 reaction has usually disappeared. 



To the hot acid solution of amino acids so obtained is added at once an excess 

 of solid sodium acetate, i.e., until the Congo red reaction for mineral acids is 

 entirely negative. A dark, heavy precipitate containing practically all the cystine 

 is obtained. After a few hours' standing at room temperature the liquid is filtered 

 off and the precipitate is washed with cold water. (From the mother liquor 

 diluted with the wash water is usually obtained on long standing a second pre- 

 cipitate consisting chiefly of tyrosine.) 



The crude cystine is then dissolved in boiling 3-5 per cent hydrochloric acid 

 and the solution is decolorized with good boneblack which should have been 

 previously thoroughly digested with hot, dilute hydrochloric acid and then washed 

 with water in order to remove the calcium phosphate. The hot filtrate from the 

 boneblack should be as clear as water. If it is not perfectly colorless the bone- 

 black treatment should be repeated and if a colorless solution is not then ob- 

 tained the fault lies with the quality of the boneblack. The last filtrate is heated 

 to boiling and the cystine precipitated by a slow addition of concentrated hot 

 sodium acetate solution. 



Large amounts of colorless cystine consisting of typical hexagonal plates can 

 thus be prepared without difficulty and with very little labor. Compare the 

 microscopical appearance of these crystals with those shown in Fig. 26, page 76. 



1 Folin and Denis: Jour. Biol. Chem., 12, 245, 1912. 



2 Abderhalden : Zeit. Physiol. Chem., 85, 91, 1913. 

 8 Folin: Jour. Biol. Chem., 8, 9, 1910. 



