vin] THE PROTEINS AND PROTEASES 123 



groupings formed by the condensation of the carboxylic group of an amino-acid with 

 the amino group of another amino-acid (see p. 119). 



(e) The sulphur reaction. Boil a few c.c. of the protein solution with some 40 / 

 sodium hydrate for two minutes, and then add a drop or two of lead acetate. The 

 solution turns black. This reaction is due to the formation of sodium sulphide by 

 the action of the strong alkali on the sulphur of the protein. On addition of the lead 

 salt, a black precipitate of lead sulphide is formed. The sulphur in the protein 

 molecule is mainly present as cystiue. 



For the following reactions, a protein solution free from other impurities is 

 required. For this purpose take 40 gms. of ground peas, add to the meal about 

 200 c.c. 10 / sodium chloride solution, and allow the mixture to stand, with occa- 

 sional stirring, for 3-12 hrs. (see p. 134). Then filter off the extract, first through 

 muslin, and, subsequently, through filter-paper. Put the extract to dialyze for 

 24 hrs. in a collodion dialyzer 1 until the protein is well precipitated. (Toluol 

 should be added to the liquid in the dialyzer.) Then filter off the protein. Reserve 

 half, and dissolve the other half in about 50 c.c. of 5 % sodium nitrate solution. 

 With this solution (after reserving a portion for Expt. 123) make the following tests : 



(/) Precipitation by alcohol. To a few c.c. in a test-tube, add excess of absolute 

 alcohol. The protein is precipitated. 



(g) Precipitation by the heavy metals. Measure out a few c.c. of the protein 

 solution into three test-tubes, and add respectively a little (1) copper sulphate 

 solution, (2) lead acetate solution, (3) mercuric chloride solution : the protein is 

 precipitated in each case. 



The following test cannot be demonstrated on the Pea protein, since carbo- 

 hydrates are absent in this case. It can, however, be demonstrated in later 

 experiments (see p. 130). 



(h) Molisch's reaction. To a few c.c. of the protein solution add a few drops of a 

 1 % solution of a-naphthol in alcohol. Mix, and then run in an equal volume of 

 strong sulphuric acid down the side of the tube. A violet ring is formed at the 

 junction of the two liquids. The reaction signifies the existence in the protein of a 

 carbohydrate group which gives rise, on treatment with acid, to furfural. The latter 

 then condenses with a-naphthol to give a purple colour (see also Expts. 38, 44, 46). 



(i) Precipitation by salts of alkaline earth metals. To a few c.c. of the protein 

 solution add a little barium chloride solution. A precipitate is formed on standing. 



(/) Precipitation by neutral salts. Saturate a few c.c. of the protein solution 

 with finely powdered ammonium sulphate. The protein is precipitated or "salted out." 



Since from a neutral salt solution the pea globulin is precipitated by acid 

 (see p. 125), the tests (k}-(ra) should be carried out with a solution of the protein in 

 dilute acid. Dissolve, therefore, the remainder of the solid pea globulin in about 

 40 c.c. of 10 / acetic acid, filter, and make the following tests : 



(k) Precipitation by tannic acid. Add a little tannic acid solution : the protein is 

 precipitated. 



1 The collodion solution is made by adding 75 c.c. of ether to 3 gms. of well-dried 

 pyroxylin, allowing it to stand for 10-15 minutes and then adding 25 c.c. of absolute 

 alcohol. The dialyzers are prepared by coating the inside of a large test-tube with the 

 solution and then filling with water, after the film is sufficiently dried so as not to be 

 wrinkled by touching with the finger. The sac can then be withdrawn from the tube. 



