IX] PROTEINS AND AMINO-ACIDS 137 



(e) The sulphur reaction. Boil a few c.c. of the protein solution with an equal 

 quantity of 40 o/q sodium hydrate for two minutes, and then add a drop or two of 

 lead acetate. The solution turns black (or brownish, if only a small amount of 

 protein is present). 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, either a black precipitate, or dark colour, due to lead sulphide is formed. 

 The sulphur in the protein molecule is mainly present as cystine. 



For the following tests, a purified protein solution is necessary, since the reactions 

 may also be given by accompanying aromatic substances, carbohydrates, etc. For 

 this purpose take 40 gms. of ground peas, add to the meal about 200 c.c. 10 ^jq sodium 

 chloride solution, and allow the mixture to stand, with occasional stirring, for 3-12 

 hrs. (see p. 147). 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^ 

 until the protein is well precipitated. (Toluol should be added to the liquid in the 

 dialyzer.) Then filter ofi" the protein. Reserve half, and dissolve the other half in 

 about 50 c.c. of 5 o/q sodium nitrate solution. With this solution (after reserving a 

 portion for Expt. 129) make the following tests : 



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

 alcohol. The protein is precipitated. 



{g) Precipitation hy the heavy metals. Measure out a few c.c. of the protein 

 solution into three test-tubes, and add respectively a little (1) 5% copper sulphate 

 solution, (2) 5 o/^ lead acetate solution, (3) 5 o/o mercuric chloride solution : the protein 

 is precipitated in each case. 



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

 are absent in this case. It can, however, be demonstrated in later experiments (see 

 p. 145. 



{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 a 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. 39, 44, 46). 

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

 solution add a little 5 <Yo 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. 139), the tests {k)-{m) should be carried out with a solution of the protein in dilute 

 acid. Dissolve, therefore, t|ie remainder of the solid pea globulin in about 40 c.c. of 

 lOo/o acetic acid, filter, and make the following tests : 



{k) Precipitation by tannic acid. Add a little 3 <>/o 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. 



