PHYSIOLOGICAL CHEMISTRY 327 



weak acid, or by saturating it with a neutral salt. (See Phospho- 

 proteins, p. 306.) 



EXPERIMENT III. Place about 5 c.c. of milk in a test tube, and 

 dilute with an equal bulk of water. To this diluted milk add, drop 

 by drop, a weak solution of acetic acid ; a precipitate of caseinogen, 

 entangling fat, falls down. Filter off this precipitate and wash it with 

 water. Now add to it a weak solution of Na 2 CO 3 ; the precipitate 

 dissolves, and an opalescent solution of caseinogen, still, however, 

 containing some fat, passes through the filter. By repeated reprecipi- 

 tation and filtration comparatively pure caseinogen can be obtained, 

 from which the last traces of fat can be removed by treating with 

 ether. 



The chief property of caseinogen is its power to clot when treated 

 with rennin (a ferment contained in gastric juice) in the presence of 

 soluble calcium salts. 



EXPERIMENT IV. Take a pure solution of caseinogen. Divide it 

 into two portions, a and b. To both add about ten drops of rennin 

 ferment. To b add also a few drops of a 5 % solution of calcium 

 chloride. Place both in the water bath at 40 C. ; after about five 

 minutes examine to see if clotting has occurred. It will be found that 

 clotting has occurred in b where both rennin and soluble Ca salts were 

 present. 



EXPERIMENT V. Make similar experiments with milk, taking five 

 tubes, a, b, c, d, e. 



a = milk + rennin only. 

 b = milk + rennin + CaCl 2 . 

 c = milk + rennin (heated). 

 d = milk + rennin + potassium oxalate solution. 

 e = milk + rennin + potassium oxalate solution (heated after 10 

 minutes). 



It will be found that a clots, but not so quickly as b ; c does not clot, 

 because the enzyme has been destroyed ; d clots only after the addition 

 of CaCl 2 ; e clots also on the addition of CaCl 2 any time even after the 

 rennin has been destroyed. 



From these experiments we learn that the clotting of milk takes 

 place in two stages. 



(1) The rearrangement of the soluble caseinogen into the form of 

 soluble casein by the enzyme rennin. 



(2) The combination of the soluble casein thus formed with calcium 

 salts to form insoluble casein or clot. 



Note that the enzyme rennin does not clot the protein; it merely 

 rearranges it in such a form that it can be clotted. 



