56 PROTEINS. 



That large amounts of the weaker acids are required is only 

 to be expected ; the behaviour of oxalic and phosphoric acids is 

 anomalous, and appears to be due to the fact that oxalic acid 

 removes the lime from the casein complex, while phosphoric 

 acid forms an acid phosphate with the tricalcium phosphate. 

 In either case the formation of an acid calcium salt of casein 

 combined with calcium phosphate is prevented. 



Revis and Payne consider that casein exists in milk com- 

 bined with calcium phosphate, and that on acidifying the milk 

 with small progressive amounts of acid the calcium phosphate 

 is removed from the combination ; when the casein is precipi- 

 tated by the acid in the cold practically all the calcium phosphate 

 is precipitated. Their results show that calcium is removed 

 from the complex in direct proportion to the acid added, 

 but their figures with regard to phosphoric acid are less 

 definite. 



They also show that it is very improbable that any appreciable 

 amount of lactates of casein are formed in milk as it turns sour, 

 as supposed by van Slyke, Hart, and Laxa. 



On boiling milk with the quantity of acid just sufficient to 

 curdle, the whole of the casein is not precipitated ; thus the 

 author found that 2-91 per cent, was thrown down out of a 

 total of 3-05 per cent. 



Casein on treatment with strong sulphuric acid gives off 1'43 

 per cent, of carbon monoxide, equal to 6 CO to 134 N. 



Preparation of Casein. Casein is prepared from milk by 

 diluting it to about five times its volume, and adding sufficient 

 acetic acid to give 0-1 per cent, of the acid in the solution ; the 

 casein is precipitated, carrying down with it the fat ; the pre- 

 cipitate is washed well by decantation some ten times, collected 

 on a cloth filter, washed on the filter, and then dried, as far as 

 possible, by pressure. This precipitate is dissolved in the least 

 possible excess of ammonia, the solution allowed to stand for 

 some time (to allow the fat to rise), then syphoned off and filtered, 

 and the filtrate precipitated, as before, by acetic acid ; the 

 precipitate washed and redissolved in ammonia ; and this treat- 

 ment repeated three or four times. Van Slyke and Bos worth 

 add a little ammonium oxalate to precipitate the last traces 

 of lime. The casein is now rubbed up in a mortar with 80 per 

 cent, alcohol, and the alcohol poured off ; the treatment with 

 alcohol is repeated several times, using, finally, absolute alcohol ; 

 it is then treated two or three times with ether which has been 

 freshly distilled from some reagent which removes aldehydes 

 (e.g., casein, sodium phenyl-hydrazine-sulphonate) in the same 

 manner, and then extracted for some hours in a Soxhlet extractor 

 to remove the fat, and the ether evaporated off at as low a 



