INDIRECT METHOD OF PRECIPITATION 71 



3. The Direct Method of Demonstrating the Existence of 

 Protein Compounds by the Solution of Otherwise Insoluble 

 Substances. — This method has been employed by W. A. 

 Osborne (30) who has shown that casein, when triturated with 

 calcium carbonate, carries a definite proportion of the calcium 

 into solution in the form of the soluble calcium caseinate with 

 the evolution of CO2. Strychnin is similarly carried into solu- 

 tion by casein. 



Osborne and Leavenworth (31) have shown that edestin will 

 hold in solution a quantity of cupric hydroxide corresponding 

 to no less than 34.67 per cent of its weight of copper, which, 

 assuming that each atom of copper is united to one nitrogen 

 atom, implies that no less than ten out of every sixteen nitrogen 

 atoms contained in the edestin molecule participate in forming 

 the compound with cupric hydroxide. This is exactly the pro- 

 portion of nitrogen atoms which is present in edestin in the 

 form of — COHN— groups. Precisely similar results were ob- 

 tained with gliadin. 



4. The Indirect Method of Precipitation. — Cohnheim and 

 Krieger (9) have elaborated an ingenious method of determining 

 the acid-binding capacity of the proteins which is based upon 

 the fact that phosphotungstic acid forms insoluble salts with 

 proteins. If the protein is combined with an acid, and calcium 

 phosphotungstate is employed, double decomposition takes place 

 as follows: 



Protein hydrochloride + calcimn phosphotungstate 



(soluble) (soluble) 



= protein phosphotungstate + CaCl2 



(insoluble) 



hence the quantity of calcium chloride present in the filtrate, 

 after filtering off the precipitate and carefully washing it (34) 

 is taken as a measure of the quantity of hydrochloric acid which 

 was bound by the protein just before precipitation. The pro- 

 cedure is as follows: Weighed amounts of protein are dissolved 

 in measured volumes of acid of known concentration. The 

 protein is then precipitated by the addition of an excess of the 

 phosphotungstate and the filtrate, plus the washings of the pre- 

 cipitate, is titrated against standard alkali. An excess of the 

 phosphotungstate is stated to be necessary because the phospho- 

 tungstate of protein is believed to undergo extensive hydrolytic 



