CHEMICAL COMPOSITION OF PROTEIN MOLECULE 67 

 B. Distribution of the Nitrogen in Seven Groups. 



Further differentiation of the units composing the protein molecule 

 into those containing amino groups and those containing nitrogen in 

 heterocyclic combination was only possible after the discovery of 

 proline and tryptophane in 1901 and of oxyproline in 1902. 



This subdivision is possible, since nitrous acid reacts only with 

 amino groups with liberation of nitrogen : 



CH . NH 2 CHOH 



+ HN0 2 =| + N 2 



COOH COOH 



the amount of nitrogen evolved being double that contained in the 

 amino acid. 



The action of nitrous acid upon amino acids and amides as a method 

 for estimating these compounds was introduced by Sachsse and Kor- 

 mann in 1875. These investigators employed an apparatus consisting 

 of a small cylinder, furnished with a rubber stopper, through which two 

 tap funnels and an exit tube for the evolved gas passed. Potassium 

 nitrite was placed in the cylinder, dilute sulphuric acid in one of the 

 tap funnels and the solution of the substance (0*6-1 *o gram) in the other. 

 The exit tube was placed under an eudiometer filled with ferrous sul- 

 phate solution to absorb the nitric oxide. Air was expelled from the 

 apparatus by the decomposition of some of the nitrite with the acid. 

 As soon as the expulsion was complete, the amide solution and more 

 acid solution were allowed to enter into the cylinder and the gas 

 collected. The ferrous sulphate solution freed the mixture from nitric 

 oxide and more was added, if necessary. Carbon dioxide was then 

 removed with potash and the remaining gas was measured. 



This method was exhaustively tested in the Guinness Research 

 laboratory by Horace Brown and J. H. Millar, who published their 

 experiences in 1903. They found that there were several serious 

 sources of error in the original method before they could apply it to 

 their own subject of investigation. These were due (i) to residual air 

 contained in the "apparatus, or in the liquids ; (2) to the difficulty of 

 absorbing the excess of nitric oxide with ferrous sulphate. Carbon 

 dioxide was used to remove the air and the evolved nitrogen, and diffi- 

 culty was experienced in obtaining a steady and constant stream of 

 this gas. Pure carbon dioxide was prepared by the action of 30 per 

 cent, sulphuric acid on normal sodium carbonate solution. A con- 

 stant evolution of gas was not obtained, when the two solutions were 

 allowed to drop separately into a flask, owing to the supersaturation of 



5* 



