GENERAL CHEMICAL CHARACTERS OF PROTEINS 69 



ferrous sulphate. In order to get the apparatus air-free the tube in 

 which the nitrous acid is allowed to act on the protein is connected 

 with the carbonic acid generating apparatus by means of a special 

 form of trap, from which the air is expelled by means of steam and 

 carbonic acid gas. The latter is evolved by the action of hydrochloric 

 acid on sodium carbonate solution in an apparatus specially designed 

 for the mixture of the liquids, in such a way as to ensure a steady 

 evolution of gas. The evolved nitrogen is collected over potash 

 solution in a modified form of Lunge nitrometer, in which it is 

 mixed with excess of oxygen, obtained by the electrolysis of water ; 

 in this way the nitrogen is freed from the nitric oxide. The excess 

 of oxygen is afterwards absorbed in alkaline pyrogallol solution, and 

 the volume of nitrogen evolved is finally measured over pure water. 

 Precautions must be taken to free all the liquids used in the ex- 

 periments from air. For full details reference must be made to the 

 original paper. 



SECTION XXI. ACTION OF FORMALDEHYDE ON PROTEINS. 



From the presence of amino and imino groups in proteins it is 

 to be expected that they would enter into reaction with formalde- 

 hyde. The first observations on such a reaction are due to Trillat 

 and Hauser. The former noticed that on addition of concentrated 

 formaldehyde solution, egg-white was converted into an opaque gelat- 

 inous mass, whilst the latter noticed that gelatin, on treatment with 

 formaldehyde, was converted into a hard, insoluble substance. Blum, 

 somewhat later, noticed that the addition of a small quantity of 

 formaldehyde to egg-white solution caused the latter to lose its 

 capacity for coagulating on heating ; it remained, however, clear after 

 the addition of the aldehyde. The observations of Blum and of Trillat 

 do not appear to be concordant ; but the apparent discrepancy was 

 later explained by Schwarz, who showed that dilute solutions, especi- 

 ally in absence of salts, remain clear on addition of formalin, and 

 lose their coagulability, whereas more concentrated solutions become 

 turbid, but can be made to coagulate by the addition of salts. These 

 phenomena indicate that formaldehyde is capable of acting on proteins. 



The reaction was studied in greater detail by Benedicenti. He 

 added dilute (2 per cent.) solutions of formaldehyde to protein solu- 

 tions, and estimated quantitatively at given intervals the amount of 

 formaldehyde which had not entered into reaction ; for this purpose 

 he employed hydroxylamine hydrochloride, which reacts with the 

 aldehyde according to the equation 



NH 2 . OH . HC1 + H . CHO = CH 2 : N . OH + HC1. 



By titrating the hydrochloric acid with standard potassium 

 hydroxide, using methyl orange as indicator, the amount of formalde- 

 hyde in a solution could be estimated. It was noticed that when 

 methyl orange was employed as indicator the proteins themselves 

 acted as bases ; a certain amount of acid was therefore necessary to 

 neutralise the solution to this indicator before the addition of the 

 hydroxylamine. This alkalinity diminished as the action of formal- 

 dehyde proceeded ; this fact indicated that the alkalinity was due to 

 the presence of amino groups, and gradually diminished, as the 

 formaldehyde condensed with them to form methylene derivatives. 



