2 3 o HAEMOGLOBIN. 



1. By the action of reagents exerting a reducing action. 

 It is an essential condition which all reagents to be employed in the 

 reduction of oxyhsemoglobin to haemoglobin must fulfil, that they do not 

 act destructively on these substances, as is the case with acids and salts 

 possessed of an acid reaction. 



Ordinary solutions of ferrous sulphate or stannous chloride cannot, 

 for instance, be employed, as they instantly lead to a decomposition 

 of the blood-colouring matter. The first and still the most generally 

 employed reducing agents, the use of which dates back to the researches 

 of Stokes on the blood-colouring matter, are ferrous and stannous salts 

 and the alkaline sulphides. Utilising the well-known property of citric 

 and tartaric acids to prevent the precipitation of the salts of iron and 

 tin by ammonia and the alkaline hydrates, Stokes indicated easy 

 methods of preparing active solutions of ferrous and stannous salts for 

 the study of the reduction of oxyhgemoglobin. 



(a) Alkaline solutions of ferrous salts (Stokes' reagent). To a solution 

 of a ferrous salt (usually ferrous sulphate or ferrous ammonium sulphate) 

 (Fe(NH 4 ) 2 (S0 4 ) 2 .6H 2 1 ), citric or tartaric acids or one of their alkaline 

 salts is added, and then ammonia, until the reaction is alkaline. A light 

 green solution is thus obtained, which rapidly darkens in the presence of 

 air by the absorption of atmospheric oxygen. Such a solution, which, 

 must be freshly prepared, exerts a powerful and exceedingly rapid 

 reducing action on oxyhsemoglobin, even in the cold. Alkaline ferrous 

 solutions possess the disadvantage, in proportion as they absorb oxygen 

 and become oxidised, of becoming coloured, and absorbing the more 

 refrangible rays of the spectrum, interfering, therefore, with the accurate 

 study of the specific absorption due to the colouring matter. 



(b) Alkaline solutions of stannous salts. These are made as described 

 under a, by substituting a stannous (usually SnCL 2 ) for a ferrous salt. 

 As they do not become coloured on salt being oxidised, these solutions 

 do not interfere with the accurate study of the absorption of the violet 

 rays. Like the analogous ferrous solutions, those containing tin rapidly 

 reduce haemoglobin even in the cold. 



(c) Solutions of the alkaline sulphides. Solutions of these salts 

 (ammonium sulphide being almost invariably employed) effect the 

 reduction of oxyhaemoglobin, but much more slowly than is the case 

 with a and ft, and their action is greatly accelerated by heat. Solutions 

 of ammonium sulphide for this purpose should be freshly prepared, and 

 be protected from the action of atmospheric oxygen and light, which 

 bring about chemical changes, and cause them to assume a yellow colour 

 and to absorb the violet end of the solar spectrum. 



Solutions of the crystalline sodium monosulphide (Na 2 S) cannot be 

 employed with advantage as reducing agents for oxyhsemoglobin, as, according 

 to my experiments, they lead at once to the formation of sulphomethsemo- 

 globin, so that the pure spectrum of reduced haemoglobin cannot be observed. 



(/) Agitation with finely -divided iron, 1 or with metallic iron reduced 

 by hydrogen the so-called officinal ferrum rcdactum. 2 



1 Rollett, "Versuche ueber thatsachliche und vermeintliclie Beziehungen d. Blutsauer- 

 stoffes," Sitzungsb. d. k. Alcad. d. Wissensch., Wien, 1866, Bel. lii. Abth. 2, S. 246 et seq. 



2 Ludwig und Schmidt, "Das Verhalten der Gase welche mit dem Blut durch den 

 reizbaren Saugethiermuskel stromen," Sitzungsb. d. k. Sachs. Gesellsch., Leipzig, 1868, Bd. 

 xx. S. 12-72. 



