THE INDIVIDUAL GROUPS OF PROTEINS 495 



(2) Blood is centrifuged at 3000-5000 revolutions per minute in a 

 Jouan centrifuge made by Leune in Paris (model C), or in a centriiuge made 

 by Heine in Viersen ; 635-660 c.c. of serum are separated in 10 minutes. 

 The blood corpuscles can be washed with -9 per cent, sodium chloride solution, 

 but this is not necessary. With the French centrifuge serum and corpuscles can 

 be separated whilst the instrument is running, but under these conditions the 

 corpuscles are broken by contact with the walls. 2 litres of blood give 7 

 litres of oxyhaemoglobin solution. The corpuscles in suspension, or the solu- 

 tion of haemoglobin, is poured slowly into 2 litres of gently boiling glacial acetic 

 acid containing 10 gm. of sodium chloride which is stirred with a mechanical 

 stirrer. Contact of the blood with the stirrer and sides of the vessel is avoided. 

 The mixture is kept boiling for 10 minutes and in the course of 15 minutes 

 1000 c.c. of water are added. Haemin crystallises out and after 24 hours is 

 filtered off on a double layer of cloth, washed with acetic acid, alcohol and ether. 

 4-6-5-2 gm. are obtained from i litre of blood. Recrystallisation is not 

 necessary. 



The products which are obtained by the various methods have been sup- 

 posed to be different, but Kiister x has shown that the differences are due to 

 impurities. 



Haemin is purified by dissolving it in chloroform containing quinine or 

 pyridine in the proportions of i gm. of haemin to i gm. of quinine and 50 c.c. 

 of chloroform, or to 2 c.c. of pyridine and 20 c.c. of chloroform, filtering and 

 adding alcohol containing hydrochloric acid or acetic acid to neutralise the 

 base. Haemin crystallises out. 



Hsemin consists of minute bluish-black crystals with a metallic lustre. 

 It is insoluble in water, alcohol and ether. It dissolves slightly in dilute 

 mineral acids and alcohol containing hydrochloric or acetic acid. It is 

 soluble in solutions of caustic alkali and alkali carbonates. 



Haemochromogen. 



A specimen of haemochromogen for microscopic examination can be 

 obtained by mixing a drop of blood with a drop of pyridine on a glass slide, 

 adding a drop of ammonium sulphide, covering with a cover slip and 

 allowing it to stand. A better method is to mix a drop of blood with a drop 

 of pyridine and a drop of 50 per cent, hydrazine hydrate, cover with a cover 

 slip, heat to nearly boiling and allow to cool. 



The crystals are delicate slender needles of a ruby- red colour, often ar- 

 ranged in rosettes. 



Haematoporphyrin. 



Haematoporphyrin can be prepared by treating blood with concentrated 

 sulphuric acid, or reduced blood with dilute acid. Haematoporphyrin is ob- 

 tained by pouring the concentrated acid solution into water. It is most con- 

 veniently prepared from its hydrochloride and is precipitated by adding sodium 

 acetate to the solution as an amorphous flocculent precipitate of a brown colour. 

 It dissolves in solutions of 'alkali hydroxide and of alkali carbonate, in dilute 

 mineral acids and alcohol ; it is not easily soluble in ether, chloroform or 

 amyl alcohol and it is insoluble in water and acetic acid. It is precipitated 

 from solutions by lead acetate and salts. 



Its solution in acid alcohol has the characteristic purple colour. Alkaline 

 solutions are red with a red fluorescence. 



t. Physiol. Chem., 40, 39*, 



