ON THE CRYSTALLOGRAPHY OF HEMOGLOBIN. 95 



crystals, but cat's blood only after a longer time. With dog's blood crystal- 

 lization proceeds from the surface. The crystalline mass may be lifted off, 

 then a new one forms, and so on. Very much more time is required for 

 human and rabbit's blood. Pig's and frog's blood gave no crystals, yet the 

 hemoglobins of these bloods are capable of being crystallized. By repeated 

 freezing and thawing of the blood all the blood corpuscles could be com- 

 pletely decolorized, but this requires larger quantities of blood, repeated 

 freezing and thawing, and much time. Moreover, it is necessary to con- 

 centrate this laked blood by evaporating at a low temperature. It is im- 

 material in this process whether or not the blood be exposed to the air 

 during the process. This method, which is particularly convenient in winter, 

 is worthy of recommendation only where absolute purity is not necessary, 

 as in making comparative crystallographic and optical investigations of 

 the hemoglobins of different animals. 



(III) Into the animal from whose blood the crystals are to be pro- 

 duced, Bottcher injected intravenously a quantity of cold water during 

 chloroform narcosis, and then the chloroform was administered until death 

 occurred. The blood is obtained immediately after death from the heart 

 and vessels, and it is readily crystallizable. If mixed with an equal volume 

 of water and alcohol, and the mixture placed in the cold, a magma of crys- 

 tals will form. This method is not recommended, because of the difficulty 

 of obtaining sufficient blood from the dead animal. 



(IV) The solvent for the hemoglobin which Kiihne recommends is the 

 taurocholate and glycocholate of sodium. Thiry also employed bile salts to 

 obtain hemoglobin crystals from bloods that crystallize with difficulty. 



600 c.c. of horse blood are collected in a cylindrical vessel and cooled. 

 As soon as the plasma has separated from the corpuscles, it is removed, 

 together with the layer of white corpuscles, and the remaining mass of red 

 corpuscles is mixed with 0.5 per cent solution of crystallized ox bile. The 

 corpuscular mass is then allowed to coagulate. The fibrin which has formed 

 has entangled the decolorized corpuscles which have not dissolved, so that 

 the deep-red lake-colored solution, which may be drained off, contains none 

 of the corpuscles. To this solution is added, during continual stirring, and 

 as long as the precipitate that forms is dissolved, 90 per cent alcohol which 

 contains a very little acetic acid. After several hours the preparation is 

 converted into a crystal pulp, which may be collected on a filter, then 

 washed with dilute alcohol, and subsequently with iced water. 



Or 100 c.c. of dog's blood are allowed to coagulate in a shallow vessel; 

 the coagulum is then detached from the walls of the vessel and allowed to 

 stand in a cool place for 24 hours, until the serum has separated as much 

 as possible. The serum is removed; the coagulum is washed with water 

 and broken up in 50 c.c. of water by means of a syringe. After 24 hours 

 it is strained through linen and the clot is washed out with 10 c.c. of water. 

 To the mixture of the washing from the clot and the fluid obtained by 

 straining are added 2 c.c. of a solution consisting of 1 part of crystallized 

 bile and 3 parts of water. After 24 hours the solution is filtered through 



