126 PREPARATION AND CRYSTALLOGRAPHY OF HEMOGLOBINS 



prisms. He states that if one observes under the microscope a drop of the 

 fluid in which the crystals are suspended, before it is covered with the cover- 

 glass, it will be seen that these hexagonal plates quickly melt, and that 

 where they dissolve bundles of fine, bright-red, prismatic crystals suddenly 

 shoot out. The dark-red hexagonal plates are crystals of reduced hemo- 

 globin, as Nencki showed several years ago, while the bright-red prisms are 

 of oxyhemoglobin. He found that horse's blood is particularly inclined to 

 give crystals of reduced hemoglobin, and that in preparing crystals of horse's 

 hemoglobin by the regular method, without particular exclusion of air, both 

 forms appear at the same time. 



Hemoglobin crystals from the bloods of the horse, ox, pig, and dog 

 were prepared by Frey (Inaug. Dissert., Wurzburg, 1894; Jahr. ii. d. Fort. d. 

 Thierchemie, 1895, xxv, 108) by means of the dialyzing method of Giirber. 

 The corpuscles were separated from the defibrinated blood by centrifu- 

 galization, mixed with 2 volumes of water, and placed in a dialyzer which 

 was suspended in 30 to 70 per cent alcohol. Beautiful crystals were obtained 

 after 3 to 24 hours. If a drop of blood be placed on a slide under a cover- 

 glass, crystals form (primary crop) which dissolve as the blood becomes 

 fully laked, when occasionally a second crop forms. By reduction the blood 

 became yellowish, and after 3 or 4 hours it was violet-red and venous, and 

 at the same time granules appear which finally separate towards the margin 

 as distinct crystals. These crystals, Frey states, are of reduced hemoglobin 

 and in addition to these are clusters of colorless crystals. Horse's blood 

 crystallized most readily, and then that of the dog, and finally that of the 

 pig with difficulty. 



Kobert (Das Wirbelthierblut in mikrokristallographischer Hinsicht, 

 1901, 25) used the Giirber method to prepare crystals from the bloods of 

 the dog and cat. Arthus (Compt. rend. soc. biolog., 1895, XLVII, 686) 

 employed a similar method to obtain crystals of the horse and dog. The 

 blood was prevented from coagulating by the addition of oxalate, and after 

 the corpuscles had been separated by decantation they were mixed with 2 

 volumes of water and placed in a Kiihne membrane dialyzer suspended in 

 90 per cent alcohol. Large masses of crystals were formed. Arthus in a 

 later research (Zeit. f. Biolog., 1897, xxxiv, 444) modified his previous 

 method: The corpuscles from oxalated blood were dissolved in 2 volumes 

 of water and filtered, and the filtrate was placed in a parchment-paper tube 

 which was suspended in 17 to 33 per cent alcohol. At room temperature 

 oxyhemoglobin crystals 7 to 8 mm. long with sharp edges were formed. 

 When stronger alcohol was used the crystals were impure owing to an 

 amorphous precipitate. 



Studies of the crystallographic characters of crystals from blood of the 

 silkworm were made by Panebianco (Zeit. f. Krystallographie, 1897, xxvin, 

 198) . These crystals were colorless and it is doubtful if they were hemoglobin. 



Crystals from horse, dog, and cat were prepared by Abderhalden (Zeit. 

 f. physiol. Chemie, 1898, xxiv, 545). Success in the production of crystals, 

 he states, depends upon using the least possible amount of water necessary 

 to dissolve the blood corpuscles which have been freed as much as possible 

 from serum. To horse's corpuscles he added 3 volumes of water; to dog's 



