124 PREPARATION AND CRYSTALLOGRAPHY OF HEMOGLOBINS 



solution is filtered in the ice-chest, a clear, dark-red filtrate is obtained, 

 which contains most of the coloring matter, and when brought to room 

 temperature soon yields a rich crystal formation, which increases if a little 

 concentrated ammonium sulphate solution is added. After several days 

 the separation is complete so complete that the filtrate appears almost 

 colorless. It is purely crystalline, without amorphous admixtures. The 

 crystals are without exception little rhombic plates, some of very consider- 

 able size, while ordinarily crystals of horse hemoglobin, produced according 

 to Hoppe-Seyler, separate in the form of long 4-sided prisms. The precipi- 

 tate is filtered on a Biichner filter by the aid of a Sprengel pump and finally 

 freed from the mother-liquor by pressing between filter-paper, then dis- 

 solved in water, and again separated by the addition of an equal volume 

 of a saturated solution of ammonium sulphate. In this way the hemoglobin 

 can be recrystallized with ease several times. Ammonium sulphate efflo- 

 resces on the surface of the firm cake that had been obtained by pressing, 

 and can easily be removed. The cake when dried in the air can be crushed 

 to a fine powder, which readily dissolves in water. The solution shows a 

 pure oxyhemoglobin spectrum. 



Schulz states that in this way the hemoglobin may be separated from 

 other proteins. Fibrinogen and serum globulin separate completely in a half- 

 saturated ammonium sulphate solution, while the albumin separates only 

 by a higher concentration of the ammonium sulphate than was used here. 

 While as mentioned the oxyhemoglobin, according to the method used by 

 Dittrich (loc. cit.}, changes to methemoglobin, even during the recrystalliza- 

 tion, a pure oxyhemoglobin can also be obtained by this method. The 

 preparation thus obtained is, however, limited in stability; in one case it 

 contained after about one year considerable methemoglobin. The limit of 

 the quantity of ammonium sulphate required for the precipitation of the 

 hemoglobin in the amorphous condition, incidentally noticed, is distinctly 

 higher than that for crystallization. An amorphous precipitate occurred 

 only when in 10 c.c. of the solution there were 6.5 c.c. of concentrated am- 

 monium sulphate solution. In the tests which contained 5, 5.5, and 6 c.c., 

 respectively, of the saturated ammonium sulphate solution in 10 c.c., no 

 amorphous separation occurred, but after longer standing crystallization 

 gradually took place. 



This method of preparation, according to Schulz, is good because of its 

 convenience for experiments not depending on preparations free from salt. 



The ammonium sulphate method was also used by Spiro (Zeit. f. 

 physiol. Chemie, 1899, xxvni, 182). The corpuscle pulp was obtained from 

 oxalated horse's blood by decantation, diluted with 2 volumes of water, 

 cooled in an ice-chest, after which the solution was agitated with ether in 

 the proportion of 1,000 c.c. of blood-corpuscle pulp to 50 to 70 c.c. of ether. 

 During continual stirring a saturated solution of ammonium sulphate in 

 the proportion of 700 c.c. to 1 liter of blood corpuscles was gradually added, 

 the ammonium sulphate solution having the same temperature as that of 

 the blood corpuscles. After 5 to 10 minutes the voluminous precipitate 

 which has formed begins to rise ; but if this does not occur more ether must 

 be added, care being exercised to avoid a great excess, since hemoglobin 



