14 DISTRIBUTION OF HEMOGLOBIN AND ALLIED SUBSTANCES 



9 per cent of hemocyanin, which would give 34.2 mg. of copper per 100 c.c. 



of blood. 



According to Henze, 1 gram of hemocyanin combines with 3 to 3.7 

 per cent of 0, or about 0.4 c.c., which is about one-fourth the capacity of 

 hemoglobin. Henze was the first to obtain crystals of hemocyanin. These 

 he prepared according to the Hofmeister (Zeit. f. physiolog. Chemie, 1892, 

 xiv, 165) and the Hopkins-Pinkus methods (Journal of Physiology, 1898- 

 99, xxm, 130) for obtaining crystals of egg albumin. In following out the 

 first method the blood of Octopus vulgaris was centrifugalized and filtered, 

 and the filtrate mixed with a sufficient amount of ammonium sulphate to 

 cause a slight precipitation. The solution was placed in shallow vessels, 

 when upon evaporation clusters of little needles were deposited. 



Much better results were obtained by the Hopkins-Pinkus method: 

 Ammonium sulphate was added to the centrifugalized blood until the 

 appearance of cloudiness; this slight precipitate was dissolved by the 

 addition of distilled water; acetic acid was then added until the appear- 

 ance of cloudiness. After standing for half a day the precipitate increased 

 very much and settled to the bottom. A crystalline mass formed at the 

 bottom of the vessel and consisted of excellently formed, doubly refrac- 

 tive, microscopic prisms. There appeared in two preparations egg-shaped 

 leaflets, some of which were 3 to 4 mm. in diameter, but which were difficult 

 to isolate from the crystalline mass owing to their fragility. Although 

 Henze found it possible to recrystallize hemocyanin, the second recrystal- 

 lization was rendered impure because of amorphous admixtures. He also 

 noted that crystallization took place perfectly only when fresh blood from 

 healthy animals was used. The mean elementary analysis he gives is 



Csa-eeHr-ssNie.ogSo.seCuo.sgC^i-e? 



(Compare with Griffiths's analysis of the amorphous hemocyanin of Sepia, 

 Homarus, and Cancer, p. 11.) In this article he reports studies of the 

 manner in which the copper is in combination in the protein molecule, of 

 the reactions, and of the O-capacity of hemocyanin, etc. He states that 

 hemocyanin behaves in certain ways differently from hemoglobin, that the 

 copper is in loose combination, that the substance behaves like a copper 

 albuminate, and that the O-capacity is only about one-fourth that of 

 hemoglobin. 



In a later contribution (Zeit. f. physiolog. Chemie, 1904-05, XLIII, 

 290) Henze reports that hemocyanin can not be separated into a protein 

 and a component free of protein, as in the case of hemoglobin, and that it 

 behaves like a copper albuminate from which the masked copper can easily 

 be separated. By hydrolysis he found tyrosin, leucin, histidin, lysin, prob- 

 ably glutaminic acid, and possibly a minimal amount of arginin. He failed 

 to find evidence of a carbohydrate group, although the fresh blood of 

 Octopus gave a positive reaction with Molisch's test. In determining the 

 distribution of its N he found the following: as mono-amino-N, 10.20 

 (63.39 per cent) ; diamino-N, 4.45 (27.65 per cent) ; humin-N, 0.43 (2.67 

 per cent); and ammonia-N, 0.93 (5.78 per cent). 



