120 PREPARATION AND CRYSTALLOGRAPHY OF HEMOGLOBINS 



the ingress of air again. It seems also that it is better to leave the bottle 

 in a room at ordinary temperature than to put it in a cool place, as advised 

 by Gamgee. After a variable time, in case of most animals at least two 

 days, a drop of blood is placed upon a slide, and when the margin of the 

 drop is slightly dry a cover-glass is gently lowered on the surface of the drop. 

 The formation of crystals will often be seen within an hour or so. Human 

 blood subjected to the same process does not usually yield crystals, but 

 when crystals do appear they invariably present the appearance of reduced 

 hemoglobin. Copemann also obtained crystals from human blood by the 

 use of bile preferably, as he states, cat's bile. 



Two methods for preparing hemoglobin crystals given by Mayet 

 (Compt. rend. soc. biol., 1890, cix, 156), and stated by him to be improve- 

 ments on the method of Hoppe-Seyler, are as follows: 



First method: The corpuscles are washed with sodium sulphate solu- 

 tion (1.5 per cent solution of the anhydrous salt) instead of sodium chloride 

 solution. To wash the corpuscles, a glass vessel having the capacity of 5 

 liters is used, the upper part of the vessel being of cylindrical form and 

 tapering conically, the lower part being in the form of a narrow cylinder 

 which holds about 80 c.c. The latter part has at the bottom an opening 

 which can be closed by a glass stopcock; a second opening, capable of 

 being closed, is located where the upper conical and the lower cylindrical 

 parts join. The treatment of the corpuscles with ether (one-fifth volume) 

 is also performed in a special vessel consisting of a cylinder 3.5 mm. in 

 diameter and 35 cm. long and extended by a conical part in the form of a 

 narrow tube provided with a glass stopcock. To the blood solution is added 

 one-fifth volume of absolute alcohol. This mixture is cooled at least 3 times 

 for 12 hours at --14. The crystals are separated and dissolved in water 

 at 35, the solution mixed with alcohol as before, and the hemoglobin at 

 least 3 times crystallized by cooling to 14. In this way crystals 1.5 mm. 

 long were obtained from the bloods of the dog, horse, and ass. 



Second method: The corpuscles are washed as above, the corpuscle 

 pulp is shaken with water (1 volume) and pure benzine (one-fifth volume), 

 and kept 24 hours at 5 to 8. Then the solution is gradually mixed with 

 one-fifth volume of absolute alcohol and treated in the usual way. The 

 yield by the second process is the greater. 



A study of the influences of various reagents upon the crystallization 

 of oxyhemoglobin and reduced hemoglobin was made by Donogany (Math- 

 ematikai es termeszettudomanyi ertesito, 1893, 11, 262; Maly's Jahr. ii. d. 

 Fort. d. Thierchemie, 1893, xxin, 126), who prepared crystals from the 

 bloods of the dog, cat, pig, mouse, ox, rabbit, duck, guinea-pig, horse, and 

 man. Donogany tested the usefulness of a number of the methods used 

 for preparing oxyhemoglobin and reduced hemoglobin crystals, and he also 

 made some examinations of the crystalline forms. Several of the methods 

 were modified. To obtain oxyhemoglobin crystals from dog's blood, the 

 "Canada balsam method" (loc. cit.) is recommended. A method of his own, 

 which he believes equally as good, is as follows: A drop of blood is treated 

 with a little ethyl bromide, methylene chloride, or ethylidene chloride. 



