23 2 HEMOGLOBIN. 
Preparation of crystallised haemoglobin (reduced haemoglobin).— 
It was shown almost simultaneously and independently by Kuhne 1 and 
by Eollett 2 that highly concentrated solutions of pure oxyhemoglobin 
may, after reduction, be made to crystallise, and that the crystals of 
reduced haemoglobin, though differing in colour and spectroscopic char- 
acters from the oxygen compound, are essentially identical with it in 
crystalline form. Kuhne explained that the difficulty which is en- 
countered, when attempting to crystallise reduced ha?moglobin, depends 
upon its very great solubility. 
Hoppe-Seyler was unable to crystallise reduced haemoglobin ; 3 and Hufner 4 
in 1880 published a note, in which he announced that he had succeeded in 
obtaining crystals of reduced haemoglobin, though he neither then nor after- 
wards referred to the much more complete account published by Iviihne fifteen 
years earlier. 
Ill order to obtain crystals of reduced haemoglobin fur microscopic 
examination, a pure and highly concentrated solution of oxyhemoglobin 
in very dilute ammonia is placed in a gas chamber, and a stream of 
chemically pure and thoroughly dried hydrogen is passed over it; as the 
solution evaporates crystals separate. 5 
Xencki and Sieber have obtained large quantities of crystals of 
reduced luemoglohin by reducing concentrated solutions of pure oxyhe- 
moglobin of the horse through the agency of putrefactive bacteria, then 
adding a sufficient epiantity of 25 per cent, alcohol and exposing to cold. 
The method which I employed more than twenty years ago, and which 
appears to me to offer some advantages, is to place a magna of pure 
oxyhemoglobin crystals with a small epiantity of the mother liquor 
from which they have separated in a glass tube, so as nearly to fill the 
latter, and then to seal it. The tube is heated for some days in an 
incubator at about 35° C, and is then set aside in a cool place. After 
some weeks of exposure to a winter temperature, the tube is found to 
contain large quantities of crystallised and' perfectly reduced haemo- 
globin. 
No one has hitherto attempted to recrystallise reduced haemoglobin, 
though, with the conveniences at present at the disposal of the scientific 
chemist, the process would present little difficulty. 
Characters of the crystals of reduced haemoglobin. — In form 
they are, as has been said, essentially identical with those of the 
oxygen compound, and like these are doubly refracting. Hiifner 
often obtained crystals 1 mm. long; and Xencki and Sieber, working 
with horses' blood, obtained crystals, mostly in the form of hexagonal 
plates, 2 or 3 mm. in diameter. They are pleochromatic, appearing 
of a dark red colour in some lights, and exhibiting a bluish or purple 
tinge in others. 
1 " Das Yorkomruen und die Ausseheidung des Hilmoglobins aus dem Blute," Virchow's 
Archiv, 1S65, Bd. xxxiv. S. 423-436. 
2 Loc. cit. 
3 Med. Chem. Untersuch., Berlin, S. 373. 
4 "Ueber krystallisehe Hamoglobin," Ztschr. f. physiol. Chem., Strassburg, 1S80, 
S. 383. It is singular that Nencki and Sieber, in an interesting and really valuable 
paper, should in 1SS7 have published again, as a new discovery, the obtaining of crystals 
of reduced haemoglobin, though they subsequently disclaimed all priority (see M. Nencki 
and X. Sieber, " Venose Hamoglobinkrystalle," Bcr. d. dcutsch. chem. Gesellsch., Berlin, 
1886, Bd. xix. S. 12S and 410). 
5 Kuhne, op. cit. 
