232 HEMOGLOBIN. 



Preparation of crystallised hsemoglobin (reduced haemoglobin). 



It was shown almost simultaneously and independently by Kiihne 1 and 

 by Rollett 2 that highly concentrated solutions of pure oxyhremoglobin 

 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. Kiihne explained that the difficulty which is en- 

 countered, when attempting to crystallise reduced hsemoglobin, depends 

 upon its very great solubility. 



Hoppe-Seyler was unable to crystallise reduced haemoglobin ; 3 and Hiifner 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 Kiihne fifteen 

 years earlier. 



In order to obtain crystals of reduced haemoglobin for microscopic 

 examination, a pure and highly concentrated solution of oxyhoemoglobin 

 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 



Nencki and Sieber have obtained large quantities of crystals of 

 reduced haemoglobin by reducing concentrated solutions of pure oxyhse- 

 moglobin of the horse through the agency of putrefactive bacteria, then 

 adding a sufficient quantity 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 

 oxyhaemoglobin crystals with a small quantity 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 hsemoglobin. 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 Nencki 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 Vorkommen und die Ausscheidung des Hamoglobins aus dem Blute," Virdiow's 

 Archiv, 1865, Bd. xxxiv. S. 423-436. 



2 Loc. cif. 



3 Med. Chem. Untersuch., Berlin, S. 373. 



4 "Ueber krystallische Hamoglobin," Ztschr. f. physiol. Chem., Strassburg, 1880, 

 S. 383. It is singular that Nencki and Sieber, in an interesting and really valuable 

 paper, should in 1887 have published again, as a new discovery, the obtaining of crystals 

 of reduced haemoglobin, though they subsequently disclaimed all priority (see M. Nencki 

 and N. Sieber, "Venose Hamoglobinkrystalle," Ber. d. deutsch. chem. Gfesellsch., Berlin, 

 1886, Bd. xix. S. 128 and 410). 



5 Ktihne, op. cit. 



