108 PEEPAEATION AND CRYSTALLOGRAPHY OF HEMOGLOBINS 



may be separated by placing the mixture in tubes and using the centrif- 

 ugal apparatus. The cakes of crystals thus obtained are mixed with water 

 holding one-fourth its volume of alcohol, and again centrifugalized. By 

 repeating this process, the crystals are said to be obtained free from serum- 

 albumin. If requisite, the crystals are recrystallized by dissolving them in 

 as small a quantity of water as possible at 25 to 30, cooling the solution to 

 0, and adding a fourth of its volume of alcohol. It is better to place the 

 fluid in a freezing mixture at a temperature of --10 to 20 for 24 hours. 



Crystals of reduced hemoglobin were prepared by Hiifner (Zeit. f. 

 physiol. Chemie, 1880, iv) from human blood, diluted or not, by placing 

 the blood in tubes from which air is excluded. After standing for a month 

 or two at summer temperature the blood became of a beautiful purple 

 color, and in many spots on the inner wall of the tubes there could be seen 

 whole layers of purple-red crystals, which upon spectroscopic examination 

 were found to give the characteristic bands of reduced hemoglobin. Wedl 

 (Archiv f. path. Anat. u. Physiologie, 1880, LXXX, 172) obtained reduced 

 hemoglobin crystals expeditiously by subjecting a solution of fresh or dried 

 blood in a confined atmosphere in the presence of a solution of pyrogallic 

 acid. The acid absorbs the oxygen and thus reduces the hemoglobin. In 

 this way crystals of reduced hemoglobin were prepared within 24 hours from 

 the blood of man, the rabbit, hare, deer, pig, and sheep. 



Crystals of reduced hemoglobin were prepared in large quantities by 

 Nencki and Sieber (Berichte d. d. chem. Ges., 1886, xix, 128, 410), who, 

 however, make the erroneous statement that no one had up to that time 

 prepared crystals of reduced hemoglobin. Kiihne (Archiv f. path. Anat. 

 u. Physiol., 1865, xxxiv, 423), and shortly after Eollett (Sitzungsb. d. k. 

 Akad. d. Wissensch., Wien, 1866, LII, 246), obtained crystals of reduced 

 hemoglobin by reduction of concentrated solutions of oxy hemoglobin. 

 Kiihne used a very concentrated solution of oxyhemoglobin in very dilute 

 ammonia, which he subjected to a stream of pure dry hydrogen in a glass 

 chamber. As evaporation proceeded crystals formed. Rollett (loc. cit.} 

 prepared reduced hemoglobin by the aid of iron filings. Gscheidlen in 1878 

 (loc. cit.) and Hiifner (toe. cit.) and Wedl (loc. cit.) in 1880 also prepared 

 crystals of reduced hemoglobin. 



Nencki and Sieber proceed in this way: Pure oxyhemoglobin crys- 

 tals from the blood of the horse are dissolved in lukewarm water; the 

 solution is then mixed with several cubic centimeters of decaying blood in 

 a flask that is provided with an india-rubber stopper having two perfora- 

 tions for tubes leading to and from the flask. The mixture is then freed 

 from air by the passage of a stream of hydrogen, after which the two tubes 

 are sealed by heat, and then the flask is set aside at a temperature of 

 20 to 25 for 8 to 14 days. After a time every trace of oxygen has dis- 

 appeared, the fluid is of a beautiful violet-red color, and contains only re- 

 duced hemoglobin. The solution is now cooled to 0, an india-rubber tube 

 is for some distance slipped over the outlet tube of the flask, and the other 

 end of the tube is dipped in cold absolute alcohol. The flask is gently 

 heated by immersing in lukewarm water, the end of the glass tube within 



