SINCE PREYER'S INVESTIGATIONS. 125 



may be precipitated by it. Within several hours a light-red deposit has 

 formed on the surface, while the fluid below appears clear and a dark 

 granite-red. The mixture is filtered, and the filtrate is kept in an ice-chest. 

 After 2 days only an insignificant quantity of crystals has formed. These 

 crystals are suspended on the top of the mixture and are filtered off. The 

 filtrate, which contains almost all of the hemoglobin, is poured into large 

 porcelain vessels and set aside at room temperature. The hemoglobin sepa- 

 rates at first as red and later as brownish crystals. After 3 days almost all 

 of the hemoglobin has crystallized, so that the filtrate appears to be colored 

 only slightly brownish. Microscopically investigated, the crystals contain 

 only slight impurities which can eventually be eliminated by recrystalliza- 

 tion. The hemoglobin is best drained on Biichner filters until the forma- 

 tion of firm cakes. To recrystallize, the crystals are dissolved in the least 

 possible amount of water and mixed with ammonium sulphate (100 c.c. of 

 the hemoglobin solution to 80 c.c. of saturated solution of ammonium sul- 

 phate). The yield from 5 liters of horse's blood was 1,500 grams. 



Fluoride of sodium was added by Arthus and Huber (Compt. rend. soc. 

 biolog., 1893, XLV, 970) to the list of inorganic salts that favor the crystal- 

 lization of hemoglobin. They found that when to normal or defibrinated 

 blood there was added an equal volume of a 2 per cent solution of fluoride 

 of sodium, and the solution allowed to stand at room temperature, crystals 

 of oxyhemoglobin could be obtained within a few days. They also state 

 that crystallization is accelerated by the addition of 0.1 to 0.5 per cent of 

 hydrochloric acid and by increasing the temperature to 40. Crystals were 

 prepared from the bloods of the dog, horse, cat, and guinea-pig. Guelfi (Rif . 

 med., 1897, No. 10; Maly's Jahr. ii. d. Fort. d. Thierchemie, 1897, xxvn, 

 149) also reports success with fluoride of sodium. He obtained crystals from 

 the bloods of the dog and guinea-pig by the addition of an equal volume 

 of a 2 per cent solution of this salt and maintaining the mixture at a tem- 

 perature of 40. This method, he states, failed in the case of both arterial 

 and venous human blood. 



The statement by Bohr (toe. cit.) of his belief that oxyhemoglobin is 

 not a homogeneous substance, and that it consists of a mixture of oxy- 

 hemoglobins which differ in elementary composition, molecular weight, 

 and combining capacity with O, has been shown by Hiifner (Archiv f. 

 Anat. u. Physiol., 1894, 130) to be untenable. Hiifner's researches proved 

 that Bohr's methods for producing the several forms of oxyhemoglobin 

 gave rise to mixtures of oxyhemoglobin with variable amounts of decom- 

 position products. Hiifner made new studies of the photometric constants 

 of oxyhemoglobin, reduced hemoglobin, and carbon-monoxide hemoglobin, 

 and determined the absorption coefficients for O and CO. He concluded, 

 from the constancy of the extinction coefficients, the O and CO capacities, 

 and the percentage of iron, that in healthy fresh bullock's blood there is 

 only one hemoglobin present, and that the blood-coloring matters of the 

 higher animals have all, when freed from water, the same molecular weight 

 and with it the same capacity for carbonic oxide and oxygen. Hiifner also 

 noted that when horse's blood is crystallized in closed cylinders there appear 

 in great abundance dark-red 6-sided plates, together with the well-known 



