5 2 



PREPARATION OF HEMOGLOBIN-CRYSTALS. 



The crystals of hemoglobin are doubly refracting and pleochromatic, 

 that is, they appear bluish red in transmitted light and scarlet red in 

 reflected light. The crystals, which contain from 3 per cent, to 9 per 



cent, of water of crystallization and 

 therefore become disintegrated from es- 

 cape of this water on exposure to the 

 air, are always soluble in water, though 

 different varieties dissolve with varying 

 degrees of facility. They are more 

 readily soluble in dilute alkali. The 

 solutions are dichroic, that is, they ap- 

 pear red in reflected light and greenish 

 in transmitted light. They are insolu- 

 ble in alcohol, ether, chloroform, and 

 fats. 



As a result of the process of crystalliza- 

 tion the hemoglobin itself appears to under- 

 go an internal change. Previous to crystal- 

 lization it does not diffuse as a true colloidal 

 body;, but it actively decomposes hydrogen 

 dioxid. Dissolved in the form of crystals, 

 however, it is slightly diffusible, and does 

 not decompose hydrogen dioxid, through the 

 action of which it is decolorized. The crys- 

 tals of hemoglobin collect like an acid at the 

 positive pole of an electric current. As the 

 hemoglobin thus exhibits alterations after its 

 separation from the erythrocytes, Hoppe- 

 Seyler believed that the oxyhemoglobin was united with lecithin within the 

 erythrocytes, and also the hemoglobin. The former 'combination he designated 

 arterin and the latter phlebin. 



FIG. 10. Hemoglobin-crystals: a b, from hu- 

 man blood; c, from the cat; d, from the 

 guinea-pig; e, from the marmot; and f, 

 from the squirrel. 



PREPARATION OF HEMOGLOBIN-CRYSTALS. 



Method of Rollett. Defibrinated blood, made lake-colored by freezing and 

 thawing, is poured into a shallow vessel, whose bottom is covered therewith to a 

 height of only i mm. Evaporation is permitted to take place slowly in a cool 

 place and as a result the crystals separate. 



Method of Hoppe-Seyler. Defibrinated blood is mixed with 10 volumes of 

 a solution of sodium chlorid or of sodium sulphate (i volume of a concentrated 

 solution to 9 volumes of water) and permitted to stand. After the lapse of two 

 days the clear supernatant layer is removed with a pipet, while the thick sediment 

 of blood-corpuscles is washed with water into a glass flask, and shaken with an 

 equal volume of ether until the blood-corpuscles are dissolved. After standing for 

 a short time the supernatant ether is removed, and the lake-colored fluid filtered 

 in the cold; then one-fourth volume of cold (o) alcohol is added. This mixture 

 is permitted to stand for several days at a temperature of 5 C. The crystals 

 that will thus have formed in abundance can be collected upon a filter and dried 

 by pressure between blotting-paper. Through the gradual action of the alcohol 

 upon the hemoglobin-solution, by introduction into a dialyzer, it is possible to obtain 

 crystals several millimeters long. 



Method of Gscheidlen. Gscheidlen obtained the largest crystals, several centi- 

 meters in length, by melting in small glass tubes defibrinated blood that had 

 been exposed to the air for 24 hours, and preserving for several days at a tem- 

 perature of 37 C. Spread upon a glass plate the crystals readily appear. 



QUANTITATIVE ESTIMATION OF THE HEMOGLOBIN. 



(a) From Its Iron-content. As in the dry state (100 C.) hemoglobin contains 

 0.42 per cent, of iron by weight, the amount of hemoglobin can be estimated 

 from the amount of iron in the blood. If m represents in percentage the weight 



