90 PREPARATION AND STUDY OF HEMOGLOBIN CRYSTALS 



ether, the whole is shaken, and then the excess of ether is poured off. 

 The solution is quickly filtered. The solution of dog, guinea-pig, squirrel, 

 and rat blood corpuscles crystallizes without further treatment. With 

 bloods that do not crystallize readily, the nitrate is cooled to and mixed 

 with one-fourth of its volume of 80 per cent alcohol which has also been 

 cooled to 0, and the mixture subjected to a temperature of 5 to 10. 

 After crystallization, the crystals are collected on a filter paper at a tem- 

 perature near 0, and washed in the cold (at 0) with a cold mixture (at 0) 

 consisting of 1 volume of alcohol to 4 volumes of water. The crystals are 

 dried between filter paper by slight pressure. To recrystallize, the crystals 

 are dissolved in 3 volumes of distilled water by heating to 30 to 40 C., 

 the solution is filtered and cooled to 0, one-fourth volume of absolute 

 alcohol cooled to is added, and the mixture subjected to 5 to 10, 

 as before. In this way crystals from the blood of man, of the pig, bullock, 

 sheep, rabbit, duck, pigeon, and goose were obtained. 



Appearing shortly after Hoppe-Seyler's second contribution, an article 

 of epochal importance in physiology was published by Stokes (Proceedings 

 of the Royal Society, London, 1864, xin, 355, June and November), to 

 whom is due the credit of the discovery of the "respiratory function" of 

 the coloring matter of the blood, and also the specific differences in the 

 spectra of oxyhemoglobin and reduced hemoglobin. Stokes writes that it 

 was to him " a point of special interest to inquire whether we could imitate 

 the change of color of arterial into that of venous blood, on the supposition 

 that it arises in reduction. He found upon adding to a solution of blood a 

 reducing agent ["Stokes's reagent"] the color almost immediately changed 

 to a much more purple red as seen in small thicknesses, and a much darker 

 red than before as seen in greater thickness. The change of color, which 

 recalls the differences between arterial and venous blood, is striking enough, 

 but the change in absorption spectrum is far more decisive." When the 

 purple solution was exposed to air in a shallow vessel it changed imme- 

 diately into its original condition. He states that the addition of a reducing 

 agent caused reduction as before and exposure to air a return to the original 

 condition, and that these phenomena could be repeated a number of times. 

 From such facts he inferred that the coloring matter of the blood, like 

 indigo, is capable of existing in two states of oxidation, distinguishable by 

 a difference in color and a fundamental difference in the action on the spec- 

 trum. Hematin having been shown by Hoppe-Seyler in his first communi- 

 cation to be a decomposition product, and Stokes being obviously unaware 

 of this communication (published in Archiv f. path. Anat. u. Physiologic 

 and elsewhere, loc. cit., several months previously), and therefore not 

 knowing that Hoppe-Seyler had proposed terms for the coloring matter 

 of the blood, proposed, at the suggestion of Dr. Sharpey, the term cruorin; 

 and in order to differentiate the two states of oxidation he suggested the 

 terms scarlet cruorin and purple cruorin. Stokes observed that the change 

 in color from arterial to venous blood is in the direction of a change from 

 scarlet to purple cruorin, and that the blood is reoxidized in passing through 

 the lungs and deoxidized while passing through the tissues generally. He 



