174 CRYSTALLOGRAPHY OF THE HEMOGLOBINS 



MARSUPIALIA. 



OPOSSUM, Didelphis virginiana. Plates 15-17. 



The first specimens were received from the Philadelphia Zoological 

 Gardens, and the blood was putrid. The blood was oxalated and ether- 

 laked, and yielded crystals very readily, the crystals forming soon after the 

 slides were covered. These crystals were reduced hemoglobin. Later, living 

 animals were procured, from which fresh blood was obtained. The animal 

 was bled into oxalate in each case, and preparations were made of the 

 whole blood, of the corpuscles alone, and of the corpuscles with various 

 diluents. From these, oxyhemoglobin was obtained; but in several cases 

 reduced-hemoglobin crystals appeared in the slides soon after the oxyhemo- 

 globin began to form, and these developed along with the crystals of the 

 first-formed oxyhemoglobin called cc-oxyhemoglobin. Later the oxyhemo- 

 globin, and eventually even the reduced hemoglobin crystals, dissolve, and a 

 second form of oxyhemoglobin called /3-oxyhemoglobin begins to appear, but 

 all three, a-oxyhemoglobin, reduced hemoglobin, and /3-oxyhemoglobin, may 

 be seen side by side in the same slide (see plates 16 and 17). These three 

 forms of crystals are produced independently of each other, and there is no 

 paramorphous change of the one into the other, as may readily be seen from 

 the difference in angles of the crystals, and also by the difference in crys- 

 tallization between the /^-oxyhemoglobin and the other two forms. These 

 three kinds of crystals were obtained in several different preparations of the 

 fresh blood. Carbon-monoxide hemoglobin was also made from the opos- 

 sum blood, and it was found to crystallize in two forms analogous to those 

 of the oxyhemoglobin a and /?. The old blood was regenerated, after it had 

 stood for some weeks and become putrid, by shaking with oxygen, and 

 also by addition of a diluted solution of commercial hydrogen peroxide. 



The treatment with oxygen was much more satisfactory, because if 

 the hydrogen peroxide is used in too concentrated a form the result is 

 methemoglobin ; and if it is very much diluted, the solution becomes too 

 dilute to crystallize well. But the corpuscles, after laking, make such a 

 thick preparation that it was usually found better to dilute somewhat with 

 normal salt solution. The procedure of regenerating was usually carried 

 out as follows: The brownish stale blood was oxidized by shaking in a 

 flask with oxygen gas until the color changed to bright oxyhemoglobin 

 red; if corpuscles alone were used, to two parts of the corpuscles laked 

 with ether was added one part of normal salt solution and one-fourth part 

 of ether, and the whole briskly shaken, and then centrifugalized in a small 

 hand machine for about 2 minutes. The solution was perfectly clear after 

 this treatment, and a thick amorphous mass rose to the top of the liquid, 

 which carried all of the precipitate and granular matter, leaving the solution 

 as clear as if centrifugalized for 2 hours in the ordinary process of treat- 

 ment. The crystals of oxyhemoglobin thus obtained were very sharp and 

 fine, and of exactly the same form as those prepared in the usual way. 



The preparations of CO-hemoglobin were made in a similar manner, 

 the blood being first shaken with oxygen gas under slight pressure, and then 

 laked with ether. After the ether-laking it was shaken with illuminating 



