128 PREPARATION AND CRYSTALLOGRAPHY OF HEMOGLOBINS 



crystallization of necturus blood is very readily obtained by the action of 

 various hemolytic agents. 



A quick method for preparing crystals of oxyhemoglobin was reported 

 by one of us (Reichert, American Journal of Physiology, 1903, ix, 97), 

 who also made studies of the effects on crystallization by mixing the bloods 

 of different species, etc. It was found that if to the blood of the dog there 

 be added, before or after laking, from 1 to 5 per cent of ammonium oxalate, 

 crystallization invariably begins immediately, and that any quantity of 

 crystals can be obtained within a few hours at ordinary room temperature. 

 If a drop of this blood be placed under the microscope, crystals will be seen 

 to form at once near the margin of the drop, and to be deposited so rapidly 

 that a solid mass is formed in a few minutes. 



The blood of the horse does not yield quite so readily to this treatment. 

 If a drop of blood so prepared be examined under the microscope, it will be 

 found that crystallization will not begin usually at room temperature until 

 after from 15 to 20 minutes or more, and that it will proceed slowly. Better 

 results can be obtained if the blood be oxalated and centrifugalized, or set 

 aside for the corpuscles to subside. The supernatant liquid is then poured 

 off, and the remaining corpuscles are laked with ether. 



Defibrinated blood of the rat, laked with water on a slide, and covered 

 with a cover-glass after the margin of the drop has become dried, usually 

 crystallizes very readily, as is well known. Quicker results can be obtained 

 if the blood be oxalated before or after laking, and even more rapid crystal- 

 lization occurs if the blood be laked with ether instead of water. Crystals 

 form so rapidly in the oxalate-ether blood that a magma is formed in the 

 test-tube within a few minutes. 



The oxalate-ether process applied to the blood of the guinea-pig gives 

 most satisfactory results. Crystallization does not proceed quite so rapidly 

 as in rat's blood, yet within a minute or two innumerable tetrahedra appear, 

 and practically complete crj'stallization can be obtained within a couple of 

 hours. The blood of the necturus crystallizes readily when so treated. 

 The crystals resemble in form those of the triple phosphates. 



The rapidity with which crystallization begins and proceeds was found 

 to be influenced decidedly both by the method of laking and the percentage 

 of oxalate. Ethyl ether is a much better laking agent than water, and acetic 

 ether is stronger than ethyl ether. The presence of any quantity of oxa- 

 late up to saturation increases crystallizability, but he found from 1 to 5 

 per cent to be the best; the larger the quantity the more is crystallization 

 hastened. When more than 5 per cent is used, the oxalate also tends to 

 crystallize upon the slide. If the blood be prevented from drying, as in the 

 test-tube, the oxalate remains in solution. Asphyxial blood yields crystals 

 more readily than normal blood. 



If to the blood of one species, the blood, plasma, or serum of another 

 species be added, the laking of the blood may be retarded, accelerated, or 

 unaffected, according to the character of the mixture. The period required 

 for laking may be prolonged 5 minutes or more. The crystallization of the 

 oxyhemoglobin may be hindered or prevented in such mixtures. Thus, 

 by varying the proportions of a mixture of the bloods of the dog and guinea- 



