A method for the preparation of crystallized oxyhemoglobin.

Available methods for the preparation of crystalline oxyhemoglobin may be divided into two groups, in the first of which alcohol or ether, or both, are used as aids to crystallization, and in the second of which the use of these solvents is avoided. Possible objections to the use of alcohol were early pointed out by Hiifner (l), and Mayet (2) emphasized the advantages of ‘(henzinc” over ether. Although these workers, as well as Bohr (3) and Torup (4)) made preparaQions in which the use of either or both of these substances was avoided, the first systematic attempt to work out a method without their aid seems to have been the recent one of Dudley and Evans (5). Their procedure involves pressure dialysis of the washed red cells of horse blood, crystallization of the oxyhemoglobin in the dialysate by oxidation, and recrystallization of the product from water by reduction in NKUO at 37” and subsequent oxidation. Data as to the yield and purity of the product are not given. In the hope of obtaining fairly large amounts of oxyhemoglobin in the highest possible state of purity, the Dudley and Evans method was first employed, using a modification suggested by Adolph and Ferry (6); namely, final dialysis against water saturated with carbon dioxide. The oxyhemoglobin obtained in this way crystallized as large plates, instead of the needles reported by Dudley and Evans. Furthermore, the insolubility of the plates and their consequent resistance to reduction not only justified the belief that isoelectric oxyhemoglobin was being dealt with, and that Dudley and Evans were probably working with a more soluble salt, but rendered the use of the method impossible for the purpose in view, and it was abandoned in favor of a different principle.