212 VI. HEMOGLOBIN 



in the refrigerator, a procedure which, however, accelerates hemt- 

 globin formation. The oxyhemoglobin must next be induced to 

 crystallize. In some species, e.g., rat and horse, this frequently takes 

 place without further treatment when the cells are hemolyzed, while 

 the pigment from ox, sh.eep, pig, or man is more difficult to crystallize. 

 With a readily crystallizable hemoglobin, the pVL is adjusted to 6.8 

 and if crystallization does not commence, alcohol is cautiously added. 

 The latter procedure should be avoided if possible, since it facilitates 

 denaturation of the protein. Crystallized human oxyhemoglobin has 

 only recently been prepared by.Drabkin {627). 



Since the above procedures destroy the reducing mechanisms 

 present in the erythrocyte, a small amount of hem/globin is generally 

 found in the preparation; this may be avoided if carboxyhemoglobin 

 is used instead of the oxyhemoglobin. In spite of all precautions, 

 however, a small amount of nonhemoglobin iron is generally found, 

 even in the best preparations. 



The preparations most frequently used are generally modifications of the 

 methods of Heidelberger {1202) or of Ferry and Green {7Jt7). Heidelberger's 

 preparation of crystalline oxyhemoglobin starts with washed horse or dog 

 erythrocytes. Toluene is added to the cells and a 4:1 mixture of carbon 

 dioxide and oxygen bubbled through the suspension. After standing and 

 centrifugation, excess toluene and a denatured stroma-toluene emulsion can 

 be removed leaving a concentrated solution of oxyhemoglobin which may 

 crystallize spontaneously. If not, alcohol is cautiously added up to 20%. 

 In the method developed by Ferry and Green, the washed cells are hemolyzed 

 by the addition of the minimum quantity of distilled water, stroma debris 

 removed in a Sharpies centrifuge and crystallization is induced by adjustment 

 of the pH. In dilute solutions, with easily soluble oxyhemoglobins, or where 

 only small amounts of a derivative are required, dialysis against saturated 

 solutions of ammonium sulfate or 2.8 M phosphate buffer of pH G.8 may be 

 used to induce crystallization. 



The crystal form is not only a function of the species (627,222^) from 

 which the hemoglobin was taken, but also of the purity of the sample, the 

 acid used in its preparation, and the anion of the anticoagulant (747). Thus 

 human oxyhemoglobin crystallizes in the tetragonal system, while horse 

 oxyhemoglobin is obtained in the form of orthorhombic crystals from citrated 

 and of monoclinic crystals from oxalated blood {627). 



When the hemoglobin derivative is prepared for respiratory physi- 

 ology experiments, crystallization is frequently not necessary; freshly 

 laked erythrocyte solutions tend to give more reproducible results 

 than solutions which have received further treatment {1286). Treat- 

 ment of solutions of partially purified hemoglobin with adsorbents 



