CRITERIA FOR PURITY 213 



to remove traces of special impurities such as catalase may also alter 

 the affinity of the pigment for oxygen (^5) (Chapter VII). Where 

 necessary the removal of salts is effected by dialysis or electrodialysis 

 (703). 



2.1.2. Myohemoglobin. The preparation of this pigment is more 

 difficult than that of oxyhemoglobin from corpuscles since the pig- 

 ment content of the starting material is much lower than in the 

 corpuscles and more foreign material is present. In addition, myo- 

 hemoglobin derivatives are much more soluble than those of hemo- 

 globin. Present methods are derived from that used by Theorell 

 {2759) in his preparation of the pigment from horse heart after 

 removal of blood by perfusion. In this preparation other soluble 

 proteins are removed by lead acetate. After removal of the lead 

 with phosphate, the pigment is crystallized by dialysis against sat- 

 urated ammonium sulfate. On account of the much greater speed 

 with which myooxyhemoglobin autoxidizes, myohemn'globin is the 

 major pigment found in the crystals; to avoid this, myocarboxy- 

 hemoglobin may be prepared. Hill {1279) has shown that the oxygen 

 capacity of the pigment is not affectetl by the lead treatment. 



Morgan {1987) has shown that horse myocarboxyhemoglobin is 

 readily soluble in 3 M phosphate solutions, in which carboxyhemo- 

 globin will dissolve only to the extent of 1 mg. per liter. Traces of 

 hemoglobin remaining after perfusion may be removed by this pro- 

 cedure. Myohemoglobin shows species differences {1987,2760), but 

 these have not so far been used for preparative purposes.* 



2.1.3. Criteria for Purity. The blood pigment was shown by 

 Bunge in 1891 to contain iron and, by 1903, investigation, in particu- 

 lar that carried out in Hufner's school {1353,1355), had established 

 values for the iron content, which have been remarkably close to 

 those of later investigators. Taken in conjunction with the measure- 

 ment of the oxygen capacity, these results showed that one molecule 

 of oxygen is combined with one atom of iron. Hlifner's figure was 

 1.34 ml. per g. hemoglobin, which gives an iron content of 0.335%. 



Hufner's proof of the stoichiometric combination between iron and 

 oxygen was not immediately accepted (c/. Barcroft's monograph, 

 llfl, for discussion). A large number of workers, however, (c/. 1989 

 and 21Jf2 for bibliography) have fully confirmed Hufner for all 



* Recently, Theorell and de Duve (27ii7a) have prepared human myohemoglobin 

 in crystalline form. 



