214 VI. HEMOGLOBIN 



species of hemoglobin so far examined, including that of invertebrates 

 {935). Their results, however, tend to give iron values slightly in 

 excess of those derived from the oxygen capacity. This discrepancy, 

 while no longer of interest for the problem of the stoichiometric 

 combination of oxygen with hemoglobin, is of considerable importance 

 in relation to hemoglobin metabolism (Chapter X, 5.3.). 



The purity of a hemoglobin preparation is therefore best defined 

 in terms of the iron content and oxygen capacity. The former should 

 lie between 0.335 and 0.340% in a salt-free sample and the amount 

 of oxygen reversibly bound should lie within 1 or 2% of the theoretical 

 figure. Further discussion on quantitative analysis of hemoglobin 

 and its derivatives is found in Section 9. 



2.2. Properties of Ferrous Compounds 



2.2.1. Oxyhemoglobin (Hb02). The absorption spectrum of oxy- 

 hemoglobin- shows two sharp bands in the green, which give its 

 solutions a characteristic bright red color. On dilution, solutions of 

 oxyhemoglobin acquire a yellowish tinge, which serves to distinguish 

 this pigment from carboxyhemoglobin, which forms a pink solution 

 when diluted. The spectrum of oxyhemoglobin is insensitive to 

 changes in pH between 5.5 and 10; outside these ranges, denaturation 

 of the protein commences. The pressure at which the pigment is 

 fully combined with oxygen, however, is extremely sensitive to 

 changes not only in pH but also in protein concentration and ionic 

 strength. The dissociation of oxyhemoglobin is not light sensitive. 

 Measurements of magnetic susceptibility {2127) show the com- 

 pound to be diamagnetic, the iron bonds being, therefore, covalent. 

 Since the oxygen molecule in its normal state contains two unpaired 

 electrons, it has undergone a profound change in electrical structure 

 on combination with hemoglobin, the system resonating between 

 structures A and B, where the electrons in the 3c? orbitals of iron are 

 represented by dots (Pauling and Coryell, 2127). The absorption 

 curve of oxyhemoglobin is shown in Figure 2, Section 2.5. 



N N N N 



globin Fe : 0=0: globin Fe = O O = 



/"^ ^ ^x. 



N N N N 



A B 



