ESTIMATION OF HEMOGLOBIN FOR CLINICAL PURPOSES 295 



interaction has also been assumed to be insensitive to changes in pH. If the 

 transmission is carried along a chain of electrostatic linkages between — C00~ 

 and — NH+, it should be unaffected between pH 4, where the carboxyl 

 groups lose their proton, and pK 10, where the basic groups become uncharged. 

 Hydrogen bond formation between one of the hydrogen atoms of the — NH + 

 groups and two adjacent oxygen atoms might also be stable in this pH 

 range, but such bifurcated hydrogen bonds are considered to occur only 

 rarely {2125). 



8.3. Hemoglobins of Different Molecular Weight 



The reactions of myohemoglobin are of considerable importance for this 

 hypothesis. So far, Kiese and Kaeske {1527) have been the only workers 

 who have reported a reaction in which myohemoglobin departs from the 

 behavior predicted for it by the Hiifner theory. Although inspection of 

 their data reveals little reason for the sigmoid curve which they draw, a 

 reinvestigation would be desirable. 



Another problem is raised by the behavior of chlorocruorin. Fox {93I^) 

 showed that its dissociation curve was sigmoid in shape and showed quali- 

 tatively the same type of pH sensitivity as does oxyhemoglobin. The 

 molecular weight of chlorocruorin is of the order of three million, and the 

 possibility exists of interaction between about two hundred hemes. In 

 view of the dissociation of this pigment into smaller fragments {cf. Chapter 

 VII) interaction may take place only between smaller groups of hemes. 

 The scheme we have presented for the transmission of interaction is quite 

 capable of extension to systems containing greater numbers of hemes than 

 the four present in oxyhemoglobin. In this connection it is of interest that 

 Ferry and Green {918) developed an empirical equation to describe the 

 dissociation curve of oxyhemoglobin in which the affinity constant was 

 related to the fraction of the hemoglobin combined with oxygen. Gaddum 

 {964b) has also drawn attention to the use of the mathematical transformation 

 known as the probit to describe the sigmoid dissociation curve of oxyhemo- 

 globin. These approaches, depending, like the earlier Hill equation on the 

 statistical behavior of a number of hemes may, perhaps, be capable of 

 inclusion in a broader treatment of interaction between many hemes. 



9. DETERMINATION OF HEMOGLOBIN 



9.1^ Estimation of Hemoglobin for Clinical Purposes 



9.1.1. Specificity. Exact methods for determining oxyhemoglobin, car- 

 boxyhemoglobin, hem/globin, sulfhemoglobin, and choleglobin in blood are 

 available. \ complete analysis involving all these procedures is laborious 

 and requires special apparatus, such as a spectrophotometer, and hence can- 

 not be applied for general routine purposes. As a rule, oxyhemoglobin forms 

 so large a percentage of the total of all hemoglobin derivatives in aerated 

 blood that its estimation will suffice for the clinical determination of total 

 hemoglobin {e.g., for gaging the intensity of an anemia). There are, however. 



