MAMMALIAN HEMOGLOBINS 311 



Alkali denaturation 



Alkali denatures hemoglobin, producing a brown, insoluble, alkaline-globin 

 hemochromogen. Korber 732 demonstrated by this method that fetal hemoglobin 

 is more resistant than adult hemoglobin to alkaline decomposition. Although fetal 

 hemoglobins of other mammals also differ in alkali resistance from their adult counter- 

 parts, greater resistance to decomposition does not apply to all fetal hemoglobins; that 

 is, the fetal hemoglobins of sheep, goat, and cow are less resistant to alkaline de- 

 naturation. 134, 669 The number and percentages of different types of hemoglobin in 

 a sample can often be determined from the rate of denaturation, since the decomposition 

 of each hemoglobin in a mixture proceeds according to first-order kinetics. 526 This 

 approach has been used in studying the hemoglobin of monkey, 1191 mouse, 1379 rat, 843 

 and sheep, 1338 as well as human hemoglobins. 202, 1385 



Crystallography and solubility 



The classical monograph of Reichart and Brown 1047 should be consulted for 

 illustrations of the various types of hemoglobin crystals that occur in different taxonomic 

 groups of mammals. Drabkin's method 286 ' 287 of crystallization of hemoglobin has 

 been used to obtain pure crystalline fractions of hemoglobins for analytical procedures, 

 on the assumption that crystalline hemoglobin was homogeneous. However, Allen 

 et al. 8 and Clegg and Schroeder 207 have shown by column chromatography that crystal- 

 line human hemoglobin is, indeed, heterogeneous. 



Crystallography has also been used to differentiate between fetal and adult 

 hemoglobins 670 ' 703 and more recently has been developed as an auxiliary method 

 for the identification of different types of murine hemoglobins (figure 43) in conjunction 

 with electrophoretic and solubility properties. 1015 These techniques have also been 

 used in studies on the mode of inheritance of some hemoglobins of the mouse, as is 

 mentioned later. 



Crystallographic and solubility studies are usually carried out in parallel; because 

 of the sensitivity of the hemoglobin molecule to physical variations, the methods used 

 separately may not be highly reliable. Determination of hemoglobin solubility is 

 based upon the determination of the amount of protein remaining in solution at a 

 series of salt concentrations. The results are influenced by the pH, ionic species, and 

 temperature of the solution and by the concentration and form of the protein. 212 

 The comparative solubilities of different hemoglobins are determined by establishing 

 salting-out curves at constant pH, temperature, and hemoglobin concentration. 261, 262 

 A number of hemoglobins have different solubilities under identical conditions and 

 may be distinguished on this basis. 



In choosing a system for salting-out, the following factors should be considered: 

 salts with a high buffering capacity are desirable, since hemoglobin solubility is greatly 

 affected by change of pH; buffers containing multivalent anions, that is, phosphate, 

 sulfate, and citrate, are more efficient for salting-out than those with univalent anions; 



