IX. GENETICS AND HUMAN HEMOGLOBIN CHEMISTRY 407 



identical in all hemoglobins (protoporphyrin IX + Fe), while tlie globin 

 varies considerably in physicochemical properties and in amino acid 

 composition in different species. In many cases more than one hemo- 

 globin has been demonstrated in individuals of the same species by the 

 electrophoretic and chromatographic methods devised for protein separa- 

 tion. That heterogeneity of hemoglobin arises as a consequence of the 

 oxidation or reduction of the heme, can usually be excluded by converting 

 the heme to the carbomonoxy or cyanmet derivative, both of which have 

 the same electrophoretic charge as oxyhemoglobin. Consequently, the 

 hemoglobin found in individuals and in species with multiple hemoglobins 

 differ somewhat in the protein part of the molecule. 



Pauling et al. discovered in 1949 the difference in electrophoretic 

 mobility between normal human hemoglobin and hemoglobin obtained 

 from sickle-cell anemic patients. This observation has been of funda- 

 mental importance in the development of the concept of gene-protein 

 relationship and has stimulated a number of workers to investigate 

 human hemoglobins. Several abnormal hemoglobins have been discovered 

 by the same method of electrophoretic analysis. The original Tiselius 

 moving boundary electrophoresis technique was found, however, to be 

 an expensive and relatively impractical method, because of the amount 

 of work involved in a single determination and because of the expensive 

 equipment. Simpler methods of analysis (such as paper or starch-gel 

 electrophoresis) have been developed for the electrophoretic analysis of 

 the human hemoglobins. These methods have been applied to the study 

 of ''normal" human hemoglobin and of the human hemoglobin variants. 



"Normal" human hemoglobin is not homogeneous in free boundary 

 electrophoresis (Hoch, 1950). Kunkel and Wallenius (1955) isolated a 

 major and two minor components by using starch block electrophoresis. 

 This method of electrophoretic analysis allows the elution from the 

 starch slab of the separated hemoglobins and the quantitative evalua- 

 tion of each component. 



The more sensitive starch-gel electrophoresis (Smithies, 1959) has 

 now become more widely used in order to reveal minor components; the 

 buffer system described by Poulik (1957) exhibits considerable sharpen- 

 ing of the bands and is greatly advantageous. The sliced starch-gel is 

 stained with the benzidine or with the o-dianisidine reagent (Smithies. 

 1959), which are catalytically oxidized by hemoproteins in the presence 

 of hydrogen peroxide. The sensitivity of the method is considerably 

 enhanced and components present in quantities of less than 1% of the 

 total hemoglobin can be detected. The use of filter paper electrophoresis 

 is confined to the screening of major abnormal components. Adsorption 



