MAMMALIAN HEMOGLOBINS 303 



analysis could become a routine laboratory procedure. Zone electrophoresis may be 

 carried out using many types of supporting media. In reviewing zone electrophoresis, 

 the methods are classified by the kind of supporting medium that is employed. Theo- 

 retical aspects of electrophoresis discussed above are also applicable to zone electro- 

 phoresis. Comparative studies on the electrophoretic mobility of mammalian hemo- 

 globins are commonly carried out with zone electrophoresis, but exact determinations 

 of electrophoretic mobilities are best determined by the moving-boundary techniques. 

 Frequently the latter method also- offers higher resolution. Because of its simplicity, 

 zone electrophoresis is generally preferred as a system when surveying populations of 

 animals for hemoglobin differences. 



1. Paper electrophoresis: Methods using paper as a supporting medium, first 

 described by Wieland and Fischer 1387 and Haugaard and Kroner, 525 developed 

 more rapidly than other methods of zone electrophoresis. Designs of several types of 

 equipment have been illustrated elsewhere 111 and the methodology has been outlined 

 very well. 203, 648 ' 76 ° 



For optimal results and reproducibility, the factors that influence the migration of 

 hemoglobin during electrophoresis should be constant throughout the paper, that is, 

 the paper porosity must be uniform and vapor pressure, quantity of hemoglobin 

 applied, electrical current, temperature, and electroosmosis should be constant. 1337 

 The filter papers commonly used as supporting media are Whatman no. 1 , 3 cm. wide, 

 3MM and 531 or Schleicher and Schuell 2043 A for hanging strip methods, and 

 Whatman 3MM, 10 to 20 cm. wide for the sandwich technique. More hemoglobin 

 can be applied to thicker papers and the presence of minor components can therefore 

 be detected more readily. 50 Moreover, thicker paper gives better reproducibility 

 among runs, but thinner paper is more often used when quantitative determinations 

 with recording instruments are to follow. Vapor-pressure equilibrium should be 

 reached before electrophoresis is begun, the case used for hanging strips should be 

 air tight to maintain the vapor pressure, and the glass plates used for the sandwich 

 technique should be sealed with silicone. Simpler instruments without cooling devices 

 may overheat at high currents, and the resulting evaporation of buffers from the 

 paper may produce distortions of electrophoretic patterns and inconsistent results. 

 The addition of inert substances, such as glycerol, 822 inhibits evaporation of buffer 

 from the paper. Pretreatment of the paper with the buffer may possibly reduce the 

 adsorption of proteins and reduce electroosmosis by neutralizing the negatively charged 

 carboxyl ions of the cellulose paper. The buffers commonly used for paper electro- 

 phoresis of hemoglobins are barbital, pH 8.6 and ionic strength of 0.025-0.06, and 

 phosphate, pH 6.5 and ionic strength of 0.1. 446 



After electrophoresis, the proteins are fixed to the paper by heat denaturation at 

 1 10-120° C. for 15-30 minutes. For quantitative analysis, the temperature and period 

 of time for heat denaturation should be controlled, since the capacity of proteins to bind 

 dyes is affected by different conditions of heat denaturation. 111 The hemoglobin on 

 the paper strips can be stained by one of the following methods: bromophenol blue, 311 



