MAMMALIAN HEMOGLOBINS 301 



value not only in characterization of proteins, but also aids in choosing physical con- 

 ditions necessary for the isolation and purification of proteins. The properties of 

 proteins that enable them to be separated in an electrical field have been discussed. 4 

 When electrophoretic analyses indicate a single component in the system, the fraction 

 may nevertheless not be pure by other criteria. 



Moving-boundary electrophoresis. — The moving-boundary apparatus developed by 

 Tiselius 1318 has been modified many times to increase its resolving power and thereby 

 its usefulness as a tool for research. An important modification has been the develop- 

 ment of an apparatus with four electrophoretic cells that can be used simultaneously. 1010 

 The essential components of the instrument, the U-shaped electrophoretic cells contain- 

 ing two electrode compartments for application of the electric field 804 and an im- 

 proved optical system for hemoglobin 648 to permit localization and visualization of the 

 shape of the moving solutes, have been described in the articles cited. The methodology 

 for moving-boundary electrophoresis is thoroughly described by Longsworth. 803 



Electrophoretic mobility, as experimentally defined, represents the distance 

 traversed by the boundary or particle under observation per unit time per unit volt. 

 The electrophoretic mobility of mammalian hemoglobins is in the order of 10 -5 cm 2 / 

 sec/volt at pH 7.0. The migration of molecules is affected by the hydrogen-ion 

 concentration, ionic strength of the buffer, chemical composition of the buffer with 

 regard to valence of the ions and viscosity of the buffer, and the surface charge density 

 of the molecule under investigation. Thus, the physical conditions should always be 

 stated precisely. Ideally, as many different moving boundaries are formed as there 

 are different types of molecules in the solution. Observations on many proteins reveal 

 that interactions between different molecular species that affect their electrophoretic 

 mobilities are relatively rare. However, components in the buffer may interact with 

 proteins, such as hemoglobins and enzymes, producing a shift in their isoelectric 

 points. 994, 1195 



Buffers used for studies on hemoglobin by the moving-boundary technique are 

 sodium phosphate with dithionite, 0.1 ionic strength, pH 5.7-8.0, 994 and cacodylate- 

 NaCl, 0.1 ionic strength, pH 6.5. 994, 1211, 1381 More rapid separations can be achieved 

 by using buffers of lower ionic strength; Beaven et al. ei used sodium phosphate, ionic 

 strength 0.05, pH 8.0, and Itano and Robinson 649 used potassium phosphate, ionic 

 strength 0.01, pH. 6.85. The lower ionic strength is satisfactory for qualitative analyses, 

 but quantitative analyses may not be highly accurate under such conditions. Differences 

 in the moving-boundary electrophoretic patterns of murine carbonmonoxyhemo- 

 globins are illustrated in figure 40. 



Moving-boundary electrophoresis is not a practical method with which to survey 

 for differences among hemoglobins. The required instrument is expensive, only a 

 few samples can be analyzed in one day, large volumes of hemoglobin solutions are 

 needed, and considerable time is required to prepare the samples. Nevertheless, 

 moving-boundary electrophoresis, along with techniques of X-ray diffraction 997, " 8 

 and chemical analysis 1053, 1054 has been a valuable analytical tool for studying problems 



