MAMMALIAN HEMOGLOBINS 309 



termined, 112 adsorption of hemoglobin below pH 7 is not due solely to electrostatic 

 forces but is influenced by hydrogen bonding between the undissociated carboxyl 

 groups of the resin and polar groups of the protein; that is, the positive charge of 

 hemoglobin realized above pH 7 becomes less important below pH 7 for specific 

 adsorption on the resin. The influence of secondary forces, such as hydrogen bonding, 

 at low pH may be helpful in aiding separation of components chromatographically 

 similar at neutral pH. As indicated by the formulae, adsorption can be reduced by 

 increasing the sodium ion concentration of the buffer. The salt concentration is gener- 

 ally increased stepwise as the fractions separate, such that elution is accomplished 

 more rapidly and with smaller volumes of effluent. The elution rate used by most 

 investigators is 4-6 ml./hr. for columns about 1 cm. in diameter. Specific conditions 

 of buffers, pH, ionic strength, and flow rate for isolation of different components of 

 human hemoglobins can be found in various articles. 8 - 207 Column chromatography 

 has also been used as a preparative method to purify hemoglobin fractions used for 

 further chemical analyses 8 ' 616 ' 1395 and to detect and isolate hybrid hemoglobin 

 molecules formed during hybridization. 1345 Column chromatography through 

 molecular sieves, such as Sephadox, is being adapted for the separation and analysis 

 of peptides of enzymatic digests. 578 



C Utracentrifugation 



The ultracentrifuge is used to enhance the migration of molecules through a solution 

 by increasing the intensity of the field of force. The sedimentation of particles in the 

 ultracentrifuge depends upon volume, shape, and density of the particle; viscosity and 

 density of the medium; and the intensity of the gravitational field. The sedimentation 

 rate, described in terms of the sedimentation constant, s, can be used to calculate 

 molecular weight by application of the formulae derived by Svedberg. 1304 The 

 molecular weights of hemoglobins (approximately 68,000) determined by this method 

 agree with those obtained by diffusion methods. 512 A description of the apparatus 

 and methodology has been presented by Pickels, 1005 and a monograph on ultra- 

 centrifugation has recently been published by Schachman. 1159 



In general, the ultracentrifuge is not useful for distinguishing between mammalian 

 hemoglobin variants since their molecular weights are usually near 68,000. A determi- 

 nation of the molecular weight of a molecule is necessary for interpretation of some 

 chemical analyses, such as amino-acid composition, peptide analysis, sulphydryl 

 analyses, or end group analyses. Popp and St. Amand 1017 and Gluecksohn-Waelsch 442 

 have reported that the diffuse-type hemoglobin of the mouse contains a component 

 of heavy molecular weight (figure 42), but additional investigation is required to ascer- 

 tain whether the heavy hemoglobin is synthesized as such or is formed in vivo by dimeri- 

 zation, including disulfide bonding. In support of the possibility that the component 

 of heavy molecular weight of certain mouse hemoglobins may be formed secondarily 

 by chemical bonding after synthesis, Gluecksohn-Waelsch 442 found that increased quanti- 

 ties of a heavy molecular component were formed during storage at 4° C, although this 



