MAMMALIAN HEMOGLOBINS 307 



phoresis affords, it might be useful for distinguishing hemoglobin from hemoglobin- 

 haptoglobin complexes through the use of the differential peroxidase activities that these 

 substances exhibit. 979, 1228 Application of starch-gel electrophoresis to the study of 

 hemoglobin differences in mammals appears in several reports. 49, 1016,1017,1072.1103 



4. Agar-gel electrophoresis: The use of agar gel in electrophoresis, like starch gel, 

 developed more slowly than paper, but agar is being used in place of starch by some 

 investigators because it makes excellent preparations for quantitative analyses. The 

 electrophoretic patterns in agar are very similar to those obtained by moving-boundary 

 electrophoresis, as 1 per cent agar gel is essentially an aqueous medium, yet agar gel 

 has sufficient structure to reduce free diffusion of macromolecules during and after 

 electrophoresis. Most agars commercially available should be purified 151 before use 

 for best reproducibility of results. 



The systems used for agar-gel and starch-gel electrophoresis are very similar with 

 one exception — liquid agar is usually layered about 3-6 mm. deep on photographic 

 plates rather than poured into plastic molds. Slits for application of filter papers 

 containing the samples are cut in the sheet of agar after it gels. Several slits can be 

 made in an agar plate and as many samples of hemoglobin can be analyzed simul- 

 taneously. The pH range in which agar gel can be used satisfactorily lies between 6 

 and 9, which is more restricted than that for starch-gel electrophoresis. Citric acid- 

 sodium citrate buffer, ionic strength 0.025, pH 6.5, 1064 and barbital buffer, ionic strength 

 0.025, pH 8.2, 471 are commonly used for hemoglobins. 



For quantitative determinations, hemoglobin can be eluted from agar gel after 

 freezing in a manner similar to that described for starch gels. The agar can also be 

 dried following electrophoresis and hemoglobin stained with a solution of Amido black 

 10B dissolved in a 0.1m sodium acetate-1.2M acetic acid buffer containing 15 per cent 

 glycerol, pH 3.5. 360 The stained, washed, and dried agar film can be separated from 

 the plate; the film is transparent and ideal for quantitative analyses in densitometric 

 devices. 



Techniques of immune electrophoresis 470 in agar gel have not been reported for 

 the study of hemoglobin, but such methods could be applied in the study of hemo- 

 globins. 



Column chromatography 



The theory of ion-exchange chromatography has been discussed by Boardman and 

 Partridge. 112 Pure ion-exchange adsorption is a function of the conditions of equili- 

 brium between (1) the protein and the buffer, (2) the protein and the resin, and (3) 

 the resin and the buffer. 



(1) Protein-NH 2 + H© ^ Protein-©NH 3 



(2) Protein-©NH 3 + Resin-COONa ^ Resin-COONH 3 -Protein + Na© 



(3) Resin-COONa + H© ^ Resin-COOH + Na© 



