Separation of neutral proteins on ion-exchange resins

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Abstract
The effect of pH and cation concentration of buffer on the adsorption of cytochrome c on the ion-exchange resin, IRC-50, was studied in some detail. The behavior of the protein on the resin is markedly different from that of ampholytes of low molecular weight such as the basic amino acids. Lysine is desorbed as the pH falls below 5 owing to suppression of the ionization of the resin carboxyl groups, but the absorption of the protein is greatly increased under acidic conditions. It is suggested that this absorption is due to a large increase in secondary short-range forces. Some closely related neutral proteins were separated by elution chromatography on columns of IRC-50. Sheep fetal CO hemoglobin was separated from sheep maternal CO hemoglobin and from bovine CO hemoglobin and bovine CO hemoglobin from bovine methemoglobin. The separation of the hemoglobins is sharply dependent on the pH and sodium ion concentration of the eluting buffer. To obtain good yields, it is necessary to work at a temperature near 0[degree] and to use freshly prepared proteins. Tests carried out on bovine CO hemoglobin eluted from the column indicated that the protein was unaltered by passage through the column. The material from the column could be readily crystallized.