Separation of neutral proteins on ion-exchange resins.

Abstract

The effect of pH of buffer on the adsorption of cytochrome C on the ion exchange resin IRC 50 has been studied in detail and the information gained has been used to predict conditions for the separation of neutral proteins such as the hemoglobins. The behavior of a protein on the resin is markedly different from that of ampholytes of low molecular weight such as the basic amino acids. The basic amino acids are desorbed as the pH falls below 5 due to suppression of the ionization of the resin carboxyl groups, but the adsorption of protein is greatly increased under acidic conditions. It is suggested that this is due to a large increase in secondary short-range forces. Sheep fetal CO hemoglobin has been separated from bovine CO hemoglobin and bovine CO hemoglobin from bovine methemoglobin. The eluting buffer was citrate of pH 5.81 and 5.92, respectively, and Na+ concentration 0.34 g. ions per liter. In initial experiments, heavy losses of the CO hemoglobins occurred which was most probably due, to a large extent, to the formation of methemoglobin. To obtain good yields, it is necessary to work at a temperature near 0°C. and to use freshly prepared proteins.