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.

Effects of salts on protein–surface interactions: applications for column chromatography

“…The nonspecific salt effects are simply due to their ionic properties. At any salt concentrations, salt ions provide either charge shielding/electrical double layer,38 often referred to as Debye length1,7 or stoichiometric ion binding8,9 on the charged protein and column surface. Almost all the chromatography uses some level of salt concentration and hence will have charge shielding effects.…”

“…Nonspecific salt effects play a major role in ion exchange chromatography (IEC), based on either charge shielding or stoichiometric ion binding for modulating protein binding and elution 1,7–9. These salt effects are independent of the type of the salts.…”

“…The nonspecific salt effects are simply due to their ionic properties 1,5,6. At any salt concentrations, salt ions provide charge shielding/electrical double layer1,7 or stoichiometric ion binding8,9 on the charged proteins and column surface. These salt effects are independent of the type of the salts.…”

Proteomics approach to investigate separation behavior of proteins in ion-exchange chromatography

“…The stoichiometric displacement model (SDM) is the earliest model to depict protein binding to ion-exchangers (Boardman and Partridge, 1955;Roth et al, 1996). The fundamental idea of SDM is that when adsorption happens, a protein molecular displaces ions stoichiometrically.…”

Charged fusions for enhanced protein purification and immobilization by ion-exchange

“…Can the extent of electrostatic interaction of each fusion with the ionexchange groups on the stationary phase be quantified using existing models? The stoichiometric displacement model (Boardman and . Partridge, 1955) was used to characterize the protein retention.…”