Hydrophobic-Ionic Chromatography

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Abstract
1. All the porcine pancreas enzymes tested, regardless of their pI's were adsorbed on Amber-lite CG-50 (a weakly acidic cation exchange resin) at pH 4, where the ion-exchange group (carboxyl group) is not dissociated. The adsorption is hardly influenced by ionic strength. 2. At pH4, the adsorbed enzymes were partially eluted by organic solvents such as 50% propanol. 3. The adsorbed enzymes were effectively eluted by increasing the pH from 4 to 6. Trypsin (pI 10.5) was eluted before carboxypeptidase A (pI 4.5 and 5.3) with 0.5 m acetate buffer, whereas the former enzyme was eluted after the latter enzyme with 0.2 m 3,3-dimethyl glu-tarate buffer. However, with either buffer, the elution order of enzymes was not always the same as the order of their pI's. 4. By a single Amberlite CG-50 column chromatography of porcine pancreas extracts, kallikrein, carboxypeptidase B, deoxyribonuclease, carboxypeptidase A, and trypsin were purified 100-fold, 16-fold, 17-fold, 8-fold, and 32-fold, respectively. 5. The adsorption-elution mechanism of proteins on Amberlite CG-50 may be as follows. At pH≤4.5, proteins are adsorbed by hydrophobic affinity on loci of the resin, each of which includes at least one carboxyl group. As the pH is increased, the carboxyl groups dissociate, resulting in a decrease of the hydrophobicity and an increase of the negative charge of the loci; thus, the proteins are now retained by the remaining hydrophobic affinity plus the increased electrostatic affinity or minus the increased electrostatic repulsion.