Britain Protein Chromatography on Adsorbents with Hydrophobic and Ionic Groups SOME PROPERTIES OF N - ( 3 - CARBOXYPROPIONYL ) AMINODECYL - SEPHAROSE

1. The charge state of two derivatives of Sepharose prepared by the CNBr activation method were studied by acid-base titration and by ion-exchange chromatography. Dodecyl-Sepharose exhibited cationic groups (21 pmol/ml of settled gel; pKa = 9.6) that were tentatively assigned to the coupling isourea group. 2. CPAD-Sepharose (N-(3carboxypropionyl)aminodecyl-Sepharose] has anionic (carboxyl) groups (pKa = 4.5) and cationic groups (pKa = 9.6) in roughly equal concentrations (13-15pmol/ml of settled gel); the cationic group was again tentatively assigned to the coupling group. CPAD-Sepharose is slightly negatively charged at pH7.0 and substantially negatively charged at pH8.5. 3. The pKa values ofdodecyl-Sepharose and CPAD-Sepharose are unaffected by a 100-fold increase in the concentration of KCI. 4. CPAD-Sepharose has considerable affinity for wheat-germ aspartate transcarbamoylase at pH 8.5 when the adsorbent and enzyme are both negatively charged. The interaction involves the C1O chain but is relatively moderate compared with CI0 chains associated only with positive charge. 5. Desorption of the enzyme adsorbed to CPAD-Sepharose can be achieved by raising the pH to increase the electrostatic repulsion, or by introducing the detergent sodium deoxycholate. Acetone and butan-1-ol also weaken the adsorption at pH 8.5. 6. High concentrations of sodium acetate or sodium phosphate induced the enzyme to bind more tightly to CPADSepharose. 7. These results are discussed in terms of a 'repulsion-controlled' model of hydrophobic chromatography.