Protein chromatography on adsorbents with hydrophobic and ionic groups. Some properties of N-(3-carboxypropionyl)aminodecyl-sepharose and its interaction with wheat-germ aspartate transcarbamoylase
- 1 November 1975
- journal article
- Published by Portland Press Ltd. in Biochemical Journal
- Vol. 151 (2), 281-290
- https://doi.org/10.1042/bj1510281
Abstract
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 (21mumol/ml of settled gel; pKa=9.6) that were tentatively assigned to the coupling isourea group. 2. CPAD-Sepharose [N-(3-carboxypropionyl)aminodecyl-Sepharose] has anionic (carboxyl) groups (pKa=4.5) and cationic groups (pKa=9.6) in roughly equal concentrations (e coupling group. CPAD-Sepharose is slightly negatively charged at pH 7.0 and substantially negatively charged at pH 8.5. 3. The pKa values of dodecyl-Sepharose and CPAD-Sepharose are unaffected by a 100-fold increase in the concentration of KCl. 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 C10 chain but is relatively moderate compared with C10 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 CPAD-Sepharose. 7. These results are discussed in terms of a ‘repulsion-controlled’ model or hydrophobic chromatography.Keywords
This publication has 27 references indexed in Scilit:
- Purification of E. coli enzymes by chromatography on amphiphilic gelsFEBS Letters, 1975
- ι-Aminoalkylagaroses in the purification of L-histidinol-phosphate aminotransferaseBiochemistry, 1974
- Affinity chromatography of β-hydroxybutyrate dehydrogenase on NAD and hydrophobic chain derivatives of sepharoseBiochimica et Biophysica Acta (BBA) - Biomembranes, 1974
- The mode of adsorption of proteins to aliphatic and aromatic amines coupled to cyanogen bromide-activated agaroseBiochimica et Biophysica Acta (BBA) - General Subjects, 1974
- Multiple forms of phosphorylase kinase in red and white skeletal muscleFEBS Letters, 1974
- Hydrophobic chromatography in the resolution of the interconvertible forms of glycogen phosphorylaseFEBS Letters, 1974
- Protein binding by agarose carrying hydrophobic groups in conjunction with chargesBiochemical and Biophysical Research Communications, 1973
- Affinity chromatography: Large‐scale purification of the soluble oestradiol‐17‐β dehydrogenase of human placentaFEBS Letters, 1972
- Chromatography of Proteins on Dipolar Ion AdsorbantsNature, 1970
- Chemical Coupling of Peptides and Proteins to Polysaccharides by Means of Cyanogen HalidesNature, 1967