Non-ionic adsorptive immobilization of proteins to palmityl-substituted Sepharose 4B.
1. Palmityl-substituted Sepharose 4B prepared by the glycidyl ether method of S. Hjertén et al. [J. Chromatogr. (1974) 101, 281-288] has been used as a non-ionic matrix for protein adsorption. A number of proteins, some of which are catalytically active, may be immobilized on this adsorbent in the form of suspension or column. 2. Of the proteins examined, bovine serum albumin, hemoglobin, myoglobin, lysozyme, glutamate dehydrogenase, and beta-galactosidase were immobilized on the adsorbent irrespective of NaCl concentration. Trypsin, alpha-chymotrypsin, papain, pepsin, and amyloglucosidase were totally adsorbed either in the absence of any additional salt or at high salt concentrations. Cytochrome c, used as a model protein, was totally immobilized only at high ionic strength and low pH. 3. Immobilization normally took place with an apparent increase in initial activity rates. In the case of trypsin using N alpha-benzoyl-DL-arginine p-nitroanilide as substrate, adsorption resulted in an increase in V (app). 4. Beef-liver glutamate dehydrogenase, used as a model allosteric enzyme, was found to retain its allosteric properties towards ADP and GTP after immobilization. 5. Results are discussed in terms of specific interactions involving a smaller number of binding sites in protein molecules as compared to the multiple attachment to highly substituted adsorbents prepared with shorter ligands. Retention of the essential properties of the proteins examined in this study are ascribed to these characteristics of the adsorbent and to its non-ionic nature. Relevance of these observations to in vivo processes and the potential use of the adsorbent for enzyme immobilization are also discussed.
D000327:Adsorption; D001665:Binding Sites; D002845:Chromatography / Q000379:methods; D004800:Enzymes, Immobilized; D005026:Ethylene Glycols; D006863:Hydrogen-Ion Concentration; D011134:Polysaccharides; D011506:Proteins / Q000302:isolation & purification*; D012492:Salts; D012685:Sepharose / Q000031:analogs & derivatives; D013696:Temperature
D004800:Enzymes, Immobilized; D005026:Ethylene Glycols; D011134:Polysaccharides; D011506:Proteins; D012492:Salts; C034543:palmityl-sepharose; D012685:Sepharose