Non‐ionic Adsorptive Immobilization of Proteins to Palmityl‐Substituted Sepharose 4B

Abstract

1. Palmityl-substituted Sepharose 4B prepared by the glycidyl ether method of S. Hjerten et al. [ J . Chromutogr. (1974) 101, 281 -2881 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 P-galactosidase were immobilized on the adsorbent irrespective of NaCl concentration. Trypsin, a-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 "-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.In recent years, much attention has been directed towards use of hydrophobic properties of proteins for their purification. In most cases, the proteins of interest are bound at high salt concentrations to agarose derivatives containing hydrophobic ligands, and are eluted at lower salt concentrations. Adsorption and desorption thus correspond to the general 'salting out' and 'salting in' properties of proteins [I]. In this so-called 'hydrophobic interaction chromatography' the degree of interaction depends not only on the ionic strength, but also on the relative hydrophobicities of the protein and the ligand, the nature of the salt in the medium, the ligand concentration, and the ambient temperature.

Adsorptive Immobilization of Acetylcholine Esterase on Octadecyl Substituted Porous Silica: Optical Bio-analysis of Carbaryl

“…Moreover, the support was found to be reusable after each run of continuous operation, through washing with organic solvents such as toluene or dioxane which eliminate adsorptive interactions between the enzyme and support thereby permitting the dissociation of the enzyme and preparing the support for re-loading by new enzyme. As reported earlier [28][29][30] relatively long alkyl chains (e.g. C18) on the surface of porous silica used in this study, ensure efficient hydrophobic interactions to take place, making the adsorption process virtually irreversible.…”

“…These kinds of strong associations are necessary to retain catalytic activity in continuous operations for the required length of time in optical bio-analysis of the carbaryl. Although warnings were made on the use of long alkyl chains for protein adsorption purposes, due to increasing the risk of denaturation 31,32 results presented here and during the last decades 28,29,33 clearly demonstrate that such process of adsorption is mild and functional properties of a protein are preserved upon adsorption and the product of adsorption is stable enough in continuous transformations or bioanalytical applications (Fig. 6).…”

Tailor-Made Enzyme Carriers: Preparation and Use of Adsorbents Specifically Designed to Immobilize Allosteric Enzymes in Activated Conformation

“…This enzyme which showed good affinities for binding to alkyl-substituted adsorbents was found to respond to all positive and negative allosteric modifiers tested, with comparable rates, in free and immobilized forms (Nemat-Gorgani and Karimian, 1982;1983;1984;Nemat-Gorgani et al, 1985). This observation suggested the possibility for immobilizing the enzyme in an allosterically-activated conformation and for design strategies regarding tailor-made enzyme carriers.…”

“…Our own previous studies involving use of hydrophobic supports indicated the possibility of protein adsorption with retention of native properties (NematGorgani and Karimian, 1982;1983;1984;NematGorgani et al, 1985). In such studies, bovine liver glutamate dehydrogenase was used as a model allosteric protein and the immobilized preparation was found to retain its response to all of the relevant allosteric modifiers tested, including L-leucine, in addition to retaining its usual catalytic activities.…”

“…X represents palmityl groups with or without the allosteric effectors which may be positioned on carbon 9 or 10 of the alkyl residues. Determination of the catalytic activity of GDH: Free GDH was assayed exactly as described previously (Nemat-Gorgani and Karimian, 1982). For determination of the activity of the immobilized enzyme, a procedure essentially as described previously (Azari and Nemat-Gorgni, 1999) was followed.…”