Chapter 20
Enzyme Immobilization on Polymerizable Phospholipid Assemblies 1
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Alok Singh , Michael A. Markowitz , Li-I Tsao , and Jeffrey Deschamps 1
Center for Bio/Molecular Science and Engineering, Code 6900, Naval Research Laboratory, Washington, DC 20375-5348 Department of Biochemistry, Georgetown University, Washington, DC 20040 Laboratory for Structure of Matters, Code 6030, Naval Research Laboratory, Washington, DC 20375 2
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Two phospholipids, one saturated and one polymerizable, containing iminodiacetic acid functionality linked to their phosphate headgroup are synthesized to build metal chelated lipid assemblies. These lipids were non-ideally miscible with analogous phosphatidylcholine as determined by monolayer studies at air/water interface. The metal chelating lipids produced vesicles when mixed with polymerizable phosphatidylcholine. These vesicles upon binding with Cu ions were used for binding enzyme - bovine carbonic anhydrase II (EC 4.2.1.1) - utilizing their affinity towards histidine present on the surface of carbonic anhydrase. The catalytic activity of surface-bound protein molecule on polymerized and nonpolymerized vesicles was measured. Only the polymerized vesicles showed improved stability and sustained activity of enzymes. 2+
The versatility of phospholipids in biological membranes has inspired researchers to explore the potential of synthetic phospholipids to build functionalized self-organized microstructures (7). Phospholipids provide a modular approach to rationally build mono- and bimolecular assemblies and to control the density of reactive functionalities accessible from the dispersion medium. The nature and number of these sites on membrane surface influence the interactions of organic, inorganic, or biological molecules at membrane interface (2,3). The formation of supramolecular assemblies from synthetic materials and their use in diverse application areas including molecular recognition, biosensors, and controlled release technology have been the area of active research (i,4-7). Polymerizable phospholipids, on the other hand, have been used in the stabilization of molecular assemblies and in the development of strategies to expand 0097-6156/94/0556-0252$08.00/0 © 1994 American Chemical Society
Usmani and Akmal; Diagnostic Biosensor Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1994.
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SINGH ET AL.
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Polymerizable Phospholipid Assemblies
the usefulness of the lipid assemblies (8,9). These strategies include the altering of surface properties of polymer films (10) and construction of polymerized lipidmembrane surface equipped with reactive sites suitable for chemical interactions (UJ2). Spontaneous formation of organized structures from lipids combined with the preservation of morphologies and functional features of the supramolecular assemblies by polymerization can lead to the development of a hybrid approach to recognizing and immobilizing macromolecules on their surface. Instead of depending on covalent bonds for immobilization, the present work relies on the hydrogen bonds or electrostatic forces in the immobilization scheme. The idea is to immobilize a macromolecule by coordinating its binding sites to a metal ion which is already complexed with ligand. This could be accomplished by incorporating a metal-chelating phospholipid in the membrane formed from phosphatidylcholine. We have focussed on iminodiacetic acid (IDA) moiety as chelating site on phospholipid headgroup because of its universal use in affinity chromatography for the separation and purification of proteins (13). The binding constant of Cu -iminodiacetate with imidazole in aqueous solutions are quite high, l O ^ M " (14). In principle, comparable binding constants can be expected for the IDA linked to phospholipid headgroups. A two fold increase in binding constants due to the presence of two histidines may provide a ground for preference in binding of macromolecules based on the number of histidines available for making complex with iminodiacetate. Thus, proteins or macromolecules can be effectively immobilized on a vesicle surface consisting of metal ions ligated to IDA functionality. Randomly distributed IDA-phospholipids in lipid membranes offer such an opportunity for binding with proteins containing multiple histidine sites. Utilizing the affinity of Cu-IDA complex with substituted bis-imidazole, Arnold and coworkers (15,16) have demonstrated specificity for bis-imidazole by matching the spatial distribution of surface coordinating group in bulk polymers with iminodiacete. In other reports, materials prepared by following similar approach have shown promise as selective supports for chromatographic separations of proteins (17) and as antibody mimics for radioassay of small molecules (18). The concept of preorganization of the lipid monomers to produce a polymer network retaining vesicular shape has been demonstrated previously by polymerizing monomer counterions present on vesicle surface (19,20). We planned to extend the utility of polymerizable lipid vesicles to protein immobilization and molecular recognition schemes by taking advantage of Cuiminodiacetate affinity towards histidines. The chemical structure of the lipids used in this work is shown in figure - 1. Vesicles are made by mixing polymerizable lipid 1,2 bis (tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (1, D C P C ) with nonpolymerizable 1,2 dipalmitoyl-sn-glycero-3-phospho-(N,N-bis carboxymethyl)-2aminoethanol (2a, DPPIDA) or polymerizable 1,2 bis (tricosa-10,12-diynoyl)-snglycero-3-phospho-(N,N-bis carboxymethyl)-2-aminoethanol (2b, D C P I D A ) . The first step involves the demonstration of the proof of principle that enzyme can be immobilized on the surface of vesicles and can maintain its catalytic activity. The n
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DIAGNOSTIC BIOSENSOR POLYMERS
binding of an enzyme to the vesicles involving more than one available binding sites will require a lipid system which facilitates the lateral mobility of lipids in the assemblies (21). The next step involves the selection of an enzyme which consist several surface-exposed histidine residues. Bovine Carbonic anhydrase Π (EC 4.2.1.1) was found to be an ideal choice, since it contains six histidine residues, four of them are available within a distance of 6 A . The four available sites may improve binding affinity by about four fold to the metal chelating sites on vesicles surface. Once the binding is achieved, the bilayers can be fixed in its morphology by cross-linking through photo-polymerization. This study includes two types of mixed lipid systems; a) non-polymerizable IDA lipid in polymerized vesicles (1 mixed with 2a) and b) polymerizable I D A lipid (2b) copolymerized with 1 in vesicles. Enzyme immobilization was carried out on copper chelated vesicles before and after polymerization following a protocol shown in Scheme-1. The binding scheme provides a straightforward route to enzyme immobilization without involving enzyme in chemical reactions. Enzyme immobilization on surfaces usually results in substantial loss of their activity (22,23).
CH OC(0)-R 2
I CHOC(0)-R Ο
I
I II CH OP-(OCH -CH )-X
I
2
2
2
o. Lipid 1 2a 2b
R
X
-(CH ) -OC-C
