When a protein is dissolved in a concentrated aqueous solution of a multifunctional organic compound, freeze-dried, and washed with an anhydrous organic solvent to remove the ligand, the resultant "imprinted" protein preparation binds up to 30-fold more of the template compound in anhydrous solvents than the nonimprinted protein in the same solvent (and both proteins in water). These artificial receptors exhibit marked ligand selectivity as well as stability in anhydrous media. This phenomenon of molecular imprinting, demonstrated for several unrelated proteins and ligands, may be helpful in the development of unique bioadsorbents and, potentially, new biocatalysts.Recent years have witnessed a surge of exciting research involving the creation of abiotic receptors for small molecules (1-6). Such artificial hosts reveal the fundamental principles of structural recognition and may be used as selective adsorbents. In addition, ligand-receptor affinity may lend itself to the induction of enzyme-like activity, as illustrated by studies on cyclodextrins (7,8) and catalytic antibodies (9,10).In addition to functioning in their natural aqueous reaction media, enzymes have recently been determined to be catalytically active in anhydrous organic solvents (11). A profound feature of enzymes in an anhydrous milieu is their high conformational rigidity, manifested in such acquired characteristics as greatly enhanced thermostability (12, 13) and ligand "memory" (14). In the present study, we have used the phenomenon of drastically lowered protein flexibility in anhydrous media (compared with water) to transform common proteins into selective artificial receptors in organic solvents. MATERIALS AND METHODSBovine serum albumin (essentially fatty acid-free), bovine erythrocyte hemoglobin (type II), chicken egg white lysozyme (EC 3.2.1.17, grade I), and poly(L-lysine) (molecular mass of -60 kDa) were obtained from Sigma. p-Hydroxybenzoic acid (Aldrich) was of 99+% purity; all other chemicals used in this study were purchased from commercial suppliers and were of analytical grade or purer.All organic solvents employed in this work were of analytical grade and were dried before use by shaking with 3-A molecolar sieves (Linde). The term anhydrous herein means that no water was detected in the solvent by the Fischer titration (15)-i.e., that the water content was below -0.01%. p-Hydroxybenzoic acid and its analogs were assayed by HPLC. Ten-microliter aliquots of ligand solutions in diisopropyl ether or another organic solvent were subjected to liquid chromatography on a Waters ,uBondapak C18 column with a mobile phase consisting of 20% acetonitrile/80% aqueous phosphate buffer (pH 2.6). The flow rate was 1.5 ml/min, and the UV absorbance of the ligands was monitored at 254 nm.L-Tartaric acid and its analogs dissolved in ethyl acetate were assayed by gas chromatography following the general method of Philip and Nelson (16). To 25 gl of a ligand in ethyl acetate was added an equal volume of bis(trimethylsilyl)trifluoroacetamide....

When the model protein bovine serum albumin (BSA) was dissolved in a concentrated aqueous solution of the multifunctional ligand L-malic acid, the solution was lyophilized, and the solid residue thoroughly washed with tetrahydrofuran to extract malic acid, then the resultant ("imprinted") protein was capable of binding 26.4 +/-0.9 mol equivalents of the ligand in anhydrous ethyl acetate. The nonimprinted BSA (i.e., that prepared in the same manner apart from the absence of malic acid) bound less then one-tenth of that amount under identical conditions. Furthermore, both imprinted and nonimprinted BSA exhibited little binding of L-malic acid in water. The imprinted BSA retained its "memory" for the ligand in ethyl acetate even after a prolonged incubation under vacuum; dissolution in water, however, eliminated the imprinted protein's binding capacity. The BSA imprinted with L-malic acid displayed affinity for this ligand not only in ethyl acetate but also in many other anhydrous solvents. It was found that the higher the solvent's propensity to form hydrogen bonds, the lower the protein-ligand binding in it, thus pointing to hydrogen bonds as the driving force of this binding. Studies with completely or partially cleaved BSA, with other globular proteins, glutathione, and poly(L-aspartic acid) revealed that the critical requirement for the imprintability is the presence of a sufficiently long polymeric chain. Moreover, many hydrogen-bond-forming macromolecules other than proteins, such as dextrans and their derivatives, partially hydrolyzed starch, and poly(methacrylic acid), also could be imprinted for subsequent binding in ethyl acetate. The mechanism of imprinting and binding inferred from these experiments involves a multipoint hydrogen bonding in water of each ligand molecule with two or more sites on the polymeric chain, thereby folding a segment of the latter into a cavity around the ligand; following lyophilization and extraction of the ligand, the cavities remain in organic solvents (but not in water) and give rise to ligand binding. This conclusion is supported by the results of binding of numerous malic acid analogs and related ligands to BSA imprinted with L-malic acid. Finally, BSA imprinted with malic acid was used as a selective adsorbent for a chromatographic separation of an equimolar mixture of maleic and acrylic acids in ethyl acetate.