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....