Molecular imprinting of proteins and other macromolecules resulting in new adsorbents

Abstract

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.

Induction of catalytic activity in proteins by lyophilization in the presence of a transition state analogue

“…The fact that ␤-elimination is a unimolecular reaction also facilitated the kinetic analysis, and the comparison with the catalytic antibodies and the polymers. BSA was selected for initial experiments as it was available in large quantities, inexpensive, and was used extensively in previous studies (Braco et al, 1990;Dabulis and Klibanov, 1992).…”

“…It can be best illustrated by (bio)molecular imprinting, an approach which enables the introduction of new binding sites into protein molecules (Braco et al, 1990;Dabulis and Klibanov, 1992) and the alteration of an enzyme's catalytic properties such as activity (Mingarro et al, 1995), substrate specificity (Johnsson et al, 1995;O'Rich, and Dordick, 1997) or enantioselectivity (Stahl et al, 1990(Stahl et al, , 1991. However, if ligands can be employed to alter protein conformations in organic solvents in a predictable manner, it should be possible to use this approach for the induction of ''de novo'' catalytic activity in existing proteins via imprinting with transition state analogues (TSA) (Lerner et al, 1991).…”

Molecular Design of Synthetic Receptors with Dynamic, Imprinting, and Allosteric Functions

“…This situation has allowed us to design a D-glucose-selective QCM resonator by a simple imprinting method. 45 More recently, it was shown that the molecular imprinting is achieved when one utilizes the reprecipitation process of functionalized ''soluble'' polymers [46][47][48] and the gel formation process of low molecular-weight gelators. 49 These new trends suggest the generality of the molecular imprinting concept: that is, the formation of 3-D cross-linked resins is not the sole way toward the molecular imprinting.…”

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“…In the case of pre cip i ta tion or liophilization of a the pro tein from aque ous en vi ron ment in the pres ence of a tem plate mol e cule, the lat ter can fur ther dem onstrate pre dict able changes of its prop er ties in or ganic sol vents [146][147][148][149][150][151][152][153][154][155][156][157], and (un der con di tion of ad di tional sta bi li za tion) in aque ous so lu tions [158]. Syn thetic recep tor sites can be also formed us ing the method of molec u lar im print ing in cross-linked car bo hy dratesstarch [159] and am y lase [160,161].…”