Microgels and nanogels with catalytic activity.

Molecular imprinting has grown considerably over the last decade with more and more applications being developed. The use of this approach for the generation of enzyme-mimics is here reviewed with a particular focus on the most recent achievements using different polymer formats such as microgels and nanogels, beads, membranes and also silica nanoparticles.

Synthesis of 1,8-naphthalimide-based probes with fluorescent switch triggered by flufenamic acid.

In this work, we report the synthesis of novel fluorescent molecules, based on 1,8-naphthalimide thio- and amino-derivatives, designed to display an OFF-ON and ON-OFF photoelectron transfer fluorescence switch upon interaction with a proton-donor drug. We have functionalized the new probes to allow easy formation of a covalent link to polymer matrices, for future applications as drug delivery sensors. We have investigated the fluorescent switch of the new probes using flufenamic acid (FA, pKa 3.65), a water insoluble, non-steroidal anti-inflammatory drug, as a model drug and proton source. The rapid interaction of the new probes with FA resulted in fluorescence enhancement or decrease and a large Stokes shift, all of which allowed the detection of the drug in the nanomolar range. The new 1,8-naphthalimide fluorescent dyes reported in this work are interesting tools for the detection and quantification of acidic drugs and biomolecules.

Fluorescent polymeric nanovehicles for neural stem cell modulation.

Nanomaterials are emerging as strong candidates for applications in drug delivery and offer an alternative platform to modulate the differentiation and activity of neural stem cells. Herein we report the synthesis and characterization of two different classes of polymeric nanoparticles: N-isopropylacrylamide-based thermoresponsive nanogels RM1 and P(TEGA)-b-P(d,lLA)2 nano-micelles RM2. We covalently linked the nanoparticles with fluorescent tags and demonstrate their ability to be internalized and tracked in neural stem cells from the postnatal subventricular zone, without affecting their proliferation, multipotency and differentiation characteristics up to 150 µg ml-1. The difference in chemical structure of RM1 and RM2 does not appear to impact toxicity however it influences the loading capacity. Nanogels RM1 loaded with retinoic acid improve solubility of the drug which is released at 37 °C, resulting in an increase in the number of neurons, comparable to what can be obtained with a solution of the free drug solubilised with a small percentage of DMSO.

Biochemical and X-ray crystallographic studies on shikimate kinase: the important structural role of the P-loop lysine.

Shikimate kinase, despite low sequence identity, has been shown to be structurally a member of the nucleoside monophosphate (NMP) kinase family, which includes adenylate kinase. In this paper we have explored the roles of residues in the P-loop of shikimate kinase, which forms the binding site for nucleotides and is one of the most conserved structural features in proteins. In common with many members of the P-loop family, shikimate kinase contains a cysteine residue 2 amino acids upstream of the essential lysine residue; the side chains of these residues are shown to form an ion pair. The C13S mutant of shikimate kinase was found to be enzymatically active, whereas the K15M mutant was inactive. However, the latter mutant had both increased thermostability and affinity for ATP when compared to the wild-type enzyme. The structure of the K15M mutant protein has been determined at 1.8 A, and shows that the organization of the P-loop and flanking regions is heavily disturbed. This indicates that, besides its role in catalysis, the P-loop lysine also has an important structural role. The structure of the K15M mutant also reveals that the formation of an additional arginine/aspartate ion pair is the most likely reason for its increased thermostability. From studies of ligand binding it appears that, like adenylate kinase, shikimate kinase binds substrates randomly and in a synergistic fashion, indicating that the two enzymes have similar catalytic mechanisms.

Critical evaluation of the applicability of capillary zone electrophoresis for the study of hapten-antibody complex formation.

The applicability of capillary zone electrophoresis (CZE) for the determination of association constants of an hapten-antibody complex with values as high as 10(7) (mol/I)-1 was investigated (dissociation constant in the nmol range). As a reference method the well known, enzyme-linked immunosorbent assay (ELISA) was selected. The study describes the optimisation of the experimental conditions of the CZE technique. The CZE measurements were optimised according to an experimental design. The results of the CZE and ELISA methods are compared giving consideration to the reproducibility (repeatability) of the two methods.

Regioselectivity and enantioselectivity in an antibody catalyzed hetero Diels-Alder reaction.

The Diels-Alder cycloadditions of trans- and cis-piperylene (1 and 2) to 4-nitroso-N-propylbenzamide (3) were selected as target reactions for the development of catalytic antibodies with regioselective and enantioselective properties (Meekel, A. A. P. Ph.D. Thesis, University of Amsterdam, 1995). The bicyclic systems 10a-c were designed as transition state analogues and employed for the immunization of mice and the generation of monoclonal antibodies. Three of the antibodies, each obtained from immunization with a different hapten, were selected for further characterization of their catalytic activities. Among these, antibody 309-1G7, raised against the protein conjugate of 10c, showed the best rate enhancement (kcat/ kuncat = 2618) in the reaction of cis-piperylene (2) with nitroso dienophile 3. Data obtained from regioselectivity and enantioselectivity analyses demonstrated that antibody 309-1G7 favors the formation of the targeted regioisomer (> 95%), with an ee of 82%.

Kinetic and titration methods for determination of active site contents of enzyme and catalytic antibody preparations.

Kinetic characterization of enzymes and analogous catalysts such as catalytic antibodies requires knowledge of the molarity of functional sites. Various stoichiometric titration methods are available for the determination of active-site concentrations of some enzymes and these are exemplified in the second part of this article. Most of these are not general in that they require the existence of certain types of either intermediate or active-site residues that are susceptible to specific covalent modification. Thus they are not readily applicable to many enzymes and they are rarely available currently for titration of catalytic antibody active sites. In the first part of the article we discuss a general kinetic method for the investigation of active-site availability in preparations of macromolecular catalysts. The method involves steady-state kinetics to provide Vmax and Km and single-turnover first-order kinetics using excess of catalyst over substrate to provide the analogous parameters k(obs)lim and K(m)app. The active-site contents of preparations that contain only active catalyst (Ea) and inert material (Ei) may be calculated as [Ea](T) = Vmax)/k(obs)lim. This is true even if nonproductive binding to E(a) occurs. For polyclonal catalytic antibody preparations, which may contain binding but noncatalytic material (Eb) in addition to Ea and Ei, the significance of Vmax/k(obs)lim is more complex but provides an upper limit to E(a). This can be refined by consideration of the relative values of Km and the equilibrium dissociation constant of EbS. Analysis of the Ea, Eb, Ei system requires the separate determination of Ei. For catalytic antibodies this may be achieved by analytical affinity chromatography using an immobilized hapten or hapten analog and an ELISA procedure to ensure the clean separation of Ei from the Ea + Eb mixture.

The kinetic basis of a general method for the investigation of active site content of enzymes and catalytic antibodies: first-order behaviour under single-turnover and cycling conditions.

The theoretical foundation has been laid for the investigation of catalytic systems using first-order kinetics and for a general kinetic method of investigation of the active site content, E(a), of enzymes, catalytic antibodies, and other enzyme-like catalysts. The method involves a combination of steady-state and single-turnover kinetics to provide Vmax and Km and k(lim)(obs) and K(app)(m), respectively. The validity of the method is shown to remain valid for two extensions of the simple two-step enzyme catalysis model (a) when the catalyst preparation contains molecules (Eb) that bind substrate but fail to catalyse product formation and (b) when the catalyst itself binds substrate non-productively as well as productively. The former is a particularly serious complication for polyclonal catalytic antibodies and the latter a potential complication for all catalysts. For the simple model and for (b) Vmax/k(lim)(obs) provides the value of [Ea]T and for (a) its upper limit. This can be refined by consideration of the relative values of Km and the equilibrium dissociation constant of EbS. For the polyclonal catalytic antibody preparation investigated, the fact that K(app/m) > Km demonstrates for the first time the presence of a substrate-binding but non-catalytic component in a polyclonal preparation. First-order behaviour in catalytic systems occurs not only with a large excess of catalyst over substrate but also with lower catalyst/substrate ratios, including the equimolar condition, when K(app)(m) >> [S]0, a phenomenon that is not widely appreciated.

A competitive immunoassay for the detection of esterolytic activity of antibodies and enzymes.

Screening of a large number of clones produced in a fusion is often the bottleneck in the isolation of catalytic antibodies. The usual approach requires two steps: clones are first selected for their high affinity to the antigen, and then the good binders are tested for their catalytic activity. To simplify this selection process, a competitive enzyme-linked immunosorbent assay (ELISA) has been developed that allows direct screening of the antibodies on the basis of their catalytic activity. In this assay, the product of the catalyzed reaction binds to an immobilized anti-product antibody in competition with a peroxidase-product conjugate. The screening assay has been developed for the antibody-catalyzed hydrolysis of esters of p-aminophenylacetic acid and has been tested on the porcine liver esterase (PLE)-catalyzed hydrolysis of the same substrates. This test allows the detection of product formation at the nanomolar level, while, in a typical assay, the catalytic activity of PLE can be traced down to 200 fmol of enzyme. Under standard conditions for the screening of hybridomas obtained from a fusion, the competitive ELISA allows detection of catalytic species with values of kcat > or = 5 x 10(-7) mol l-1 s-1 and kcat/kuncat > or = 50. While the assay has been designed for the selection of catalytic antibodies, other potential applications of this methodology are in the screening of libraries of engineered and designed enzymes and, in general, in the quantitative measurement of enzyme activity.

A general kinetic approach to investigation of active-site availability in macromolecular catalysts.

A potentially general kinetic method for the investigation of active-site availability in preparations of macromolecular catalysts was developed. Three kinetic models were considered: (a) the conventional two-step model of enzyme catalysis, where the preparation contains only active catalyst (E(a)) and inert (i.e. non-binding, non-catalytic) material (E(i)); (b) an extension of the conventional model (a) involving only E(a) and E(i), but with non-productive binding to E(a) (in addition to productive binding); (c) a model in which the preparation contains also binding but non-catalytic material (E(b)), predicted to be present in polyclonal catalytic antibody preparations. The method involves comparing the parameters V(max) and K(m) obtained under catalytic conditions where substrate concentrations greatly exceed catalyst concentration with those (klim/obs, the limiting value of the first-order rate constant, k(obs), at saturating concentrations of catalyst; and Kapp/m) for single-turnover kinetics, in which the reverse situation obtains. The active-site contents of systems that adhere to model (a) or extensions that also lack E(b), such as the non-productive binding model (b), may be calculated using [E(a)](T)=V(max)/klim/obs. This was validated by showing that, for alpha-chymotrypsin, identical values of [E(a)](T) were obtained by the kinetic method using Suc-Ala-Ala-Pro-Phe-4-nitroanilide as substrate and the well-known 'all-or-none' spectroscopic assay using N-trans-cinnamoylimidazole as titrant. For systems that contain E(b), such as polyclonal catalytic antibody preparations, V(max)/klim/obs is more complex, but provides an upper limit to [E(a)](T). Use of the kinetic method to investigate PCA 271-22, a polyclonal catalytic antibody preparation obtained from the antiserum of sheep 271 in week 22 of the immunization protocol, established that [E(a)](T) is less than approx. 8% of [IgG], and probably less than approx. 1% of [IgG].

Characterization of the hydrolytic activity of a polyclonal catalytic antibody preparation by pH-dependence and chemical modification studies: evidence for the involvement of Tyr and Arg side chains as hydrogen-bond donors.

The hydrolyses of 4-nitrophenyl 4'-(3-aza-2-oxoheptyl)phenyl carbonate and of a new, more soluble, substrate, 4-nitrophenyl 4'-(3-aza-7-hydroxy-2-oxoheptyl)phenyl carbonate, each catalysed by a polyclonal antibody preparation elicited in a sheep by use of an analogous phosphate immunogen, were shown to adhere closely to the Michaelis-Menten equation, in accordance with the growing awareness that polyclonal catalytic antibodies may be much less heterogeneous than had been supposed. The particular value of studies on polyclonal catalytic antibodies is discussed briefly. Both the kcat and kcat/K(m) values were shown to increase with increase in pH across a pKa of approx. 9. Group-selective chemical modification studies established that the side chains of tyrosine and arginine residues are essential for catalytic activity, and provided no evidence for the involvement of side chains of lysine, histidine or cysteine residues. The combination of evidence from the kinetic and chemical modification studies and from studies on the pH-dependence of binding suggests that catalysis involves assistance to the reaction of the substrate with hydroxide ions by hydrogen-bond donation at the reaction centre by tyrosine and arginine side chains. This combination of hydrogen-bond donors appears to be a feature common to a number of other hydrolytic catalytic antibodies. High-pKa acidic side chains may be essential for the effectiveness of catalytic antibodies that utilize hydroxide ions.