Electrochemistry of immobilized redox enzymes: kinetic characteristics of NADH oxidation catalysis at diaphorase monolayers affinity immobilized on electrodes

In the class of NADH:acceptor oxidoreductases, the diaphorase from Bacillus stearothermophilusis a particularly promising enzyme for sensing NADH, and indirectly a great number of analytes, when coupled with a NAD-dependent dehydrogenase as well as for the design of mono- and multienzyme affinity sensors. The design and rational optimization of such systems require devising immobilization procedures that prevent dramatic losses of the enzymatic activity and a full kinetic characterization of the immobilized enzyme system. Two immobilization procedures are described, which involve recognition of the biotinylated diaphorase by a monolayer of neutravidin adsorbed on the electrode surface either directly or through the intermediacy of a monolayer of biotinylated rabbit immunoglobulin. Thorough kinetic characterization of the two systems is derived from cyclic voltammetric responses. A precise estimate of the enzyme coverages is obtained after comparing the enzyme kinetics of the immobilized and the homogeneous system.     

Experimental/theoretical electrostatic properties of a styrylquinoline-type HIV-1 integrase inhibitor and its progenitors

We have established that polyhydroxylated styrylquinolines are potent inhibitors of HIV-1 integrase (IN). Among them, we have identified (E)-8-hydroxy-2-[2-(4,5-dihydroxy-3-methoxyphenyl)-ethenyl]-7-quinolinecarboxylic acid (1) as a promising lead. Previous molecular dynamics simulations and docking procedures have shown that the inhibitory activity involves one or two metal cations (Mg2+), which are present in the vicinity of the active center of the enzyme. However, such methods are generally based on a force-field approach and still remain not as reliable as ab initio calculations with extended basis sets on the whole system. To go further in this area, the aim of the present study was to evaluate the predictive ability of the electron density and electrostatic properties in the structure-activity relationships of this class of HIV-1 antiviral drugs. The electron properties of the two chemical progenitors of 1 were derived from both high-resolution X-ray diffraction experiments and ab initio calculations. The twinning phenomenon and solvent disorder were observed during the crystal structure determination of 1. Molecule 1 exhibits a planar s-trans conformation, and a zwitterionic form in the crystalline state is obtained. This geometry was used for ab initio calculations, which were performed to characterize the electronic properties of 1. The electron densities, electrostatic potentials, and atomic charges of 1 and its progenitors are here compared and analyzed. The experimental and theoretical deformation density bond peaks are very comparable for the two progenitors. However, the experimental electrostatic potential is strongly affected by the crystal field and cannot straightforwardly be used as a predictive index. The weak difference in the theoretical electron densities between 1 and its progenitors reveals that each component of 1 conserves its intrinsic properties, an assumption reinforced by a 13C NMR study. This is also shown through an excellent correlation of the atomic charges for the common fragments. The electrostatic potential minima in zwitterionic and nonzwitterionic forms of 1 are discussed in relation with the localization of possible metal chelation sites.     

Solvent, anion, and structural effects on the redox potentials and UV-visible spectral properties of mononuclear manganese corroles

A series of manganese(III) corroles were investigated as to their electrochemistry and spectroelectrochemistry in nonaqueous solvents. Up to three oxidations and one reduction were obtained for each complex depending on the solvents. The main compound discussed in this paper is the meso-substituted manganese corrole, (Mes 2PhCor)Mn, and the main points are how changes in axially coordinated anion and solvent will affect the redox potentials and UV-vis spectra of each electrogenerated species in oxidation states of Mn(III), Mn(IV), or Mn(II). The anions OAc (-), Cl (-), CN (-), and SCN (-) were found to form five-coordinate complexes with the neutral Mn(III) corrole while two OH (-) or F (-) anions were shown to bind axially in a stepwise addition to give the five- and six-coordinate complexes in nonaqueous media. In each case, complexation with one or two anionic axial ligands led to an easier oxidation and a harder reduction as compared to the uncomplexed four-coordinate species.     

Theory and practice of enzyme bioaffinity electrodes. Chemical, enzymatic, and electrochemical amplification of in situ product detection

The two articles in this series are dedicated to bioaffinity electrodes with in situ detection of the product of the enzyme label after recognition by its conjugate immobilized on the electrode. Part 1 was devoted to direct electrochemical detection, whereas the present contribution deals with homogeneous chemical and enzymatic amplification of the primary electrochemical signal. The theoretical relationships that are established for these modes of amplification are applied to the avidin-biotin recognition in a system that involves alkaline phosphatase as enzyme label and 4-amino-2,6-dichloro-phenyl phosphate as substrate, generating 2,6-dichloro-4-aminophenol as electrochemically active product. Chemical amplification then results from the addition of NADH, which reduces the 2,6-dichloro-quinonimine resulting from the electrochemical oxidation of 2,6-dichloro-4-aminophenol. An increased amplification is obtained when the reduction of 2,6-dichloro-quinonimine involves diaphorase in solution with NADH as substrate. The excellent agreement between theoretical predictions and experimental data required a detailed theoretical analysis and the independent determination of the key kinetic parameters of the system. The theoretical analysis was extended to monolayer and multilayered films of auxiliary enzyme as well as to electrochemical amplification by means of closely spaced dual electrodes so as to offer a rational comparative panorama of the amplification capabilities of the various possible strategies. Confinement of the profile of the product, and/or its oxidized form, in the vicinity the electrode surface appears as a key parameter of amplification.     

Theory and practice of enzyme bioaffinity electrodes. Direct electrochemical product detection

The use of enzyme labeling techniques to convert biorecognition events into high sensitivity electrochemical signals may follow two different strategies. One, in which the current is the electrocatalytic response of a redox couple serving as cosubstrate to a redox enzyme label and another that consists in the detection of an electrochemically active product of the enzyme label. The theoretical relationships that link, in the latter case, the electrochemical current response to the amount of recognized labeled target analyte are established for steady-state diffusion-convection chronoamperometric regimes. Two governing parameters thus emerge. One measures the Michaelis-Menten competition in the enzyme kinetics. The other characterizes the competition between the enzymatic kinetics and the diffusion of the substrate. The electrochemical response is finally related to the labeled target analyte concentration in solution through the recognition isotherm. The direct electrochemical product detection thus provides a route to the characteristics of the recognition isotherm, which serves as a calibration curve in analytical applications. The establishment of further theoretical relationships allows one to surmise the increase in sensitivity that may be obtained by using cyclic voltammetry instead of steady-state chronoamperometry in standard electrochemical cells or by accumulation of the enzyme-product in cells of small volume/surface ratios. The theoretical predictions are tested with the example of the avidin-biotin recognition process in a system that involves alkaline phosphatase as enzyme label and 4-amino-2,6-dichlorophenyl phosphate as substrate, generating 4-amino-2,6-dichlorophenol as electrochemically active product. The advantages of the dichloro-substitution are discussed. The theoretical analysis is a requisite for a rational and realistic discussion of the analytical performances of the steady-state chronoamperometric and cyclic voltammetric approaches. These are shown to compare favorably with the best heterogeneous bioaffinity assays so far reported.     

Ultrafast dynamics of acetylacetone (2,4-pentanedione) in the S2 state

The dynamics of the enolic form of acetylacetone (E-AcAc) was investigated using a femtosecond pump-probe experiment. The pump at 266 nm excited E-AcAc in the first bright state, S2(pi pi*). The resulting dynamics was probed by multiphoton ionization at 800 nm. It was investigated for 80 ps on the S2(pi pi*) and S1(n pi*) potential energy surfaces. An important step is the transfer from S2 to S1 that occurs with a time constant of 1.4 +/- 0.2 ps. Before, the system had left the excitation region in 70 +/- 10 fs. An intermediate step was identified when E-AcAc traveled on the S2 surface. Likely, it corresponds to an accidental resonance in the detection scheme that is met along this path. More importantly, some clues are given that an intramolecular vibrational energy relaxation is observed, which transfers excess vibrational energy from the enolic group O-H to the other modes of the molecule. The present multistep evolution of excited E-AcAc probably also describes, at least qualitatively, the dynamics of other electronically excited beta-diketones.     

The sticking of H and D atoms on a graphite (0001) surface: the effects of coverage and energy dissipation

Classical trajectory methods are used to examine the trapping and sticking of H and D atoms on the graphite (0001) surface. Total energy calculations based on density functional theory are used to construct the model potential energy surface, and graphite clusters of up to 121 atoms are considered. For hydrogen to chemisorb, the bonding carbon must pucker out of the surface plane by roughly 0.4 A. For incident energies above the 0.2 eV barrier, any trapped H atoms must rapidly dissipate their excess energy into the surrounding lattice within a few vibrations of the C-H stretch in order to remain bound. For sufficiently large clusters, the C-H bond stabilizes within about 0.1 ps. The sticking probability for D at 150 K is in the range of 5%-10%, more-or-less consistent with the most recent measurements in the limit of zero coverge. Variation with isotope and substrate temperature is weak. We estimate that the sticking cross section for adsorption at the para site, directly across the sixfold carbon ring from an already adsorbed H atom, can be four or more times larger that the zero coverage sticking cross section.     

Evidence for concerted proton-electron transfer in the electrochemical oxidation of phenols with water as proton acceptor. Tri-tert-butylphenol

Proton-coupled electron transfer oxidation of phenols play a prominent role in several natural processes, and one may wonder if their high efficiency is related to the possibility that the electron and proton transfer steps are concerted. The cyclic voltammetric observation of the electrochemical oxidation and reverse reaction has allowed, with the example of 2,4,6-tri-tert-butylphenol in nonbuffered aqueous media, the clear identification of a pathway in which a phenol is directly and reversibly converted into the phenoxyl radical while the generated proton is accepted by a water molecule in a concerted manner. In very basic media, a stepwise mechanism takes place in which the phenol is deprotonated by OH- and the resulting phenoxide ion rapidly oxidized into the phenoxyl radical. As the pH decreases, this pathway progressively shuts down to the advantage of the concerted pathway. The latter assignment is confirmed by the observation of a substantial H/D kinetic isotope effect. At moderately basic pH 10.5, the contributions of the two pathways are about equal and the occurrence of the two competing routes is directly visualized in the cyclic voltammetry response.     

Physical Gels of a Syndiotactic Polystyrene Derivative with a Large Side-Chain Group

Summary: Gelation of syndiotactic poly(p-tert-butylstyrene) (sPTBS), a syndiotactic polystyrene (sPS) derivative having a larger side-chain group, was first examined with several solvents. The temperature-concentration phase diagram of sPTBS/trans-decalin gel clearly exhibited that sPTBS formed a polymer-solvent molecular compound with a ratio of 2.7 trans-decalin per one monomer unit. Our polarized fluorescence technique demonstrated that there appeared to exist more spacious free volume among sPTBS chains than sPS in a gel state. A cause determining the morphology of sPTBS with organic solvents was discussed in the present paper.