Electrochemical Characteristics of Nitro-Heterocyclic Compounds of Biological Interest. II. Nitrosochloramphenicol

The electrochemical Characteristics of nitrosochloramphenicol have been studied in aqueous buffer systems (pH 7.1) using direct current (d.c.) and differential pulse polarography. cyclic voltammetry and coulometric techniques. Up to 4 charge-transfer steps can be identified. The first reduction step is reversible both chemically and electrochemically. the charge-transfer product showing no tendency to undergo further reaction on the electrochemical time-scale. In contrast, the second reduction step is irreversible, with the product undergoing a fast following reaction to yield a redox-active species which was detected by cyclic voltammetry. From the data and by comparison with related systems. two reduction mechanisms are possible and are discussed.     

Electrochemical Characteristics of Nitroheterocyclic Compounds of Biological Interest. VIII Stability of Nitro Radical Anions from Cyclic Voltammetric Studies

The stability of the one electron addition product of four biologically important nitroheterocyclic compounds has been examined electrochemically. Using cyclic voltammetry the tendency of the nitro radical anion to undergo disproportionation was studied by two methods of analysis. The first was based on determining the voltammetric time-constant required for half of the reduction product, RNO₂, to react further. The second concerned the minimum volume of dimethylformamide which had to be added to the aqueous electrolytic medium to give a specific cyclic voltammetric response. Both methods were found to compare well with the results obtained for RNO₂T stabilities using a theoretically derived procedure for a second order reaction following a charge-transfer step. The use of these alternative approaches for quantifying the reactivity of reduction products is discussed. The time-constant method in particular may be useful in studying complex reaction pathways.     

Electrochemical study of triscyclopentadienyluranium complexes

The electrochemical behaviour of Cp₃UBH₄ and Cp₃UNEt₂ in THF has been studied using cyclic voltammetry and chronoamperometry. Cp₃UBH₄ undergoes a simple one-electron quasi-reversible reduction, while chemical reactions between the product of the charge-transfer reaction and the starting electroactive species occur both after oxidation and reduction of Cp₃UNEt₂.     

Simulation of the electrochemical behavior of multi-redox systems. Current potential studies on multiheme cytochromes.

The direct unmediated electrochemical response of the tetrahemic cytochrome c3 isolated from sulfate reducers Desulfovibrio baculatus (DSM 1743) and D. vulgaris (strain Hildenborough), was evaluated using different electrode systems [graphite (edge cut), gold, semiconductor (InO2) and mercury)] and different electrochemical methods (cyclic voltammetry and differential pulse voltammetry). A computer program was developed for the theoretical simulation of a complete cyclic voltammetry curve, based on the method proposed by Nicholson and Shain [Nicholson, R.S. & Shain, I. (1964) Anal. Chem. 36, 706-723], using the Gauss-Legendre method for calculation of the integral equations. The experimental data obtained for this multi-redox center protein was deconvoluted in to the four redox components using theoretically generated cyclic voltammetry curves and the four mid-point reduction potentials determined. The pH dependence of the four reduction potentials was evaluated using the deconvolution method described.     

Selective interaction of tirapazamine with DNA bases and DNA

An electrochemical model has been used to study the reductive activation of the hypoxic cell cytotoxin tirapazamine (TPZ, 3-amino-1,2,4-benzotriazine-1,4-dioxide). Cyclic voltammetry and controlled potential electrolysis have been used to generate and study the 1-electron reduction product, the assumed biologically active species. Cyclic voltammetry of tirapazamine in dimethylformamide shows a quasi-reversible 1-electron reduction with the product showing a tendency to participate in a following chemical reaction. Controlled potential electrolysis to generate the 1-electron reduction product was unsuccessful due to the formation of a new redox-active species at less negative reduction potentials. However, the cyclic voltammetry of tirapazamine in the presence of E. coli DNA shows a decrease in the lifetime of the radical anion, signifying direct interaction with the DNA. The radical lifetime also decreased in the presence of adenine, thymine and guanine, but increased upon addition of cytosine and ribose. The study shows that cyclic voltammetry is an extremely useful tool for investigating the interaction between bio-reductive drugs and biological target molecules.     

Selective interaction of tirapazamine with DNA bases and DNA

An electrochemical model has been used to study the reductive activation of the hypoxic cell cytotoxin tirapazamine (TPZ, 3-amino-1,2,4-benzotriazine-1,4-dioxide). Cyclic voltammetry and controlled potential electrolysis have been used to generate and study the 1-electron reduction product, the assumed biologically active species. Cyclic voltammetry of tirapazamine in dimethylformamide shows a quasi-reversible 1-electron reduction with the product showing a tendency to participate in a following chemical reaction. Controlled potential electrolysis to generate the 1-electron reduction product was unsuccessful due to the formation of a new redox-active species at less negative reduction potentials. However, the cyclic voltammetry of tirapazamine in the presence of E. coli DNA shows a decrease in the lifetime of the radical anion, signifying direct interaction with the DNA. The radical lifetime also decreased in the presence of adenine, thymine and guanine, but increased upon addition of cytosine and ribose. The study shows that cyclic voltammetry is an extremely useful tool for investigating the interaction between bio-reductive drugs and biological target molecules.     

The electrochemical preparation of FAD/ZnO with hemoglobin film-modified electrodes and their electroanalytical properties

Flavin adenine dinucleotide (FAD)-modified zinc oxide self-assembly films were prepared using repeated cyclic voltammetry. The electrochemical reaction of the hemoglobin with the FAD/ZnO self-assembly film-modified electrodes and their electrocatalytic properties were investigated. This paper describes the successful loading of the electrochemically active molecules of hemoglobin and FAD along with ZnO by electrochemical method. In addition to the cyclic voltammetry, an electrochemical quartz crystal microbalance was used to study the growth mechanism and the properties of the films. The FAD/zinc oxide films exhibited a single redox couple, which corresponded to the FAD redox couple. The electrocatalytic properties of the O2, H2O2, trichloroacetic acid and SO(3)2- were studied by the FAD/zinc oxide films in the absence or in the presence of hemoglobin. The electrocatalytic reduction current had been developed from the cathodic peak of the FAD/zinc oxide redox couple. The electrocatalytic process involved an interaction of hemoglobin and FAD/GC film-modified electrode to increase the electrocatalytic reduction current. The electrocatalytic reduction of O2 using the FAD/zinc oxide films was investigated by cyclic voltammetry and rotating ring-disk electrode methods.     

Electrochemical Characteristics of Nitro-Heterocyclic Compounds of Biological Interest. III. Nitroso Derivative Formation

Upon electrolytic reduction of a range of nitro-aromatic complexes (including imidazoles. benzenoids. furans and pyrazoles) an associated oxidation-reduction process is observed at more positive potentials with respect to nitro group reduction when using repeat scan cyclic voltammetry. This new couple has been identified as the reversible first reduction of the nitroso derivative for chloramphenicol, by the addition of a genuine sample of nitrosochloramphenicol to the electrochemical cell. We have failed to observe formation of the new redox-active species for five 5-nitroimidazoles examined.

Possible reaction schemes for nitroso formation under electrolytic reduction conditions and the importance of the nitroso redox couple with respect to the cytotoxic action of the parent drug are discussed. The applicability of nitrosochloramphenicol as a model for the behaviour of nitroso-heterocycles in general is shown.     

Electrochemical Characteristics of Nitro-Heterocyclic Compounds of Biological Interest. III. Nitroso Derivative Formation

Upon electrolytic reduction of a range of nitro-aromatic complexes (including imidazoles. benzenoids. furans and pyrazoles) an associated oxidation-reduction process is observed at more positive potentials with respect to nitro group reduction when using repeat scan cyclic voltammetry. This new couple has been identified as the reversible first reduction of the nitroso derivative for chloramphenicol, by the addition of a genuine sample of nitrosochloramphenicol to the electrochemical cell. We have failed to observe formation of the new redox-active species for five 5–nitroimidazoles examined.

Possible reaction schemes for nitroso formation under electrolytic reduction conditions and the importance of the nitroso redox couple with respect to the cytotoxic action of the parent drug are discussed. The applicability of nitrosochloramphenicol as a model for the behaviour of nitroso-heterocycles in general is shown.     

Flow-injection analysis with electrochemical detection of reduced nicotinamide adenine dinucleotide using 2,6-dichloroindophenol as a redox coupling agent

The determination of reduced nicotinamide adenine dinucleotide (NADH) by electrochemical oxidation requires a more positive potential than is predicted by the formal reduction potential for the NAD+/NADH couple. This problem is alleviated by use of 2,6-dichloroindophenol (DCIP) as a redox coupling agent for NADH. The electrochemical characteristics of DCIP at the glassy carbon electrode are examined by cyclic voltammetry and hydrodynamic voltammetry. NADH is determined by reaction with DCIP to form NAD+ and DCIPH2. DCIPH2 is then quantitated by flow-injection analysis with electrochemical detection by oxidation at a detector potential of +0.25 V at pH 7. NADH is determined over a linear range of 0.5 to 200 microM and with a detection limit of 0.38 microM. The lower detection potential for DCIPH2 compared to NADH helps to minimize interference from oxidizable components in serum samples.     

Electrochemical Studies of Tirapazamine: Generation of the One-Electron Reduction Product

The electrochemical properties of the benzotriazine di-N-oxide, tirapazamine (SR4233), and the mono-and zero-N-oxides, SR4317 and SR4330 respectively, have been investigated in dimethylformamide and acetonitrile. The voltammetry of tirapazamine is complicated, with up to 6 reduction steps being identified, depending on the solvent. Both SR4317 and SR4330 show two reduction steps. The first reduction of all three compounds is a reversible or quasi-reversible step, which is assigned to a 1-electron addition. Cyclic voltammetric studies show that the anion radical product is stable, although the tirapazamine 1-electron addition product shows a tendency to participate in a chemical following reaction. Subsequent reduction steps are all highly irreversible in nature. The 2nd electron transfer of SR4317 results in the formation of the free base, SR4330, which is identified voltammetrically. Comparison is made with the voltammetric behaviour of quinoline and quinoline-oxide.     

Structure-function correlation of fatty acyl-CoA dehydrogenase and fatty acyl-CoA oxidase

We have employed a new pseudosubstrate, beta-(2-furyl)propionyl coenzyme A (FPCoA), to study the functional properties of two enzymes, fatty acyl-CoA dehydrogenase from porcine liver and fatty acyl-CoA oxidase from Candida tropicalis, involved in the oxidation of fatty acids. Previous studies from our laboratory have shown that the dehydrogenase exhibits oxidase activity at the rate of dissociation of the product charge-transfer complex. This raises the question of the difference in functionality between these two flavoproteins. To investigate these differences, we have compared the pH dependence of product formation, the isotope effects using tetradeuterio-FPCoA, and the spectral properties and chemical reactivity of the product charge-transfer complexes formed with the two enzymes. The pH dependencies of the reaction of FPCoA with electron-transfer flavoprotein (ETF) for the dehydrogenase and of the reaction of FPCoA with O2 for the oxidase are quite similar. Both reactions proceed more rapidly at basic pH values while substrate binds more tightly at acidic pH values. These data for both enzymes are consistent with a mechanism in which enzyme is involved in protonation of the carbonyl group of substrate followed by base-catalyzed removal of the C-2 proton from substrate. The C-2 anion of substrate may then serve as the active species in reduction of enzyme-bound flavin. The deuterium isotope effects for both enzyme systems are primary across the entire pH range, assuring that the chemically important step of substrate oxidation is rate limiting in these steady-state kinetic experiments. The two enzymes differ in the chemical reactivity of their product charge-transfer complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrochemical Properties As A Function of Ph for the Benzotriazine DI-N-Oxides

The electrochemistry of five benzotriazine di-N-oxides has been examined by cyclic voltammetry and differential pulse and dc polarographies as a function of pH. Between the pH range 8.5 and 2 the trend to less negative potentials with lowering of pH can be described by an equation of the type Ep = - apH + b. Comparison has been made with the mono- and zero-N-oxides which were found to show virtually identical trends in electron affinity with pH. The general electrochemical characteristics for the di- and mono-N-oxides under acidic conditions were found to be comparable with the zero-N-oxide. This was particularly the case on repeat scanning in the cyclic voltammetric mode. The redox mechanism involved reduction by a 4-electron addition step and subsequent loss of the A-oxide group(s) yielding the intact benzotriazine heterocycle. The helerocycle was also redox active, involving a reversible 2-electron reduction. for the di-N-oxides these two stages could be identified as separate processes at alkaline pH, but ony a single step at acidic values. The mono-N-oxide in which the electrochemical behaviour was dominated by the triazine, showed only a single reduction step, although the single N-oxide group was redox active.     

Development of a molecular diagnosis assay based on electrohybridization at plastic electrodes and subsequent PCR

Technologies enabling specific recognition of medically relevant nucleic acid sequences will play a pivotal role in future medical diagnosis. Whereas many approaches to molecular diagnosis systems include DNA microarrays on chips and fluorometric detection, the basis of our approach is the use of inexpensive components like plastic or metal thin film electrodes with low multiplexing and an electrochemical detection unit. To increase the sensitivity, PCR can be used as an intermediate step. For selective enrichment, specific nucleic acid probes were covalently attached at their 5′-ends to conducting polycarbonate/carbon fiber electrodes. Complementary oligonucleotides were enriched at the electrodes by cyclic inversion of an electrochemical potential, transferred into a PCR vial and thermally or electrochemically desorbed. The analysis of the PCR product shows the efficiency and selectivity of the electrochemical enrichment. Hybridization of DNA was shown by electrochemical methods, in this work especially by differential pulse voltammetry (DPV) using the single strand specific hybridization redox indicator osmium(VIII)-tetroxide, and potentiometric stripping analysis (PSA). This combination of experimental methods is the basis for a molecular diagnosis system including a disposable nucleic acid modified working electrode for specific enrichment, detection and quantification, and an optional capillary PCR module for fast amplification.     

Label-free detection of DNA hybridization based on a novel functionalized conducting polymer

A new functionalized pyrrole monomer, 3-pyrrolylacrylic acid (PAA) was synthesized. It was used to prepare a copolymer with pyrrole, poly(Py-co-PAA), which was investigated by reflective FT-IR, UV-vis spectroscopy and cyclic voltammetry. A label-free DNA sensor was prepared based on a poly(Py-co-PAA) film. Hybridization with complementary and non-complementary DNA targets was studied by electrochemical impedance spectroscopy. Results show a significant increase in the charge-transfer resistance upon addition of complementary target. The impedance spectra were analyzed by using a modified Randles and Ershler equivalent circuit model. The change in charge-transfer resistance that was used as an index of sensor response was found to be linear with logarithmic target concentration in the range of 2 x 10(-9) to 2 x 10(-7)M. The detection limit was 0.98 nM.     

Time course of release of catecholamines from individual vesicles during exocytosis at adrenal medullary cells.

The time course of extrusion of the vesicular contents during exocytosis has been examined at adrenal medullary cells with carbon-fiber microelectrodes. Two electrochemical techniques were used: cyclic voltammetry and amperometry. Spikes obtained by amperometry had a faster time course than those measured by cyclic voltammetry, consistent with the different concentration profiles established by each technique. However, the experimental data obtained with both techniques were temporally broadened with respect to dispersion of an instantaneous point source by diffusion. Measurements with the electrode firmly pressed against the cell surface established that the temporal broadening is a result of a rate-limiting kinetic step associated with extrusion of the vesicular contents at the cell surface. The data do not support a rate-limiting process due to restricted efflux from a small pore. When combined with previous results, the data suggest that the rate-limiting step for chemical secretion from adrenal medullary cells during exocytosis is the dissociation of catecholamines from the vesicular matrix at the surface of the cell.     

Kinetic studies of the electron exchange reaction between the octaheme cytochrome c3 (Mr 26000) and the hydrogenase from Desulfovibrio desulfuricans Norway.

The octaheme cytochrome c3 (Mr 26000) from Desulfovibrio desulfuricans Norway was studied using cyclic voltammetry at the pyrolytic graphite electrode. The kinetics of reduction of the octaheme cytochrome c3 (Mr 26000) from D. desulfuricans Norway by the Ni-Fe-Se hydrogenase purified from the same organism was investigated by an electrochemical method. From cyclic voltammetry experiments a value of 8.108M-1S-1 was obtained for the second order homogenous rate constant of the electron transfer between the two proteins. Results are compared with similar experiments performed on the electron exchange between the tetrahemic cytochrome c3 (Mr 13000) and hydrogenase.     

基于“酶-邻氨基苯酚”体系的电化学免疫传感方法及其应用

本研究建立了一种新的电化学免疫传感方法,并将其应用于实际样本的检测。采用辣根过氧化物酶(horseradish peroxidase,HRP)的新型底物邻-氨基苯酚(O-aminophenol,O-AP)构建了基于"酶-邻氨基苯酚"体系的电化学免疫传感方法,并用于血吸虫抗体检测。邻-氨基苯酚在辣根过氧化物酶催化双氧水(H2O2)时被氧化,生成的产物3-氨基吩噁嗪能通过电化学方法检测。实验结果验证了基于"酶-邻氨基苯酚"的新型蛋白检测体系用于检测人血清中血吸虫抗体的可操作性,实现了实际样本的检测。并且,采用方波伏安法作为最终检测方法,具有更高的灵敏性。     

Mechanism of flavin reduction in the alkanesulfonate monooxygenase system

The alkanesulfonate monooxygenase system from Escherichia coli is involved in scavenging sulfur from alkanesulfonates under sulfur starvation. An FMN reductase (SsuE) catalyzes the reduction of FMN by NADPH, and the reduced flavin is transferred to the monooxygenase (SsuD). Rapid reaction kinetic analyses were performed to define the microscopic steps involved in SsuE catalyzed flavin reduction. Results from single-wavelength analyses at 450 and 550 nm showed that reduction of FMN occurs in three distinct phases. Following a possible rapid equilibrium binding of FMN and NADPH to SsuE (MC-1) that occurs before the first detectable step, an initial fast phase (241 s(-1)) corresponds to the interaction of NADPH with FMN (CT-1). The second phase is a slow conversion (11 s(-1)) to form a charge-transfer complex of reduced FMNH(2) with NADP(+) (CT-2), and represents electron transfer from the pyridine nucleotide to the flavin. The third step (19 s(-1)) is the decay of the charge-transfer complex to SsuE with bound products (MC-2) or product release from the CT-2 complex. Results from isotope studies with [(4R)-(2)H]NADPH demonstrates a rate-limiting step in electron transfer from NADPH to FMN, and may imply a partial rate-limiting step from CT-2 to MC-2 or the direct release of products from CT-2. While the utilization of flavin as a substrate by the alkanesulfonate monooxygenase system is novel, the mechanism for flavin reduction follows an analogous reaction path as standard flavoproteins.     

Electrochemical Properties As A Function of Ph for the Benzotriazine DI-N-Oxides

The electrochemistry of five benzotriazine di-N-oxides has been examined by cyclic voltammetry and differential pulse and dc polarographies as a function of pH. Between the pH range 8.5 and 2 the trend to less negative potentials with lowering of pH can be described by an equation of the type Ep = - apH + b. Comparison has been made with the mono- and zero-N-oxides which were found to show virtually identical trends in electron affinity with pH. The general electrochemical characteristics for the di- and mono-N-oxides under acidic conditions were found to be comparable with the zero-N-oxide. This was particularly the case on repeat scanning in the cyclic voltammetric mode. The redox mechanism involved reduction by a 4-electron addition step and subsequent loss of the A-oxide group(s) yielding the intact benzotriazine heterocycle. The helerocycle was also redox active, involving a reversible 2-electron reduction. for the di-N-oxides these two stages could be identified as separate processes at alkaline pH, but ony a single step at acidic values. The mono-N-oxide in which the electrochemical behaviour was dominated by the triazine, showed only a single reduction step, although the single N-oxide group was redox active.