naphthoquinones and 2-dimethylamino-3-chloro-1-4-naphthoquinone

The electron donor-acceptor (EDA) interaction between 2,3-dicyano-1,4-naphthoquinone (DCNQ) and 3,4-dimethylaniline (3,4-DMA) is studied in chloroform, dichloromethane and 1:1 (v/v) mixture of chloroform and dichloromethane. The rate of formation of the product was measured as a function of time using UV-vis spectrophotometer. The formation constant (K) and molar extinction coefficient (ɛ) values for the formation of EDA complex were evaluated in the temperature range of 20-35°C. The pseudo-first-order rate constant (k1) and the second-order rate constant (k2) for the disappearance of EDA complex and for the formation of product were evaluated. The activation parameters (ΔH#, ΔS# and ΔG#) of the reaction were determined by temperature dependence of rate constants using the Arrhenius plots. The effect of relative permittivity of the medium on the reaction is discussed. The observed results indicate that formation of final product proceeds through initial formation of EDA complex as an intermediate. The product of the reaction was purified by column chromatography method and identified as 3-(N-3,4-dimethyl-phenylamino)-2-cyano-1,4-naphthoquinone by elemental analysis, IR and NMR spectroscopy. On the basis of kinetic, analytical and spectroscopic results, a plausible mechanism for the formation of EDA complex and its transformation into product is proposed.

Topics: Aniline Compounds; Electrons; Entropy; Kinetics; Naphthoquinones; Spectrum Analysis; Time Factors

The inhibition of jack bean urease by 2,3-dichloro-1,4-naphthoquinone (DCNQ) was studied at ambient temperature in 20 mM phosphate buffer, pH 7.8. The process was investigated by incubation procedure in the absence of substrate. It was found that DCNQ acted as a time- and concentration-dependent inactivator of urease. The time course of the reaction displayed a biphasic mode. Each phase followed a pseudo-first-order kinetics, however the inactivation rate at the first phase was significantly faster than at the next one. The biphasity indicated the complex mechanism of DCNQ action on urease. Quinones action on proteins has been elucidated as at least two processes: direct arylation of essential protein thiols and/or indirect oxidation of essential thiols by reactive oxygen species (ROS) realising during quinone reduction to semiquinones. The next evidence of the studied mechanism was provided by the reactivation experiment that showed the participation of reversible and irreversible processes in the inactivation. The application of dithiothreitol (DTT) into DCNQ blocked-urease solution resulted in an effective enzyme activity regain which quickly returned to 70 +/- 10%. The irreversible inactivation of urease was attributed to DCNQ arylation of thiol residues in the protein. On the other hand, it was assumed that the reversible inactivation was a result of the action of ROS such as H(2)O(2). Presence of H(2)O(2) in the incubation system was proved by an experiment with the use of catalase. The enzyme by the elimination of H(2)O(2) decreased DCNQ inactivating influence on urease. The comparison of participation of the fast and slow phase in the inactivation with the percentage of the process reversibility was assumed that the fast period was a result of the arylation mechanism while the slow phase was related to the oxidative influence of H(2)O(2).

Topics: Canavalia; Enzyme Inhibitors; Hydrogen-Ion Concentration; Kinetics; Molecular Structure; Naphthoquinones; Oxidation-Reduction; Sulfhydryl Compounds; Urease

Two new EPR approaches were developed for determination of rate constants of reaction glutathione (GSH), N-(2-mercaptopropionyl) glycine (MPG), dihydrolipoic acid (BNL), and tetranor-dihydrolipoic acid (TNL) with superoxide radical. In both cases the competition between thiols and spin-trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) for superoxide radical was used. In the first method the dependence of amplitude of EPR spectrum of DMPO-OOH spin adduct on concentration of thiols in a superoxide-generating system was studied. In the second approach the changes in reduced thiol concentration due to reaction with superoxide radical were measured by nitroxide biradical containing disulfide bond. Observed rate constants were the following: GSH, 1.8 x 10(5) M-1s-1; MPG, 2.2 x 10(5) M-1s-1; TNL, 1.2 x 10(5) M-1s-1; BNL, 2.5 x 10(5) M-1s-1; DHL, 4.8 x 10(5) M-1s-1. The determination of the rate constants of reaction of superoxide radical with thiols by spectrophotometrical cytochrome C assay could result in an underestimation of the values due to the reduction of cytochrome C by thiols. Use of epinephrine for this purpose could lead to an overestimation of experimental rate constants because the adrenochrome formed in the reaction of epinephrine with superoxide radical reacts with thiols.

Topics: Adrenochrome; Cyclic N-Oxides; Cytochrome c Group; Electron Spin Resonance Spectroscopy; Evaluation Studies as Topic; In Vitro Techniques; Kinetics; NADPH-Ferrihemoprotein Reductase; Naphthoquinones; Oxidation-Reduction; Spectrophotometry; Spin Labels; Sulfhydryl Compounds; Superoxides

Using ESR and spin-trapping techniques, it was found that synthetic 2-dimethylamino-3-chloro-1,4-naphthoquinone and the natural anticancer quinone daunomycin, when added to a system containing purified NADPH-cytochrome P-450 reductase, NADPH, ferric ions, and oxygen, (i) generated hydroxyl radicals and (ii) caused single-strand scission of supercoiled DNA of the plasmic pBR322. Since these two effects of the quinones were correlated to each other, we propose that potential anticancer quinones can be effectively screened by measuring their ability to form hydroxyl radicals in the above system.

Topics: Daunorubicin; DNA Damage; DNA, Bacterial; Electron Spin Resonance Spectroscopy; Ferric Compounds; Free Radicals; Hydroxides; In Vitro Techniques; NADPH-Ferrihemoprotein Reductase; Naphthoquinones; Plasmids

2-Dimethylamino-3-chloro-1,4-naphthoquinone (DCNQ) is bound to microsomal cytochrome P-450 as a type I substrate (lambda max = 391 nm, lambda min = 420 nm). The Ks is 40.5 microM. In a rat-liver microsomal system, the N-demethylation of DCNQ produces formaldehyde (rate 225 pmol/min per mg of protein). Induction by phenobarbital increases the rate of formation, while addition of metyrapone and SKF-525A into the system decreases the rate by 52% and 35%, respectively. The microsomal N-demethylation of DCNQ is not inhibited by CO. Under full anaerobiosis, the microsomal oxidation of DCNQ again gives formaldehyde (rate 416 pmol/min per mg of protein). The anaerobic oxidation of DCNQ is inhibited by metyrapone and SKF-525A. The microsomal, chemical and electrochemical reduction of DCNQ to the corresponding semiquinones and hydroquinones have been studied. Non-enzymic DCNQ reduction is insufficient for the formation of formaldehyde. Under anaerobic conditions the microsomal DCNQ oxidation is assumed to occur via the intramolecular oxazole bond which is then hydrolysed, yielding formaldehyde. This may be a new example of substrate activation by cytochrome P-450.

Topics: Aerobiosis; Aminopyrine; Anaerobiosis; Animals; Cytochrome P-450 Enzyme System; Enzyme Activation; In Vitro Techniques; Male; Microsomes, Liver; Naphthoquinones; Oxidation-Reduction; Photochemistry; Rats; Rats, Inbred Strains; Substrate Specificity