ACTION SPECTRA FOR LETHALITY IN RECOMBINATION-LESS STRAINS OF SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI*

Abstract— Action spectra for lethality of both stationary and exponentially growing cells of recombinationless (recA) mutants of Salmonella typhimurium and Escherichia coli were obtained. Maximum sensitivity was observed at 260nm which corresponds to the maximum absorbance of DNA. However, a shoulder occurred in the 280–300 nm range that departed significantly from the absorption spectrum of DNA. At wavelengths longer than 320nm, the shapes of inactivation curves departed significantly from those at wavelengths shorter than 320nm and survival curves at wavelengths longer than 320nm had a large shoulder. A small peak or shoulder occurred in the 330–340nm region of the action spectra. The special sensitivity of recA mutants to broad spectrum near-UV radiation may be due to synergistic effects of different wavelengths. Parallels between the inactivation of recA mutants and the induction of a photoproduct of l -tryptophan toxic for recA mutants (now known to be H₂O₂) suggest that H₂O₂ photoproduct from endogenous tryptophan may be involved in the high sensitivity of these strains to broad spectrum near-UV radiation.     

Separation-free amperometric enzyme immunoassay

A new technique for conducting a separation-free amperometric enzyme immunoassay is described using DNP-aminocaproic acid as the analyte. The technique is based on the combined use of a recently described separation-free enzyme immunoassay (19) and an electrode system that senses H₂O₂. Oxidation of glucose to gluconate and H₂O₂ by the enzyme reconstituted from DNP-conjugate apoglucose oxidase (DPN-CAGO) and FAD was continuously measured amperometrically. The reconstitution was inhibited by preincubation with anti-DNP antibody before adding FAD. This antibody-induced inhibition of the reconstituting of the holoenzyme was reversed by adding DNP-amino caproic acid to DNP-CAGO before adding the antibody to DNP-CAGO. Based on (a) the antibody-induced inhibition of holoenzyme reconstitution, (b) a specific ligand-induced reversal of the inhibition, and (c) an electrochemical system that measures H₂O₂, we developed a separation-free (homogeneous) amperometric enzyme immunoassay.     

A Fast and Direct Amperometric Determination of Hg2+ by a Bienzyme Electrode Based on the Competitive Activities of Glucose Oxidase and Laccase

A new concept of enzyme inhibition‐based biosensor involving the appearance of an amperometric signal for an inhibition by mercury was developed. The bienzyme sensor was composed of two layers of clay materials. The inner layer was constituted of layered double hydroxides entrapping laccase wired by ABTS. The outer laponite layer contained glucose oxidase (GOD). GOD catalyzed the glucose oxidation with the reduction of O₂ into H₂O₂. This induced a drastic decrease of the biosensor response to O₂ by the electrically wired laccase. HgCl₂ inhibited the O₂ consumption by GOD leading to a signal increase of the electroenzymatic reduction of O₂.     

A new oxygen sensitivity and its potential application in photosynthetic H2 production

We have discovered a new competitive pathway for O₂ sensitivity in algal H₂ production that is distinct from the O₂ sensitivity of hydrogenase per se. This O₂ sensitivity is apparently linked to the photosynthetic H₂ production pathway that is coupled to proton translocation across the thylakoid membrane. Addition of the proton uncoupler carbonyl cyanide-p-trifluoromethoxy-phenylhydrazone eliminates this mode of O₂ inhibition on H₂ photoevolution. This newly discovered inhibition is most likely owing to background O₂ that apparently serves as a terminal electron acceptor in competition with the H₂ production pathway for photosynthetically generated electrons from water splitting. This O₂-sensitive H₂ production electron transport pathway was inhibited by 3[3,4-dichlorophenyl]1,1-dimethylurea. Our experiments demonstrated that this new pathway is more sensitive to O₂ than the traditionally known O₂ sensitivity of hydrogenase. This discovery provides new insight into the mechanism of O₂ inactivation of hydrogenase and may contribute to the development of a more-efficient and robust system for photosynthetic H₂ production.     

Electrochemical Monitoring of the Early Events of Hydrogen Peroxide Production by Mitochondria

Mitochondria consume oxygen in the respiratory chain and convert redox energy into ATP. As a side process, they produce reactive oxygen species (ROS), whose physiological activities are still not understood. However, current analytical methods cannot be used to monitor mitochondrial ROS quantitatively and unambiguously. We have developed electrochemical biosensors based on peroxidase‐redox polymer‐modified electrodes, providing selective detection of H₂O₂ with nanomolar sensitivity, linear response over five concentration decades, and fast response time. The release of H₂O₂ by mitochondria was then monitored under phosphorylating or inhibited respiration conditions. We report the detection of two concomitant regimes of H₂O₂ release: large fluxes (hundreds of nM ) under complex III inhibition, and bursts of a few nM immediately following mitochondria activation. These unprecedented bursts of H₂O₂ are assigned to the role of mitochondria as the hub of redox signaling in cells.     

Ultraviolet molar absorptivities of aqueous hydrogen peroxide and hydroperoxyl ion

Molar absorptivities are measured at 11 wavelengths between 200 and 300 nm for H₂O₂(aq) with an average error of 8.5% (95% confidence limit), and at 10 wavelengths in the same range for HO₂^? (aq), from absorbance vs. concentration plots. Results are compared to previous measurements of both aqueous and vapor-phase molar absorptivities.     

Manganese-doped ZnS quantum dots as a phosphorescent probe for use in the bi-enzymatic determination of organophosphorus pesticides

We present a sensitive and selective method for the determination of organophosphorus pesticides (OPs) based on the inhibition of the enzyme acetylcholinesterase (AChE). It is making use of quantum dots QDs of the type Mn: ZnS that display long-lived phosphorescence emission and act as optical probes for hydrogen peroxide (H₂O₂). In this assay, acetylcholine (ACh) is first hydrolyzed by AChE, and the enzyme choline oxidase (ChOx) further oxidizes choline under the formation of H₂O₂ which quenches the phosphorescence of the QDs. If, however, OPs are added to the solution, the rate of enzymatic hydrolysis by AChE is retarded. This reduces the rate of production of H₂O₂ and results in a reduced quenching efficiency. The slow decay time of the phosphorescence of the QDs also allows time-resolved luminescence intensity to be measured. This can eliminate background fluorescence from the sample and therefore improves analytical accuracy and the signal-to-noise ratio. Under optimized conditions, there is a linear relationship between luminescence intensity and the concentration of paraoxon in the 1 pM to 1 μM range, with an ~0.1 pM limit of detection which is much lower than that of most existing methods. The phosphorescent probe was applied to determine OPs in spiked real samples. Figure

We present a sensitive and selective method for the determination of organophosphorus pesticides (OPs) based on the inhibition of the enzyme acetylcholinesterase (AChE). It is making use of quantum dots QDs of the type Mn-doped ZnS that display long-lived phosphorescence emission and act as optical probes for hydrogen peroxide (H₂O₂).     

Decolorization kinetics of the azo dye drimaren blue X3LR by laccase

Summary An extracellular Drimaren Blue X3LR decolorizing enzymatic activity was found in the crude filtrate of Funalia trogii grown by solid-state fermentation using wheat bran and soya bean waste. Decolorization of the azo dye Drimaren Blue X3LR by the crude filtrate and partially purified enzyme of Funalia trogii were investigated and compared. In the absence of additional redox mediator, maximum decolorization ratios of 81.33 % and 77.4 % were observed for Drimaren Blue X3LR using crude filtrate and partially purified enzyme respectively. Decolorization yield was found to be higher with crude enzyme preparations. Na₂S₂O₅ inhibited laccase and dye decolorizing enzyme activities but a significant peroxidase activity inhibition was not observed. Since the reaction was catalyzed in the absence of H₂O₂ as co-substrate, it could be concluded that this enzyme is not a peroxidase but may be a laccase.. The kinetic parameters of decolorization were calculated according to Michaelis constant (Km of 1.700 x 10^-5 mol dm^-3 and W_max = 8.02 x10^-7 mol dm^-3 sec^-1).     

Further Insight into the Mechanism of the Irreversible Inhibition of Histidine Ammonia‐Lyase by L‐Cysteine and Dioxygen

Histidine ammonia‐lyase (HAL) was irreversibly inhibited by L ‐cysteine at pH 10.5 under aerobic conditions. The inhibited enzyme, still in its intact conformation, showed an absorption maximum at 338 nm. Upon denaturation, followed by pronase digestion, two main chromophoric products 1 and 2 (Figs. 4 and 5, resp.) could be isolated with absorption maxima at 335 and 332 nm, respectively. As determined by MALDI‐TOF mass spectrometry and ¹H‐NMR spectroscopy, in product 1 one of the methylidene H‐atoms of the 3,5‐dihydro‐5‐methylidene‐4H‐imidazol‐4‐one (formerly called 4‐methylideneimidazol‐5‐one; MIO) prosthetic group was substituted by one of the amino groups of L ‐ cystine, while in product 2 the ε‐amino group of L ‐lysine was the analogous substituent. Acid‐catalyzed hydrolysis of product 1 gave compound 3 whose chromophore (λ_max 310 nm) was that of 3,5‐dihydro‐5‐(4‐hydroxymethylidene)‐4H‐imidazol‐4‐one, i.e., of a vinylogous acid. These results support our previous proposal that, in the first step, the L ‐cysteine S‐atom attacks the prosthetic electrophile (Scheme 2). The resulting nucleophilic enolate captures O₂ to form a peroxide. On the basis of the present results, we postulate that the observed products 1 – 3 arise from a vinylogous thioester 4 , which is formed in the conformationally intact inhibited enzyme by an electrocyclic reaction eliminating H₂O₂.     

Photoinduced formation of hydrogen peroxide in aqueous solutions of adenine derivatives at 77 K

The amount of hydrogen peroxide in aqueous solutions of adenine (A), adenosine (Ado), cytidine (Cyt), and thymine (T) containing 0.1 M NaCl and irradiated with near-UV light at 77 K is determined. It is established by comparing the results to data obtained earlier that the amount of H₂O₂ detected in the defrosted samples following identical irradiation falls in the order Ado > adenosine-5′-diphosphate (ADP) > A >> Cyt. The formation of H₂O₂ was not detected for T. The formation of H₂O₂ in solutions of adenine derivatives was observed when the samples were irradiated with light having wavelengths in the ranges λ = 240–400 nm and 290–450 nm. The latter covers only the long wave absorption range of these compounds. It is shown that the change in the intensity of irradiation that strongly affected the intensity оf EPR signals of irradiated samples prior to defrosting affected the amount of detected H₂O₂ only slightly, and the effect was not unidirectional. The results from determining H₂O₂ in the samples of adenine derivatives are compared to estimates of the content of free peroxyl radicals, obtained by analyzing EPR spectra. Plausible mechanisms of the processes are discussed.     

Preparation,photocatalytic activity and mechanism of nano-Titania/Nafion hybrid membrane

Nano-Titania/Nafion (TiO₂/Nafion) hybrid membranes were prepared by recasting, using Nafion solution and TiO₂ anatase hydrosol as the raw materials. The microstructure of the hybrid membrane was characterized by X-ray diffraction, high-resolution transmission electron microscopy (HR-TEM), X-ray Photoelectron Spectroscopy and Fourier Transform Infrared Spectroscopy (FT-IR). The photocatalytic properties of TiO₂/Nafion hybrid membranes were evaluated. Furthermore, endurance of photocatalytic activity of the hybrid membrane was investigated. The results indicate that the TiO₂ Nanoparticles are bounded to Nafion molecule via Ti-O-S bonds and the formed flocculates are distributed homogeneously throughout the recasting Nafion membrane, while the initial pure anatase TiO₂ nanoparticles remain intact in re-crystallized membrane. The hybrid membranes possessed excellent photocatalytic activities with and without H₂O₂. Moreover, the degradation of photocatalytic activities has been better controlled with the presence of H₂O₂.     

Electrochemical study of butylate: application to the analysis of water

A biochemical gas-sensor (bio-sniffer) was constructed for convenient measurement of odourless hydrogen peroxide (H₂O₂) vapour, which is harmful to skin and mucous membranes. An enzyme-immobilized membrane was fabricated by spreading the mixture of catalase and photo-crosslinkable polymer on a dialysis membrane. An H₂O₂ biosensor was constructed by attaching this catalase-immobilized membrane to the sensitive top of a Clark-type oxygen electrode, and the oxygen generation from the decomposition of H₂O₂ catalysed by catalase was measured amperometrically. This biosensor was first applied to the measurement of H₂O₂ solution and was able to quantify the concentrations of H₂O₂ solution from 0.02 to 10.0?mmol?L^?1. Then, this biosensor was applied to gaseous phase as a bio-sniffer and was able to detect the odourless H₂O₂ vapour with the calibration range from 0.5 to 30?ppm, where the threshold limit value assigned by the American Conference of Governmental Industrial Hygienists (1?ppm) is covered.     

Fluorescence assay of catecholamines based on the inhibition of peroxidase-like activity of magnetite nanoparticles

We report a fluorescence approach for the highly selective and sensitive detection of catecholamines using magnetite nanoparticles (Fe₃O₄ NPs) in the presence of Amplex UltraRed (AUR) and H₂O₂. Fe₃O₄ NPs catalyze H₂O₂-mediated oxidation of AUR. The resulting product fluoresces (excitation/emission maxima, ca. 568/587 nm) more strongly, relative to AUR. When catecholamines bind to Fe₃O₄, the complexes that are formed induce decreased activity of Fe₃O₄ NPs, mediated through the coordination between Fe³⁺ on the NP surface and the catechol moiety of catecholamines. As a result, Fe₃O₄ NPs-catalyzed H₂O₂-mediated oxidation of AUR is inhibited by catecholamines. The limits of detection for dopamine (DA), l-DOPA, norepinephrine, and epinephrine were 3 nM, 3 nM, 3 nM, and 6 nM, respectively. The Fe₃O₄ NPs-H₂O₂-AUR probe exhibited high selectivity (>1000-fold) toward catecholamines over other tested biomolecules that commonly exist in urine. Four catecholamines had similar sensitivity because the inhibition of the Fe₃O₄ NPs activity relies on the presence of the catechol moiety. This approach also allowed the determination of tyrosinase activity because tyrosinase catalyzes the conversion of l-tyrosine to l-DOPA. We validated the practicality of the use of the Fe₃O₄ NPs-H₂O₂-AUR probe for the determination of the concentrations of DA in urine samples.     

Synthesis,crystal structure,enzyme inhibition,DNA protection,and antimicrobial studies of di- and triorganotin(IV) derivatives of 2-thiopheneacetic acid

A series of organotin(IV) complexes, [Bu₂Sn(C₆H₅O₂S)₂] (1), [Bu₃Sn(C₆H₅O₂S)] (2), [Oct₂Sn(C₆H₅O₂S)₂] (3) of 2-thiopheneacetic acid (HL) have been prepared and characterized through FT-IR and NMR spectroscopy. The crystal structure of 2 has been confirmed by X-ray single crystal analysis, in which tin adopts a trigonal bipyramidal geometry. The synthesized complexes have been screened for antibacterial, DNA protection, and enzyme inhibition activities against acetylcholinesterase as well as butylcholenesterase.     

Mesoporous cadmium sulfide nanoparticles derived from a new cadmium anthranilato complex: Characterization and induction of morphological abnormalities in pathogenic fungi

A cadmium complex of the general formula Cd(C₁₃H₉O₂NCl)₂(H₂O)₂ {C₁₃H₉O₂NCl = 2-(4-chlorophenylamino)benzoate} was synthesized and characterized regarding its CHN data, solution molar conductivity and spectroscopic (UV–Vis. and IR) properties. Cadmium sulfide nanoparticles (CdS NPs) were grown form the microcrystalline complex and thiourea via a hydrothermal route. The as-prepared NPs were assigned based on X-ray powder diffraction (PXRD), transmission electron microscopy (TEM), field emission scanning electron microscopy (FE-SEM) and Brunauer – Emmett – Teller (BET) surface area measurements. The CdS absorption and emission spectra were also recorded that revealed an energy gap of 2.47 eV and large Stokes shift of 130 nm. For the as-prepared NPs, the measurements have also indicated a mesoporous structure and an average particle size of 20–28 nm associated with an average pore diameter of 11.21 nm. The as-synthesized CdS NPs acted as antifungal controlling agent against human and plant pathogenic fungi of serious environmental and health concerns. The NPs at concentration of 200 ppm inhibited several fungi with inhibition efficiency of 100% against Aspergillus ustus Au-28. The nanoparticles induced morphological abnormalities in fungal mycelia, conidia and vesicle. Additionally, they inhibited the conidia septum formation, accelerated the chlamydospores generation and enlarged the yeast cells.     

Analytical use of a biochemical luminous membrane

Luminous membranes were prepared by immobilizing peroxidase (POD) to collagen matrix. The POD luminous membrane generated luninescence in the presence of luminol and H₂O₂, and the peroxide was determined in the concentration range 10-⁶-10-³ M by following luminescence emitted from the membrane. Glucose was determined using a luminous membrane in which POD and glucose oxidase (GOD) were coimmobilized. The luminous membranes appear to be feasible for the determination of enzyme substrates and enzyme activity.     

A Combined Experimental and Theoretical Approach toward the Development of Optimized Luminescent Carbostyrils

The synthesis and photophysical data of new carbostyrils (quinoline‐2(1H)‐ones) with the longest hitherto observed absorption‐ and emission wavelengths are described. Introduction of 6‐amino, 7‐MeO, and 4‐(CF₃) substituents enabled us to rise the absorption and fluorescence maxima up to 414 and 557 nm, respectively. Supported by semi‐empirical and ab initio calculations, the 6,7‐(1,4‐diazine)‐fused carbostyril 23b displayed absorption maxima at up to 440 nm, with quantum yields of up to 0.9 and large Stokes shifts (>100 nm), comparable to the best coumarin chromophores known. The new fluorophore is neither pH‐sensitive between pH 6 and 10 nor susceptible to O₂ quenching. At pH 3, the emitted light appears greenish‐white, which arises from three different stages of protonation.     

Porous Molybdenum Carbide Nanostructured Catalyst toward Highly Sensitive Biomimetic Sensing of H2O2

There are great challenges to fabricate a highly selective and sensitive enzyme‐free biomimetic sensor. Herein for the first time a unique nanostructure of porous molybdenum carbide impregnated in N‐doped carbon (p‐Mo₂C/NC) is synthesized by using SiO₂ nanocrystals‐templating method and is further used as an enzyme‐free electrochemical biosensor toward highly selective, sensitive detection of H₂O₂, of which the limit of detection, dynamic detection range and sensitivity accomplish as 0.22 μM, 0.05–4.5 mM and 577.14 μA mM^?1 cm^?2, respectively, and are much superior to the non‐porous molybdenum carbide impregnated in N‐doped carbon (Mo₂C/NC). The sensor is also used to monitor H₂O₂ released from A549 living cells. This work holds a great promise to be used to monitor the presence of H₂O₂ in biological research while offering an important knowledge to design a highly selective and sensitive biomimetic sensor by synthesizing a porous catalyst to greatly improve the reaction surface area rather than conventionally only relying on dispersing the catalyst material into porous carbon substrate.     

Bioimprinted protein exhibits glutathione peroxidase activity

A strategy for design of bioimprinted proteins with glutathione peroxidase (GPX) activity has been proposed. The proteins imprinted with a glutathione derivative were converted into selenium-containing proteins by chemical modifying the reactive hydroxyl groups of serines followed by sodium hydrogen selenide displacement. These selenium-containing proteins exhibited remarkable GPX activities and the GPX activities of reduction of H₂O₂ by glutathione (GSH) were found to be 101-817 U μmol⁻¹, which approaches the activity of a selenium-containing catalytic antibody elicited by a hapten similar to our template. The steady state kinetic study for imprinted protein catalysis revealed Michaelis-Menten kinetics for both H₂O₂ and GSH, e.g. the pesudo-first-order rate constant k_cat (H₂O₂) and the apparent Michaelis constant K_m (H₂O₂) at 1 mM GSH were calculated to be 784 min⁻¹ and 1.24×10⁻³ M, respectively, and the apparent second-order rate constant k_cat (H₂O₂)/K_m (H₂O₂) was determined to be 6.33×10⁵ (M min)⁻¹. The kinetics and the template inhibition showed that the strategy might be a remarkably efficient one for generating artificial enzyme with GPX activity.