Pyrophosphate as substrate for alkaline phosphatase activity: A convenient flow‐injection chemiluminescence assay

A sensitive and convenient flow‐injection chemiluminescence (FI‐CL) turn‐on assay for alkaline phosphatase (ALP) activity without any label and synthesis is developed. Cu²⁺ can catalyze the luminol–H₂O₂ CL reaction. Pyrophosphate (PPi) can chelate Cu²⁺ and therefore the Cu²⁺‐mediated luminol‐H₂O₂ CL reaction is inhibited. The addition of ALP can catalyze the hydrolysis of PPi into phosphate ions, Cu²⁺ is released and the chemiluminescence recovers. A detection limit of 1 mU/mL ALP is obtained.     

What do we measure with luminol-, lucigenin- and penicillin-amplified chemiluminescence? 1. Investigations with hydrogen peroxide and sodium hypochlorite

Evidence is provided that the amplifiers luminol and lucigenin react with different reactive oxygen species (ROS), depending on the ROS-generating system used. H₂O₂ is used to produce calibration curves for luminol- and lucigenin-amplified chemiluminescence. With this chemiluminescence generator we characterized the specificity and sensitivity of luminol- and lucigenin-amplified chemiluminescence and also studied penicillin G, a known enhancer of luminol-amplified chemiluminescence. The combination of luminol and lucigenin in reciprocally changing concentrations is effective in an additive manner, but the weak amplifier penicillin increases luminol-amplified chemiluminescence distinctly more than in an additive manner in different combinations. Lucigenin-amplified chemiluminescence is increased by penicillin at about 1% of the optimum concentration of penicillin; increasing concentrations of penicillin are less and less effective. On the other hand, low lucigenin concentrations enhance penicillin-amplified chemiluminescence at optimum penicillin concentrations more than in an additive manner. Fe²⁺ does not alter luminol-, lucigenin- or penicillin-amplified chemiluminescence. Co²⁺ increases luminol-amplified chemiluminescence by a factor of 100. Lucigenin- and penicillin-amplified chemiluminescence are minimally enhanced by Co²⁺. Cu²⁺ enhances luminol-amplified chemiluminescence with increasing concentrations by a factor of 1000. Lucigenin-amplified chemiluminescence increases also by the factor of 1000, but the concentration–reaction curve is not as steep. NaOCl enhances H₂O₂/Fe²⁺-driven luminol-amplified chemiluminescence in a concentration-dependent manner by a factor of 10⁴ (in the highest concentration of 10 mmol/L) and lucigenin amplified chemiluminescence only by a factor of about 25. Catalase (CAT) abolishes luminol-, lucigenin- and penicillin-amplified chemiluminescence completely, whereas superoxide dismutase (SOD) has no effect on luminol- or penicillin-amplified chemiluminescence, but enhances lucigenin-amplified chemiluminescence five-fold increasingly with increasing SOD activity. © 1998 John Wiley & Sons, Ltd.     

Chemiluminescence of human bloodstream monocytes and neutrophils: An unusual oxidant(s) generated by monocytes during the respiratory burst

Maximal rates of O and H₂O₂ production by human bloodstream monocytes activated during the respiratory burst by phorbol ester were only about 10% of those of neutrophils. Furthermore, monocytes possess only about 5% of the myeloperoxidase activity of neutrophils and so can only produce low levels of HOCI and related compounds. These combined reductions in O generating ability and lower myeloperoxidase levels result in low luminol chemiluminescence stimulated during the respiratory burst of monocytes. However, although monocytes generate much lower levels of O and H₂O₂ than neutrophils, these cells produce comparable rates of PMA-stimulated lucigenin chemiluminescence. Hence, this assay does not accurately reflect the production of either of these two oxidants by activated phagocytes, and further lucigenin must react with some other oxidant(s) via a process which leads to photon emission. This oxidant(s) is not O, H₂O₂, · OH, ¹O₂ or NO, but is derived from O generated during the respiratory burst and is generated in greater quantities by activated monocytes compared with neutrophils. Thus, lucigenin chemiluminescence is an indirect measure of superoxide release.     

Chemiluminescence of lucigenin by electrogenerated superoxide ions in aqueous solutions

The chemiluminescence reaction of lucigenin (Luc²⁺?2NO₃^?, N,N′‐dimethyl‐9,9′‐biacridinium dinitrate) at gold electrodes in dioxygen‐saturated alkaline aqueous solutions (pH 10) was investigated in detail by the use of electrochemical emission spectroscopy. We noted that both O₂ and Luc²⁺ are reduced on a gold electrode in aqueous solution of pH 10 in almost the same potential region. From this fact, we expected chemiluminescence based on a radical–radical coupling reaction of superoxide ion (O₂·^?) and one‐electron reduced form of Luc²⁺ (Luc·⁺, a radical cation). Chemiluminescence was actually observed in the potential range where O₂ and Luc²⁺ were simultaneously reduced at the electrodes. The effects were examined upon addition of enzymes, i.e. superoxide dismutase (SOD) and catalase, into the solution and the substitution of heavy water (D₂O) for light water (H₂O) as a solvent on the chemiluminescence. In the presence of native and active SOD, chemiluminescence was completely absent. On the other hand, chemiluminescence was observed, unchanged in the presence of either denatured and inert SOD or catalase. In addition, the amount of chemiluminescence in D₂O solution was about three times greater than that in H₂O solution. These results, together with cyclic voltammetric results, suggest that O₂·^? participates directly in the chemiluminescence but H₂O₂ does not, and the chemiluminescence results from the coupling reaction between O₂·^? and Luc^·+ under the present experimental conditions. These chemically unstable species, O₂·^? and Luc·⁺, are produced during the simultaneous electroreduction of O₂ and Luc²⁺. The coupling reaction between those radical species would lead to the formation of a dioxetane‐type intermediate and, finally, to chemiluminescence. The chemiluminescence reaction mechanism is discussed. Copyright © 2002 John Wiley & Sons, Ltd.     

STUDIES ON NITRIC OXIDE FREE RADICALS GENERATED FROM POLYMORPHONUCLEAR LEUKOCYTES (PMN) STIMULATED BY PHORBOL MYRISTATE ACETATE (PMA)

The interaction of NO and O^?₂free radicals generated from PMA (phorbol myristate acetate)-stimulated PMN (polymorphonuclear leukocytes) was studied by a nitroxide spin trap, DMPO (5,5-dimethyl-1-pyrroline-1-oxide). It was found that addition of L-arginine to the system would significantly decrease the trapped O^?₂by DMPO and addition of N^G-monomethyl-arginine (NGMA) would significantly increase the trapped O^?₂by DMPO. It was proved that the formation of ONOO^?by the reaction of NO and O^?₂was the main reason for the decrease of trapped O^?₂in the experiment with xanthine/xanthine oxidase and irradiation of riboflavin systems. The yield of NO during this process was calculated. The generation dynamic of NO was studied by a luminol-dependent chemiluminescence technique and it was found that after stimulation of PMN by PMA, there would be an immediate, significant chemi-luminescence, which came mainly from the active oxygen free radicals generated by PMN. If L-arginine was added to this system, the chemiluminescence would increase about 100-fold, but NGMA inhibited the increase of the chemiluminescence. Ten minutes after addition of L-arginine, this increase did not change, the chemiluminescence peak decreased gradually, but the half life increased. The ESR and chemiluminescence properties of NO and ONOO^?synthesized were also studied in model systems.     

Light generation with Fenton's reagent its relationship to granulocyte chemiluminescence

A simple chemical system consisting of FeSO₄ and H₂O₂ (Fenton's reagent) was shown to emit light (chemiluminescence). The addition of tryptophan to the reaction markedly enhanced light production. Very little chemiluminescence was observed when H₂O₂ was omitted from the reaction and when ferric, instead of ferrous, ions were used. Hydroxyl radical (OH^.) and singlet oxygen (¹Δ_gO₂) quenchers suppressed chemiluminescence of the FeSO₄ + tryptophan + H₂O₂ system; and, deuterium oxide (²H₂O) enhanced chemiluminescence of both FeSO₄ reactions. These observations suggest that a radical chain reaction involving both OH^. and ¹Δ_gO₂ is responsible for the chemiluminescent reactions. Six iron-containing proteins, some of which are located within granulocytes, all emitted light in the presence of H₂O₂. Since iron and H₂O₂ are present in metabolically stimulated granulocytes, it is likely that chemiluminescent reactions similar to the ones demonstrated in this study account for part of the chemiluminescence of activated granulocytes.     

Bio-synthesis of PGD2 and TXB2 by rabbit blood monocytes

A method for the preparation of a highly purified sample of rabbit blood monocytes is described. The metabolism of arachidonic acid (AA) in these cells was studied. Mononuclear cells were prepared by centrifugation on Ficoll-Paque gradients and the monocytes were obtained by further centrifugation and adherence onto plastic culture dishes. These procedures provided a preparation which contained 95% monocytes (non-specific esterase positive). Incubation of [1-¹⁴C]-AA with these cells produced four major metabolites which were separated by TLC; these corresponded to prostaglandin (PG) D₂, thromboxane (TX) B₂, 12-hydroxyheptadecatrienoic acid (HHT) and 12-/15- hydroxyeicosatetraenoic acid (HETE). A minor product which co-migrated with PGE₂ was also detected but neither 6-keto-PGF_1α nor PGF_2α were detected. Also, there was no evidence of the formation of 5-lipoxygenase products (5-HETE and LTB₄) by rabbit monocytes with or without calcium-ionophore A23187-stimulation. The production of PGD₂, TXB₂ and PGE₂ was further confirmed by analyzing [³H]-AA metabolites using high-performance liquid chromatography (HPLC) with tritiated standards as references. The biosynthesis of these compounds from endogenous substrate in A23187-stimulated monocytes was confirmed by specific radioimmunoassays with or without prior HPLC separation. The synthesis of immunoreactive LTB₄ and LTC₄ by A23187-stimulated cells was also monitored and found to be relatively low. The synthesis of PGD₂, TXB₂ and PGE₂ from both exogenous and endogenous substrate was suppressed by treatment of the monocytes with indomethacin (10⁻⁶ M).     

Pharmacological characterization of metabotropic glutamate receptors in cultured cerebellar granule cells

A detailed pharmacological characterization of metabotropic glutamate receptors (mGluR) was performed in primary cultures of cerebellar granule cells at 6 days in vitro (DIV). The rank order of agonists induced polyphosphoinositide (PPI) hydrolysis (after correcting for the ionotropic component in the response) was as follows: in terms of efficiency, Glu>quisqualate (quis)=ibotenate (ibo)>(1_S,3_R)-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD)>-methyl-amino-l-alanine (BMAA) and in terms of potency, quis>ACPD>Glu>ibo=BMAA. Ionotropic excitatory amino acid (EAA) receptor agonists, such as -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) were relatively inactive (in the presence of Mg²⁺). Quis and ACPD-induced PPI hydrolysis was unaffected by ionotropic Glu receptor antagonists, but was inhibited, in part by L-2-amino-3-phosphonopropionate (AP3). In contrast, Glu-or ibo- induced PPI hydrolysis was reduced, in part, by both AP3 and NMDA receptor antagonists. Characteristic interactions involving different transmitter receptors were noted. PPI hydrolysis evoked by quis and 1_S,3_R-ACPD was not additive. In contrast, PPI hydrolysis stimulated by quis/ACPD and carbamylcholine was additive (indicating different receptors/transduction pathways). In the presence of Mg²⁺, the metabotropic response to quis/AMPA and NMDA was synergistic (this being consistent with AMPA receptor-induced depolarization activating NMDA receptor). On the other hand, in Mg²⁺-free buffer the effects of quis and NMDA, at concentrations causing maximal PPI hydrolysis, were additive (indicating that PPI hydrolysis was effected by two different mechanisms). Thus, in cerebellar granule cells EAAs elicit PPI hydrolysis by acting at two distinct receptor types: (i) metabotropic Glu receptors (mGluR), with pharmacological characteristics suggesting the expression of a unique mGluR receptor that shows certain similarities to those observed for the mGluR1 subtype (Aramori and Nakanishi, 1992) and (ii) NMDA receptors. The physiological agonist, Glu, is able to stimulate both receptor classes.Abbreviations ACPD (1_S,3_R)-1-amino-cyclopentane-1,3-dicarboxylic acid - AMPA -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid - AP₃ L-2-amino-3-phosphono-propionate - AP₅ D-2-amino-5-phosphonopentenoate - BMAA -methyl-amino-L-alanine - DIV days in vitro - DNOX 6,7-dinitroouinoxoline-2,3-dione - EAA excitatory amino acids - Glu glutamate - InsP inositol monophosphate - mGluR metabotropic glutamate receptors - MK-801 (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohept-5,10-imine hydrogen maleate - NMDA N-methyl-D-aspartate - PPI polyphosphoinositide - quis quisqualate     

A comparative study of ethylene oxidation inVicia faba andMycobacterium paraffinicum

Vicia faba L. ‘Herz Freya’ (fababean) cotyledons andMycobacterium paraffinicum Bardane strain (MPB) cells were studied to describe and compare physiological and biochemical factors regulating ethylene oxidation. Both organisms demonstrated a linear rate of ethylene uptake as a function of concentration from 1 ppm to 1,000 ppm. CO₂ did not influence ethylene oxidation by either organism. Zero degree temperatures and CO inhibited ethylene oxidation by fababeans but not by MPB. An N₂ gas phase blocked ethylene consumption by fababeans. In contrast, MPB continued to consume ethylene at a reduced rate under anaerobic conditions. Hydrocarbon oxidation was limited to alkenes. Alkanes were not oxidized by either organism. Both organisms were sensitive to diethyldithiocarbamic acid, o-phenanthroline, carbonyl cyanidem-chlorophenyl hydrazone, and CS₂. The possibility that CS₂ acted as a suicide substrate is discussed. Evidence is presented that hydrocarbon gas oxidation by fababeans is not a part of, or reflection of, the way ethylene acts as a hormone.     

Methylglyoxal-modified arginine residues — a signal for receptor-mediated endocytosis and degradation of proteins by monocytic THP-1 cells

Non-enzymatic glycosylation or glycation of proteins to form advanced glycation endproducts (AGE) has been proposed as a process which provides a signal for the degradation of proteins. Despite this, the AGE which act a recognition factor for receptor-mediated endocytosis and degradation of glycated proteins by monocytes and macrophages has not been identified. Methylglyoxal, a reactive α-oxoaldehyde and physiological metabolite, reacted irreversibly with arginine residues in proteins to form N_δ-(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine and N_δ-(5-methyl-4-imidazolon-2-yl)ornithine residues. Human serum albumin minimally-modified with methylglyoxal (MG_min-HSA) was bound by cell surface receptors of human monocytic THP-1 cells in vitro at 4°C: the binding constant K_d value was 377±35 nM and the number of receptors per cell was 5.9±0.2×10⁵ (n=12). N_α-Acetyl-N_δ-(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine displaced MG_min-HSA from THP-1 cells, suggesting that the N_δ-(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine residue was the receptor recognition factor. At 37°C, MG_min-HSA was internalised by THP-1 cells and degraded. Similar binding and degradation of human serum albumin modified by glucose-derived AGE was found but only when highly modified. MG_min-HSA, therefore, is the first example of a protein minimally-modified by AGE-like compounds that binds specifically to monocyte receptors. The irreversible modification of proteins by methylglyoxal is a potent signal for the degradation of proteins by monocytic cells in which the arginine derivative, N_δ-(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine, is the receptor recognition factor. This factor is not present in glucose-modified proteins.     

Determination of H2O2-dependent generation of singlet oxygen from human saliva with a novel chemiluminescence probe

Singlet oxygen (¹O₂) has been shown to play an important role in salivary defense system, but its generation process and level from human saliva remain uncertain due to the lack of a reliable detection method. We have previously reported 4,4′(5′)-bis[2-(9-anthryloxy)ethylthio]tetrathiafulvalene (BAET) as a novel chemiluminescence probe for ¹O₂. In this work, the probe is successfully used to characterize H₂O₂-dependent generation of ¹O₂ from saliva in real time. However, the yield of ¹O₂ is found to be very low, for example, being about 0.13 nmol from 200 μL saliva in the presence of 1 mM of hydrogen peroxide over a 5-s reaction period. The result is also compared with that obtained with another ¹O₂ probe 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (CLA), demonstrating that, besides ¹O₂, the other reactive oxygen species such as hydroxyl radical may also be involved in the reaction of saliva with H₂O₂. Furthermore, the present study shows that the selectivity of BAET for ¹O₂ is much higher than that of CLA and thus BAET is highly suited for the detection of ¹O₂ in the presence of other reactive oxygen species in biological systems.     

A comparative study of ethylene oxidation inVicia faba andMycobacterium paraffinicum

Vicia faba L. Herz Freya (fababean) cotyledons andMycobacterium paraffinicum Bardane strain (MPB) cells were studied to describe and compare physiological and biochemical factors regulating ethylene oxidation. Both organisms demonstrated a linear rate of ethylene uptake as a function of concentration from 1 ppm to 1,000 ppm. CO₂ did not influence ethylene oxidation by either organism. Zero degree temperatures and CO inhibited ethylene oxidation by fababeans but not by MPB.An N₂ gas phase blocked ethylene consumption by fababeans. In contrast, MPB continued to consume ethylene at a reduced rate under anaerobic conditions. Hydrocarbon oxidation was limited to alkenes. Alkanes were not oxidized by either organism. Both organisms were sensitive to diethyldithiocarbamic acid, o-phenanthroline, carbonyl cyanidem-chlorophenyl hydrazone, and CS₂. The possibility that CS₂ acted as a suicide substrate is discussed. Evidence is presented that hydrocarbon gas oxidation by fababeans is not a part of, or reflection of, the way ethylene acts as a hormone.     

A study on the enzyme catalysed enantioselective hydrolysis of methyl 2-methyl-4-oxopentanoate,a precursor of chiral γ-butyrolactones

Porcine pancreas lipase (PPL) resolution of the α-methyl group of racemic methyl 2-methyl-4-oxopentanoate, a valuable synthetic precursor of fragrances and marine natural products, was enhanced by salt modulation of the enzymatic hydrolysis. For the enantioselective hydrolysis of the title ester, PPL was selected from a series of esterases and lipases, and its enantioselectivity was evaluated by changing the reaction medium parameters. The use of 1.6?mol L^–1 sodium sulfate in phosphate buffer (pH 7.2) improved the enantioselectivity allowing the formation of methyl (2R)-(+)-2-methyl-4-oxopentanoate and (2S)-(–)-2-methyl-4-oxopentanoic acid with an enantiomeric excess of >99% and 71%, respectively. The study showed that a modulation of PPL enantioselectivity could be achieved by using kosmotropic salts in the reaction media. The present method consists of a practical and low-cost option to improve enzymatic kinetic resolution reactions.     

Interaction of trout hemoglobin with H2O2: a chemiluminescence study

Erythrocytes from trout Salmo irideus are characterized by four different hemoglobin components (HbI, HbII, HbIII and HbIV), HbI and HbIV being predominant. In this study we describe the interaction between trout hemoglobin (HbI and HbIV) and H₂O₂ using a chemiluminescence assay. Our data show that the reaction of hemoglobins with H₂O₂ produces a time-limited and significant increase of chemiluminescence signal. The half-life of the decay of this chemiluminescence signal was characteristic for each type of hemoglobin used. These results indicate the formation of excited molecules related to the interaction between trout hemoglobin and H₂O₂. © 1997 John Wiley & Sons, Ltd.     

New type of arrangement of rare-earth quinolinolate. Molecular structure of scandium 2-methyl-8-quinolinolate

The tetrakis 2-methyl-8-quinolinolate scandium complex [Sc(q^Me)₄(H)] (1) have been prepared by the reaction of ScCl₃ with 2-methyl-8-quinolinol (Hq^Me) in methanol in the presence of aqueous ammonia. The X-ray diffraction analysis has shown that a molecule of (1) has a propeller like shape herein the Sc(III) ion is surrounded by four methylquinolinolate ligands two of which are chelate but the other two are monodentate and one of these monodentate methylquinolinolate ligands contains hydrogen atom at nitrogen atom. Furthermore there are two molecules of water per one complex molecule not coordinated to the Sc cation that is not typical for rare-earth compounds.     

The effect of dielectric polarization of the electrode on anomalous temperature effects in the electrical double layer

Monte Carlo simulations and a modified Poisson–Boltzmann (MPB) theory are used to investigate the temperature dependence of the capacitance (around the potential of zero charge) of an electric double layer in the presence of surface polarization due to a dielectric boundary. Within the context of the restricted primitive model planar double layer, whose solvent dielectric constant is ε₂, the cases when the electrode is an insulator (ε₁ = 1), when the electrode and the electrolyte have the same permittivity (ε₁ = ε₂, no polarization), and when the electrode is a conductor (ε₁ → ∞) are studied for the case where the electrolyte concentration is 0.1 M. The simulations reveal a capacitance anomaly, that is, a positive temperature dependence of the capacitance at low temperatures for the former two situations. The MPB theory also shows this effect for these two situations and is in qualitative or better agreement with the simulation data. In these two cases, both the simulations and theory show a dramatic increase of the diffuse layer potential in the temperature regime where capacitance anomaly occurs. However, in the latter situation, where the electrode is metallic, the capacitance always has a negative temperature derivative for the MPB theory and probably also for the simulation data.     

Determination of horserdish peroxidase concentration using the chemiluminescence of Cypridina luciferin analogue, 2 methyl-6-(p-methyoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one

The chemiluminescence of the Cypridina luciferin analogue, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (MCLA) was observed at 462nm in the presence of horseradish peroxidase (HRP) and the total spectrum of light emitted was found to depend linearly on HRP concentration. Methods for the determination of HRP concentration using the chemiluminescence was investigated. HRP could be detected in the range from 100 pmol/L to 100nmol/L under the optimum condition, H₂O₂ (10mmol/L) and MCLA (10μmol/L) at pH 5.8.     

Quantitative analysis of CL-20 explosive by smartphone-based chemiluminescence method

A new smartphone-based chemiluminescence method has been introduced for the quantitative analysis of CL-20 (Hexanitroazaisowuertzitan) explosive. The solvent mixture, oxidizer agent, and concentration of the reactants were optimized using statistical procedures. CL-20 explosive showed a quenching effect on the chemiluminescence intensity of the luminol−NaClO reaction in the solvent mixture of DMSO/H₂O. A smartphone was used as a detector to record the light intensity of chemiluminescence reaction as a video file. The recorded video file was converted to an analytical signal as intensity luminescence–time curve by a written code in MATLAB software. Dynamic range and limit of detection of the proposed method were obtained 2.0–240.0 and 1.1 mg⋅L⁻¹, respectively, in optimized concentrations 1.5 × 10⁻³ mol⋅L⁻¹ luminol and 1.0 × 10⁻² mol⋅L⁻¹ NaClO. Precursors TADB, HBIW, and TADNIW in CL-20 explosive synthesis did not show interference in measurement the CL-20 purity. The analysis of CL-20 spiked samples of soil and water indicated the satisfactory ability of the method in the analysis of real samples. The interaction of CL-20 molecules and OCl⁻ ions is due to quench of chemiluminescence reaction of the luminol−NaClO.     

Inhibition of monoamine oxidase B by N-methyl-2-phenylmaleimides

Based on a recent report that 1-methyl-3-phenylpyrrolyl analogues are moderately potent reversible inhibitors of the enzyme monoamine oxidase B (MAO-B), a series of structurally related N-methyl-2-phenylmaleimidyl analogues has been prepared and evaluated as inhibitors of MAO-B. In general, the maleimides were more potent competitive inhibitors than the corresponding pyrrolyl analogues. N-Methyl-2-phenylmaleimide was found to be the most potent inhibitor with an enzyme–inhibitor dissociation constant (K_i value) of 3.49 μM, approximately 30-fold more potent than 1-methyl-3-phenylpyrrole (K_i = 118 μM). This difference in activities may be dependent upon the ability of the maleimidyl heterocyclic system to act as a hydrogen bond acceptor. This is in correspondence with literature reports which suggest that hydrogen bond formation is involved in stabilizing inhibitor–MAO-B complexes. Also reported here is a brief kinetic study of the hydrolysis of the N-methyl-2-phenylmaleimidyl analogues in aqueous solution. The findings of the inhibition studies are discussed with reference to the rate and extent of hydrolysis.     

The antioxidant action of 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-α]pyrazin-3-one (MCLA), a chemiluminescence probe to detect superoxide anions

The antioxidant effect of 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-α]pyrazin-3-one (MCLA), a Cypridina luciferin analog that acts as a chemiluminescence probe to detect O^⋅−₂, was investigated. MCLA produced a lag in oxygen consumption induced by cumene hydroperoxide in microsomes or by 2,2′-azobis (2-amidinopropane) dihydrochloride in liposomes and disappeared during the duration of the lag. MCLA profoundly inhibited the propagation reaction in Fe²⁺-dependent lipid peroxidation in liposomes, and MCLA disappearance accompanied by suppression of oxygen consumption markedly occurred in liposomes susceptible to peroxidation. Thiobarbituric acid-reactive substances in all systems used were also suppressed by MCLA dose dependently. These results indicate that MCLA has an antioxidant property through scavenging free radicals.