Evidence for a free radical chain mechanism in the reaction between peroxidase and indole-3-acetic acid at neutral pH

The oxidation of indole-3-acetic acid (IAA) catalyzed by horseradish peroxidase (HRP) in the absence of added H2O2 was studied at pH 7.4 using spectral and kinetic approaches. Upon addition of a hundred-fold excess of IAA to HRP the native enzyme was rapidly transformed to compound II (HRP-II). HRP-II was the predominant catalytic enzyme species during the steady state. No compound III was observed. HRP-II was slowly transformed to the stable inactive verdohemo-protein, P-670. A precursor of P-670, so-called P-940 was not detected. After the cessation of IAA oxidation there was neither oxygen consumption nor P-670 formation; the remaining HRP-II was spontaneously reduced to native enzyme. Single exponential kinetics were observed in the steady state for IAA oxidation, oxygen consumption and P-670 formation yielding identical first order rate constants of about 6 . 10(4) s(-1). A comparison of the rate of IAA oxidation by HRP-II in the steady state and in the transient state indicated that more than 1 3 of the IAA was oxidized non-enzymatically during the steady state, confirming that a free radical chain reaction is involved in the peroxidase-catalyzed oxidation of IAA. IAA oxidation stopped before IAA was completely consumed, which cannot be ascribed to enzyme inactivation because 30-50% of the enzyme was still active after the end of the reaction. Instead, incomplete IAA oxidation is explained in terms of termination of the free radical chain reaction. Bimolecular rate constants of IAA oxidation by HRP-I and HRP-II determined under transient state conditions were (2.2 +/- 0.1) x 10(3) M(-1) s(-1) and (2.3 +/- 0.2) x 10(2) M(-1) s(-1).     

Hydrogen peroxide is a mediator of indole-3-acetic acid/horseradish peroxidase-induced apoptosis

Recently, we reported that a combination of indole-3-acetic acid (IAA) and horseradish peroxidase (HRP) induces apoptosis in G361 human melanoma cells. However, the apoptotic mechanism involved has been poorly studied. It is known that when IAA is oxidized by HRP, free radicals are produced, and since oxidative stress can induce apoptosis, we investigated whether reactive oxygen species (ROS) are involved in IAA/HRP-induced apoptosis. Our results show that IAA/HRP-induced free radical production is inhibited by catalase, but not by superoxide dismutase or sodium formate. Furthermore, catalase was found to prevent IAA/HRP-induced apoptotic cell death, indicating that IAA/HRP-produced hydrogen peroxide (H2O2) may be involved in the apoptotic process. Moreover, the antiapoptotic effect of catalase is potentiated by NADPH, which is known to protect catalase. On further investigating the IAA/HRP-mediated apoptotic pathway, we found that the IAA/HRP reaction leads to caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage, which was also blocked by catalase. Additionally, we found that IAA/HRP produces H2O2 and induces peroxiredoxin (Prx) sulfonylation. Consequently, our results suggest that H2O2 plays a major role in IAA/HRP-induced apoptosis.     

Role of Phenolic Inhibitors in Peroxidase-mediated Degradation of Indole-3-acetic Acid

7-Hydroxy-2,3-dihydrobenzofuran derivatives, metabolites of a carbamate insecticide carbofuran, and five other phenolic inhibitors of indoleacetic acid (IAA) oxidase interfered with IAA-induced spectral change in the Soret band of horseradish peroxidase (HRP). The onset of IAA degradation required transformed HRP intermediates. The inhibitors, when added before IAA, protected HRP from reacting with IAA, thus preventing formation of highly reactive enzyme intermediates, and consequently, IAA degradation. When added after IAA, the inhibitors quickly reversed the IAA-induced spectral change of HRP and inhibited further IAA degradation.     

Intermediates formed by laser flash photolysis of [PtCl(6)](2-) in aqueous solutions.

The stationary photolysis of [PtCl(6)](2-) in aqueous solutions (10(-5)-10(-4) M) at the region of 313 nm leads to its photoaquation with a quantum yield of 0.19. Laser flash photolysis experiments (308 nm) provided evidence of the formation of Pt(iii) intermediates, namely [PtCl(4)(OH)(H(2)O)](2-) and [PtCl(4)](-), and Cl(2) (-) radical anions. The Pt(iii) complexes formed as a result of an intrasphere electron transfer from Cl(-) ligands to the excited Pt(iv) ion. However, the main ( approximately 90%) photolysis channel was not accompanied by the transfer of Cl atoms to the solvent bulk. The photoaquation of [PtCl(6)](2-) results from the back electron transfer in the secondary geminate pair, [PtCl(5)(H(2)O)](2-)-Cl. The relative yield of Pt(iii) intermediates, recorded after the completion of all processes in the geminate pair, was less than 10% of the number of disappearing initial [PtCl(6)](2-) complexes.     

Exposure to hypoxia primes the respiratory and metabolic responses of the epaulette shark to progressive hypoxia

The majority of vertebrates are not tolerant to hypoxia but epaulette sharks (Hemiscyllium ocellatum) living on shallow reef platforms appear to tolerate hypoxic periods during tidal fluctuations. The effects of progressive hypoxia on the metabolic and ventilatory responses of these elasmobranchs were examined in a closed respirometer. In order to determine whether repeated exposure to hypoxia primes these sharks to alter their metabolism, one group of sharks was exposed to repeated sub-lethal hypoxia, at 5% of air saturation, prior to respirometry. In response to falling oxygen concentration [O(2)], the epaulette shark increased its ventilatory rate and maintained its O(2) consumption rate (VO(2)) down to 2.2 mg O(2) l(-1) at 25 degrees C. This is the lowest critical [O(2)] ([O(2)](crit)) ever measured for any elasmobranch. After reaching the [O(2)](crit), the shark remained in the respirometer for a further 4-5 h of progressive hypoxia. Only after the [O(2)] fell to 1.0 mg l(-1) was there a decrease in the ventilatory rate followed by a rise in blood lactate levels, indicating that the epaulette shark responds to severe hypoxia by entering a phase of metabolic and ventilatory depression. Interestingly, hypoxia tolerance was dynamic because hypoxic pre-conditioning lowered the VO(2) of the epaulette shark by 29%, which resulted in a significantly reduced [O(2)](crit) (1.7 mg O(2) l(-1)), revealing that hypoxic pre-conditioning elicits an enhanced physiological response to hypoxia.     

Diffusion-based process for carbon dioxide uptake and isoprene emission in gaseous/aqueous two-phase photobioreactors by photosynthetic microorganisms

Photosynthesis for the generation of fuels and chemicals from cyanobacteria and microalgae offers the promise of a single host organism acting both as photocatalyst and processor, performing sunlight absorption and utilization, as well as CO(2) assimilation and conversion into product. However, there is a need to develop methods for generating, sequestering, and trapping such bio-products in an efficient and cost-effective manner that is suitable for industrial scale-up and exploitation. A sealed gaseous/aqueous two-phase photobioreactor was designed and applied for the photosynthetic generation of volatile isoprene (C(5)H(8)) hydrocarbons, which operates on the principle of spontaneous diffusion of CO(2) from the gaseous headspace into the microalgal or cyanobacterial-containing aqueous phase, followed by photosynthetic CO(2) assimilation and isoprene production by the transgenic microorganisms. Volatile isoprene hydrocarbons were emitted from the aqueous phase and were sequestered into the gaseous headspace. Periodic replacement (flushing) of the isoprene (C(5)H(8)) and oxygen (O(2)) content of the gaseous headspace with CO(2) allowed for the simultaneous harvesting of the photoproducts and replenishment of the CO(2) supply in the gaseous headspace. Reduction in practice of the gaseous/aqueous two-phase photobioreactor is offered in this work with a fed-batch and a semi-continuous culturing system using Synechocystis sp. PCC 6803 heterologously expressing the Pueraria montana (kudzu) isoprene synthase (IspS) gene. Constitutive isoprene production was observed over 192 h of experimentation, coupled with cyanobacterial biomass accumulation. The diffusion-based process in gaseous/aqueous two-phase photobioreactors has the potential to be applied to other high-value photosynthetically derived volatile molecules, emanating from a variety of photosynthetic microorganisms.     

Redox and ligand exchange reactions of potential gold(I) and gold(III)-cyanide metabolites under biomimetic conditions

Biomimetic pathways for the oxidation of [Au(CN)(2)](-), a gold metabolite, and further cyanation of the gold(III) products to form Au(CN)(4)(-) were investigated using 13C NMR and UV-Visible spectroscopic methods. Hypochlorite ion, an oxidant released during the oxidative burst of immune cells, was employed. The reaction generates mixed dicyanoaurate(III) complexes, trans-[Au(CN)(2)X(2)](-), where X(-) represents equilibrating hydroxide and chloride ligands, and establishes the chemical feasibility of dicyanoaurate oxidation by OCl(-) to gold(III) species. This oxidation reaction suggests a new procedure for synthesis of H[Au(CN)(2)Cl(2)]. Reaction of trans-[Au(CN)(2)X(2)](-) (X(-)=Cl(-) and Br(-)) or [AuCl(4)](-) with HCN in aqueous solution at pH 7.4 leads directly to [Au(CN)(4)](-) without detection of the anticipated [Au(CN)(x)X(4-x)](-)intermediates, which is attributed to the cis- and trans-accelerating effects of the cyanides. The reduction of [Au(CN)(4)](-) by glutathione and other thiols is a complex, pH-dependent process that proceeds through two intermediates and ultimately generates [Au(CN)(2)](-). These studies provide further insight into the possible mechanisms of an immunogenically generated gold(I)/gold(III) redox cycle in vivo.     

Activation of indole-3-acetic acid oxidase from horseradish and Prunus by phenols and H2O2

The enzyme-catalysed oxidation of indole-3-acetic acid (IAA) was sytematically investigated with respect to enzyme source and cofactor influence using differential spectrophotometry and oxygen uptake measurement. Commercially-available horseradish peroxidase (HRP) and a peroxidase preparation from Prunus phloem showed identical catalytic properties in degrading IAA. There was no lag phase of IAA oxidation with any of the reaction mixtures tested. Monophenols exhibited a much stronger stimulatory effect than inorganic cofactors, but during the incubation of IAA the phenols were also gradually oxidised. Hydrogen peroxide (H₂O₂) in combination with monophenols accelerated peroxidation of the monophenol and IAA oxidation simutaneously. Since photometric determination of IAA was affected by oxidation products of dichlorophenol or phenol contamination of the enzyme preparation used, the standard IAA absorption measurements appear to be susceptible to methodological errors. Under certain incubation conditions a catalase-like activity of HRP during the course of IAA oxidation was noted and substrate inhibition was observed above 1.5 × 10^\s-4 M IAA. Some concepts concerning the mode of activation of the enzyme-catalysed IAA oxidation are deduced from the experimental results.     

Enzymatic oxidation of dipyridamole in homogeneous and micellar solutions in the horseradish peroxidase-hydrogen peroxide system

Enzymatic oxidation of dipyridamole (DIP) by horseradish peroxidase-hydrogen peroxide system (HRP-H2O2) in aqueous and micellar solutions was carried out. The reaction was monitored by optical absorption and fluorescence techniques. In aqueous solution at pH 7.0 and pH 9.0, the disappearance of the characteristic bands of DIP centered at 400 nm and 280 nm was observed. A new strong band at 260 nm is observed for the oxidation product(s) with shoulders at 322 nm and 390 nm. A non-fluorescent product is formed upon oxidation. In cationic cethyl trimethyl-1-ammonium chloride (CTAC) and zwitterionic 3-(N-hexadecyl-N,N-dimethylammonium) propane sulfonate (HPS) micellar solutions the same results are observed: three, well-defined, isosbestic points in the optical spectra suggest the transformation between two species. In anionic micellar sodium dodecylsulfate solution (SDS), the appearance of a new band centered around 506 nm was observed, associated to a solution color change from the usual yellow to deep blue/violet, characteristic of a radical species associated to the one-electron oxidation of DIP to its cation radical (DIP*+), observed previously in electrochemical oxidation. Experiments of radical decay kinetics monitoring the absorbance change at 506 nm were performed and analyzed in the frame of a kinetic model taking into account the species both in homogeneous and micellar media. The reaction medium is composed of bulk solution, SDS micelle/solution interface and enzyme catalytic site(s). The variation of DIP*+ concentration was analyzed assuming: (1) synthesis of DIP*+ by HRP through one-electron oxidation; (2) decomposition of DIP*+ by further one-electron oxidation; (3) direct two-electron oxidation of DIP by HRP; (4) bimolecular DIP*+ disproportionation. The main results of the analysis are as follows: (1) kinetic data can be divided in two phases, an HRP active phase and another phase which proceeds in the absence of enzyme activity due to consumption of all H2O2; (2) the reactions of DIP*+ formation, DIP*+ decomposition and DIP two-electron oxidation are HRP concentration dependent; (3) since DIP*+ formation constant seems to be overestimated, it is proposed that two-electron oxidation is another source of DIP*+, through the comproportionation reaction. Evidences for this reaction were also observed previously in electrochemical experiments; and (4) the kinetic analysis provides evidences that the bimolecular reaction of DIP*+ takes place mainly in the absence of active HRP and in this phase the combination of, at least, two second-order kinetic processes is needed to model the experimental data. Our data suggest that HRP oxidizes DIP in general by a two-electron process or that the cation radical is very unstable so that the one-electron process is only detected in the presence of anionic surfactant, which stabilizes significantly the DIP*+ intermediate.     

Effect of yeast growth limitation on the respiratory chain, the economic coefficient and the critical oxygen concentration for respiration

The effect of the yeast growth limitation by oxygen on the economical coefficient (EC), the operation of the cyanide resistant electron transport pathway (CrETP), and the critical for respiration oxygen concentration concentration ([O2]cr) was studied. The operation of CrETP was found to differ among various yeasts growing on glucose: it could function during both the exponential phase and limitation of growth (Torulopsis candida), or only in the conditions of growth limitation (Candida tropicalis, C. mycoderma, C. lipolytica), sometimes for a very long period (Endomyces geotrichum); in certain cases (C. utilis), it cannot be detected at all. If the main respiratory chain is inhibited by cyanide (i. e. if only CrETP operates), the value of [O2]cr sharply increases; such an increase can be also found in the absence of cyanide but in the conditions of active operation of CrETP. Apparently, the value of [O2]cr is higher for cyanide resistant oxydase of the studied organisms than for cytochrome oxydase. A decrease in EC observed upon the limitation of yeast growth by oxygen (Lozinov et al., 1974) correlates with the appearance or intensification of CrETP. Therefore, the decrease of EC can be attributed to the operation of non-phosphorylating CrETP which occurs in all the studied yeasts (with an exception of C. utilis) when their growth is limited by oxygen.     

Correlated oscillations in glucose consumption, oxygen consumption, and intracellular free Ca(2+) in single islets of Langerhans

Micron-sized sensors were used to monitor glucose and oxygen levels in the extracellular space of single islets of Langerhans in real-time. At 10 mM glucose, oscillations in intraislet glucose concentration were readily detected. Changes in glucose level correspond to changes in glucose consumption by glycolysis balanced by mass transport into the islet. Oscillations had a period of 3.1 +/- 0.2 min and amplitude of 0.8 +/- 0.1 mM glucose (n = 21). Superimposed on these oscillations were faster fluctuations in glucose level during the periods of low glucose consumption. Oxygen level oscillations that were out of phase with the glucose oscillations were also detected. Oscillations in both oxygen and glucose consumption were strongly dependent upon extracellular Ca(2+) and sensitive to nifedipine. Simultaneous measurements of glucose with intracellular Ca(2+) ([Ca(2+)](i)) revealed that decreases in [Ca(2+)](i) preceded increases in glucose consumption by 7.4 +/- 2.1 s during an oscillation (n = 9). Conversely, increases in [Ca(2+)](i) preceded increases in oxygen consumption by 1.5 +/- 0.2 s (n = 4). These results suggest that during oscillations, bursts of glycolysis begin after Ca(2+) has stopped entering the cell. Glycolysis stimulates further Ca(2+) entry, which in turn stimulates increases in respiration. The data during oscillation are in contrast to the time course of events during initial exposure to glucose. Under these conditions, a burst of oxygen consumption precedes the initial rise in [Ca(2+)](i). A model to explain these results is described.     

UNDAMPED OSCILLATIONS OF PYRIDINE NUCLEOTIDE AND OXYGEN TENSION IN CHEMOSTAT CULTURES OF KLEBSIELLA AEROGENES

Klebsiella aerogenes was grown in chemostat culture with the pH controlled to ±0.01 and temperature to ±0.1°C. The oxygen tension of the culture was regulated by changing the partial pressure of oxygen in the gas phase and recorded by means of an oxygen electrode. Reduced pyridine nucleotide was monitored continuously in the culture by means of direct fluorimetry. On applying an anaerobic shock to the culture, damped oscillations in pyridine nucleotide fluorescence were obtained. Further anaerobic shocks decreased the damping and eventually gave rise to undamped oscillations of a 2–3 min period which continued for several days. These oscillations were paralleled by oscillations of the same frequency in respiration rate. The amplitude of the oscillations in the respiration rate was equivalent to only 1% of the total steady-state respiration, whereas that of pyridine nucleotide oscillations was equivalent to 10% of the total aerobic/anaerobic fluorescence response. The oscillations ceased on interrupting the glucose feed but restarted on adding excess glucose to the culture. Addition of succinate also restarted the oscillations so that they appear not to be of glycolytic origin. The frequency of oscillations varied with growth rate and conditions. Oscillations of much lower frequency were obtained under limited-oxygen and anaerobic conditions than under fully aerobic conditions. Under glucose-limited conditions, fluctuations were found in adenosine triphosphate (ATP) content which were in phase with the pyridine nucleotide oscillations, but under nitrogen-limited growth conditions no such fluctuations in ATP were observed. The primary oscillating pathway could not be identified but the mechanism would appear to be quite different from that involved in oscillations observed in yeast cells. The synchronization of oscillations and observations of negative damping could be explained by a syntalysis effect.     

Different pathways of the oxygen supply in the sapwood of young<Emphasis Type="Italic"> Olea europaea</Emphasis> trees

Gaseous transport through lenticels is widely accepted to be the main pathway for oxygen supply to the parenchymatous tissues of the wood. Circumstantial evidence exists that the oxygen required for respiration by these living cells can be obtained from the transpiration stream. However, there has been no functional confirmation of this role. To address this problem and to quantify the contribution of the different pathways to the oxygen supply of the sapwood, we have developed a three-electrode miniaturized oxygen-selective sensor to be implanted into the sapwood for long-term determination of the oxygen concentration. In spring, during the active growing season, the oxygen concentration of the sapwood of young olive (Olea europaea L.) trees changed from 80-90 micromol O(2) l(-1) around midday to 20-30 micromol O(2) l(-1) in the night. These concentrations correspond to a deficit of oxygen for the sapwood between 65-70% and 88-90% of an aqueous solution saturated with air. In the daylight hours, almost all the oxygen present in the sapwood was delivered by the transpiration stream, driven by the soil-plant-atmosphere water-potential gradient. During the night the diffusion of oxygen via the sap-filled lumina of the tracheids and vessels (xylary diffusion in the aqueous phase) accounted for about 87% of all the oxygen present, whereas only the remaining 13% was assessed as supplied by radial diffusion in the aqueous or gaseous phase.     

Detailed methods for the quantification of nitric oxide in aqueous solutions using either an oxygen monitor or EPR

The interest in nitric oxide has grown with the discovery that it has many biological functions. This has heightened the need for methods to quantify nitric oxide. Here we report two separate methods for the quantification of aqueous stock solutions of nitric oxide. The first is a new method based on the reaction of nitric oxide with oxygen in liquid phase (*NO + O2 + 2H2O --> 4HNO2); an oxygen monitor is used to measure the consumption of oxygen by nitric oxide. This method offers the advantages of being both simple and direct. The presence of nitrite or nitrate, frequent contaminants in nitric oxide stock solutions, does not interfere with the quantification of nitric oxide. Measuring the disappearance of dissolved oxygen, a reactant, in the presence of known amounts of nitric oxide has provided verification of the 4:1 stoichiometry of the reaction. The second method uses electron paramagnetic resonance spectroscopy (EPR) and the nitric oxide trap [Fe2+-(MGD)2], (MGD = N-methyl-D-glucamine dithiocarbamate). The nitrosyl complex is stable and easily quantitated as a room temperature aqueous solution. These two methods are validated with Sievers 280 Nitric Oxide Analyzer and cross-checked with standards using UV-Vis spectroscopy. The practical lower limits for measuring the concentration of nitric oxide using the oxygen monitor approach and EPR are approximately 3 microM and 500 nM, respectively. Both methods provide straightforward approaches for the standardization of nitric oxide in solution.     

Reactions of e(-)(aq), CO(2)(*)(-), HO(*), O(2)(*)(-) and O(2)((1)delta(g)) with a dendro[60]fullerene and C(60)[C(COOH)(2)](n) (n = 2-6)

Using pulse radiolysis and laser flash photolysis, we have investigated the reactions of the deleterious species, e(-)(aq), HO&z.rad;, O(2)(*)(-) and O(2)((1)Delta(g)) with 10 water-soluble cyclopropyl-fused C(60) derivatives including a mono-adduct dendro[60]fullerene (d) and C(60) derivatives based on C(60)[C(COOH)(2)](n=2-6), some of which are known to be neuroprotective in vivo. The rate constants for reactions of e(-)(aq) and HO&z.rad; lie in the range 0.5-3.3 x 10(10) M(-1) s(-1). The d and bis-adduct monoanion radicals display sharp absorption peaks around 1000 nm (epsilon = 7 000-11 500 M(-1) cm(-1)); the anions of the tris-, tetra-, and penta-adduct derivatives have broader, weaker absorptions. The monohydroxylated radicals have their most intense absorption maxima around 390-440 nm (epsilon = 1000-3000 M(-1) cm(-1)). The anion and hydroxylated radical absorption spectra display a blue-shift as the number of addends increases. The radical anions react with oxygen (k approximately 10(7)-10(9) M(-1) s(-1)). The reaction of O(2)(*)(-) with the C(60) derivatives does not occur via an electron transfer. The rate constants for singlet oxygen reaction with the dendrofullerene and eee-derivative in D(2)O at pH 7.4 are k approximately 7 x 10(7) and approximately 2 x 10(7) M(-1) s(-1) respectively, in contrast to approximately 1.2 x 10(5) M(-1) s(-1) for the reaction with C(60) in C(6)D(6). The large acceleration of the rates for electron reduction and singlet oxygen reactions in water is due to a solvophobic process.     

Biodegradation processes in a laboratory-scale groundwater contaminant plume assessed by fluorescence imaging and microbial analysis

Flow reactors containing quartz sand colonized with biofilm were set up as physical model aquifers to allow degrading plumes of acetate or phenol to be formed from a point source. A noninvasive fluorescent tracer technique was combined with chemical and biological sampling in order to quantify transport and biodegradation processes. Chemical analysis of samples showed a substantial decrease in carbon concentration between the injection and outflow resulting primarily from dilution but also from biodegradation. Two-dimensional imaging of the aqueous oxygen [O2(aq)] concentration field quantified the depletion of O2(aq) within the contaminant plume and provided evidence for microbial respiration associated with biodegradation of the carbon source. Combined microbiological, chemical, and O2(aq) imaging data indicated that biodegradation was greatest at the plume fringe. DNA profiles of bacterial communities were assessed by temperature gradient gel electrophoresis, which revealed that diversity was limited and that community changes observed depended on the carbon source used. Spatial variation in activity within the plume could be quantitatively accounted for by the changes observed in active cell numbers rather than differences in community structure, the total biomass present, or the increased enzyme activity of individual cells. Numerical simulations and comparisons with the experimental data were used to test conceptual models of plume processes. Results demonstrated that plume behavior was best described by growth and decay of active biomass as a single functional group of organisms represented by active cell counts.     

The time evolution of sequential enzyme reactions: A singular perturbation approach

A perturbative procedure is developed to describe the entire time course of a sequential enzyme reaction occurring in a closed system. The perturbation parameter is the ratio of the enzyme concentration to a reference concentration defined in analogy with the Michaelis-Menten constant. It is shown that damped oscillations may occur about the quasi-steady state for certain realistic values of the parameters. Even under the most favourable circumstances, the damping is exceptionally heavy. In contrast, the evolution to the equilibrium is always monotonic.     

Analysis and computer simulation of aerobic oxidation of reduced nicotinamide adenine dinucleotide catalyzed by horseradish peroxidase.

Under suitable experimental conditions the aerobic oxidation of NADH catalyzed by horseradish peroxidase occurred in four characteristic phases: initial burst, induction phase, steady state, and termination. A trace amount of H2O2 present in the NADH solution brought about initial burst in the formation of oxyperoxidase. About 2 mol of oxyperoxidase was formed per mol of H2O2. When a considerable amount of the ferric enzyme still remained, the initial burst was followed by an induction phase. In this phase the rate of oxyperoxidase formation from the ferric enzyme increased with the decrease of the ferric enzyme and an approximately exponential increase of oxyperoxidase was observed. A rapid oxidation of NADH suddenly began at the end of the induction phase and the oxidation continued at a relatively constant rate. In the steady state, oxygen was consumed and H2O2 accumulated. A drastic terminating reaction suddenly set in when the oxygen concentration decreased under a certain level. During the reaction, H2O2 disappeared accompanying an accelerated oxidation of NADH and the enzyme returned to the ferric form after a transient increase of peroxidase compound II. Time courses of NADH oxidation, O2 consumption, H2O2 accumulation, and formation of enzyme intermediates could be simulated with an electronic computer using 11 elementary reactions and 9 rate equations. The results were also discussed in relation to the mechanism for oscillatory responses of the reaction that appeared in an open system with a continuous supply of oxygen.     

Effects of amine ligand bulk and hydrogen bonding on the rate of reaction of platinum(II) diamine complexes with key nucleotide and amino acid residues

NMR spectroscopy has been used to observe the effects of the amine ligand on the rate of reaction of platinum diamine and triamine complexes with DNA and protein residues. Whereas [Pt(dien)Cl]Cl and [Pt(dien)(D(2)O)](2+) have been known to react faster with thioether residues such as N-AcMet than with 5'-GMP, we found that [Pt(Me(4)en)(D(2)O)(2)](2+) appeared to react faster with 5'-GMP. To quantitatively assess the factors influencing the rates of reaction, rate constants at pH 4 were determined for the reactions of [Pt(en)(D(2)O)(2)](2+) [en = ethylenediamine] and [Pt(Me(4)en)(D(2)O)(2)](2+) with N-AcMet, N-AcHis, 5'-GMP, and Guo (guanosine). In each case the less bulky complex ([Pt(en)(D(2)O)(2)](2+)) reacts more quickly than does the bulkier [Pt(Me(4)en)(D(2)O)(2)](2+), as expected. Both complexes reacted faster with 5'-GMP; however, analysis of the rate constants suggests that the [Pt(en)(D(2)O)(2)](2+) complex favors reaction with 5'-GMP due to hydrogen bonding with the 5'-phosphate, whereas [Pt(Me(4)en)(D(2)O)(2)](2+) disfavors reaction with N-AcMet due to steric clashes. Bulk had relatively little effect on the rate constant with N-AcHis, suggesting that peptides or proteins that coordinate via His residues would not have their reactivity affected by bulky diamine ligands.     

Reactive oxygen species in the aerobic decomposition of sodium hydroxymethanesulfinate.

Sodium hydroxymethanesulfinate, (HOCH2SO2Na, HMS) is relatively stable in aqueous alkaline environments, but rapidly decomposes in acidic medium to give a variety of products that include sulfur dioxide. A detailed kinetic and mechanistic study of the decomposition of HMS in slightly acidic medium has shown a process that produces dithionite, S2O2-4, which is preceded by an induction period which persists for as long as molecular oxygen is present in the reaction solution. The complete consumption of molecular oxygen is a prerequisite for the formation of S2O2-4. Among some of the intermediates detected in the decomposition of HMS is the sulfite radical, SO-3. Comparisons are made between the decomposition mechanisms of thiourea dioxide (aminoiminomethanesulfinic acid) and HMS.