Photoproduction of hydrated electron from constituents of natural waters.

The wavelength dependence for the photoproduction of the hydrated electron (e-(aq)) from various humic and fulvic acids and from natural waters was determined, employing a method that converts e-(aq) to a methyl radical that is detected fluorimetrically as the O-methylhydroxylamine of a stable nitroxide. Quantum yields for e-(aq) production from potassium ferrocyanide and N,N-dimethylaniline are in agreement with previously reported values. The quantum yields for production of e-(aq) from colored dissolved organic matter (CDOM) decrease precipitously with increasing wavelength with the rate of decline increasing in the order: humic acid < fulvic acid < natural water in the UV-B region. For Suwannee River fulvic acid, quantum yields ranged from 7.9 x 10(-6) at 366 nm to 1.9 x 10(-4) at 296 nm indicating that previously reported values for e-(aq) production from CDOM involving laser sources of irradiation are high due to experimental artifact. Apparent natural water quantum yields at 296 nm are higher than those for humic substances, ranging from 9.4 x 10(-5) to 3.7 x 10(-3) depending on location. The highly absorbing waters of the Delaware and Chesapeake Bays show insignificant production of e-(aq). These results indicate that the hydrated electron, through its reaction with dioxygen, is not a significant source of hydrogen peroxide in many natural waters and that humic substances may not be the principal source of e-(aq) production.

Trace detection of hydroxyl radicals during the redox cycling of low concentrations of diaziquone: a new approach.

Quantifying oxygen radicals that arise during the redox cycling of quinone-containing anticancer agents such as diaziquone (AZQ) has been difficult, as has been their detection at low drug concentrations. This is due to the fact that EPR spin trapping, the method most often used for *OH detection, requires the use of high drug concentrations. Using a new highly sensitive technique that employs a fluorescamine-derivatized nitroxide, we show that low levels of NADPH-cytochrome P450 reductase (4.25 microg/ml) catalyze the production of hydroxyl radicals at very low, clinically relevant AZQ concentrations. Thus, at this enzyme concentration, we were able to detect a rate of 0.10 nM s(-1) hydroxyl radical production by 5 microM AZQ, a clinically relevant concentration. The Michaelis-Menten constants for AZQ-mediated hydroxyl radical production are: K(M) = 10.7 +/- 1.4 microM, and V(max) = 5.2 +/- 0.9 x 10(-8) M s(-1) (mg protein)(-1). Experiments employing catalase, superoxide dismutase, and NADPH-cytochrome P450 reductase, confirm the previously deduced conclusions from high drug concentrations, that is, that at low concentrations, AZQ acts to shuttle reducing equivalents from the enzyme to oxygen, thus generating the redox cycle. The data presented here suggest that the levels and locations of redox active metal ions may be the principal controlling factor in the pathway of AZQ activity that involves oxidative stress.

Hydroxyl radical production by mouse epidermal cell lines in the presence of quinone anti-cancer compounds.

The effect of quinone anti-cancer compounds, 3,6-diaziridinyl-2, 5-bis(carboethoxyamino)-1,4-benzoquinone (diaziquone or AZQ) and 3, 6-dihydro-2,5-bis(aziridinyl)-1,4-benzoquinone (DZQ), on the production of hydroxyl radical ((*)OH) in a series of mouse epidermal cell lines was examined using a new, highly sensitive method that employs a fluorescamine-derivatized nitroxide probe. Cell lines that were examined included the mouse epidermal cell line, JB6 clone 41, and JB6 cells transfected with the human Cu-Zn superoxide dismutase (SOD) genes (SOD3 and SOD15) and human catalase (CAT) genes (CAT13 and CAT10). Bioreduction of the nitroxide probe by these cell lines was insignificant at the cell densities employed in these experiments, and thus did not interfere with the (*)OH measurements. In the presence of low concentrations of AZQ or DZQ (20-200 microM), (*)OH production rates were highest in the JB6 cells, intermediate in the SOD-transfected cells, and lowest in the CAT-transfected cells, illustrating that both superoxide and hydrogen peroxide are involved in the production of (*)OH in these systems. Further experiments in which the addition of exogenous SOD and CAT was employed, as well as measurements of probe incorporation into the cells, indicated that this probe can cross cell membranes and detect (*)OH generated intracellularly. In the presence of 100 microM diethylenetriaminepentaacetic acid (DTPA), the rate of (*)OH production in the presence of 100 microM AZQ is approximately 4.7 x 10(4) molecules s(-)(1) cell(-)(1); as much as 45% of this production appears to originate within the cells. This new method should be broadly applicable to the rapid screening of compounds or treatments thought to induce oxidative stress in mammalian cells.

Trace determination of hydroxyl radical in biological systems.

A simple and highly sensitive method to quantify the rates of production of OH in biological systems is described. This method employs the reaction between OH and dimethyl sulfoxide to generate quantitatively a methyl radical, which then reacts with a fluorescamine-derivatized nitroxide to produce the stable O-methylhydroxylamine. This O-methylhydroxylamine is separated by reversed-phase high-performance liquid chromatography and quantified fluorometrically. The estimated detection limit of the O-methylhydroxylamine is 3.5 nM for a 50 microL injection at a signal to noise ratio of 2. The method is applied to the determination of the rates of OH production in a biologically relevant model system and in a mouse epidermal cell line treated with a quinone anticancer compound.

Combined liquid chromatography/mass spectrometry of the radical adducts of a fluorescamine-derivatized nitroxide.

Dynamic liquid secondary ion mass spectrometry (dyn-LSIMS) was employed to acquire continuous, on-line mass spectral data from the effluent of a reversed-phase high-performance liquid chromatograph (HPLC) used to separate a broad suite of carbon-centered radicals trapped as the O-alkylhydroxylamine adducts of an amino nitroxide that was subsequently derivatized with fluorescamine. Data obtained by the use of these combined techniques (LC/MS) can be employed to elucidate radical adduct structures; elemental compositions of the adducts can be confirmed by acquiring mass spectra at high resolution. At low resolution, introduction into the source of < 1 pmol of adduct yielded usable spectra. The first application of this technique to the identification of photochemically generated radicals in natural water samples is presented.

Inherent optical properties of the ocean: retrieval of the absorption coefficient of chromophoric dissolved organic matter from airborne laser spectral fluorescence measurements.

The absorption coefficient of chromophoric dissolved organic matter (CDOM) at 355 nm has been retrieved from airborne laser-induced and water Raman-normalized CDOM fluorescence. Four combined airborne and ship field experiments have demonstrated that (1) the airborne CDOM fluorescence-to--water Raman ratio is linearly related to concurrent quinine-sulfate-standardized CDOM shipboard fluorescence measurements over a wide range of water masses (coastal to blue water); (2) the vicarious calibration of the airborne fluorosensor in units traceable to a fluorescence standard can be established and then maintained over an extended time period by tungsten lamp calibration; (3) the vicariously calibrated airborne CDOM fluorescence-to-water Raman ratio can be directly applied to previously developed shipboard fluorescence-to-absorption algorithms to retrieve CDOM absorption; and (4) the retrieval is not significantly affected by long-path multiple scattering, differences in attenuation at the excitation and emission wavelengths, or measurement in the 180° backscatter configuration. Airborne CDOM absorption measurements will find immediate application to (a) forward and inverse modeling of oceanic water-leaving radiance and (b) validation of satellite-retrieved products such as CDOM absorption.

Multiple N-acyl-L-homoserine lactone autoinducers of luminescence in the marine symbiotic bacterium Vibrio fischeri.

In Vibrio fischeri, the synthesis of N-3-oxohexanoyl-L-homoserine lactone, the autoinducer for population density-responsive induction of the luminescence operon (the lux operon, luxICDABEG), is dependent on the autoinducer synthase gene luxI. Gene replacement mutants of V. fischeri defective in luxI, which had been expected to produce no autoinducer, nonetheless exhibited lux operon transcriptional activation. Mutants released into the medium a compound that, like N-3-oxohexanoyl-L-homoserine lactone, activated expression of the lux system in a dose-dependent manner and was both extractable with ethyl acetate and labile to base. The luxI-independent compound, also like N-3-oxohexanoyl-L-homoserine lactone, was produced by V. fischeri cells in a regulated, population density-responsive manner and required the transcriptional activator LuxR for activity in the lux system. The luxI-independent compound was identified as N-octanoyl-L-homoserine lactone by coelution with the synthetic compound in reversed-phase high-pressure liquid chromatography, by derivatization treatment with 2,4-dinitrophenylhydrazine, by mass spectrometry, and by nuclear magnetic resonance spectroscopy. A locus, ain, necessary and sufficient for Escherichia coli to synthesize N-octanoyl-L-homoserine lactone was cloned from the V. fischeri genome and found to be distinct from luxI by restriction mapping and Southern hybridization. N-Octanoyl-L-homoserine lactone and ain constitute a second, novel autoinduction system for population density-responsive signalling and regulation of lux gene expression, and possibly other genes, in V. fischeri. A third V. fischeri autoinducer, N-hexanoyl-L-homoserine lactone, dependent on luxI for its synthesis, was also identified. The presence of multiple chemically and genetically distinct but cross-acting autoinduction systems in V. fischeri indicates unexpected complexity for autoinduction as a regulatory mechanism in this bacterium.

Fluorescence detection of carbon-centered radicals in aqueous solution.

A simple, highly sensitive method for the simultaneous determination of arrays of carbon-centered radicals in aqueous systems is described. Radicals are efficiently trapped by an amino-nitroxide to form stable products which are then reacted with fluorescamine to produce highly fluorescent adducts. The adducts are easily separated by reversed-phase high performance liquid chromatography. The detection limit for individual radical adducts (0.5 to 2 nM) is two to three orders of magnitude lower than those of current methods employing electron paramagnetic resonance detection. Results on the photolysis of ketones and alpha-keto acids demonstrate the potential of this technique. This approach should be widely applicable to the study of radical processes in biological and chemical systems.

Fluorescence detection of free radicals by nitroxide scavenging.

The fluorescence quantum yield of 4-(1-napthoyloxy)-2,2,6,6-tetramethylpiperidine-1-oxyl (I) in acetonitrile and hexane is 55 and 30-fold lower, respectively, than those of diamagnetic analogs. Experiments described herein demonstrate that this property makes possible the fluorescence detection of radical scavenging reactions in which the paramagnetic nitroxide-substituted naphthalene is converted to a diamagnetic N-alkoxy derivative. 2-Cyanopropyl free radicals were generated by the thermal decomposition of azobisisobutyronitrile (AIBN) in cyclohexane or in acetonitrile containing I. The fluorescence intensity of the sample increased proportionally to the decrease in its ESR signal intensity, indicating the conversion of the paramagnetic nitroxide to the diamagnetic product. The linear relationship between the increase in fluorescence intensity and decrease in ESR signal intensity shows that the changes in the fluorescence intensity can serve as a sensitive means for optically detecting radicals.

X-ray diffraction studies of a partially liganded hemoglobin, [alpha(FeII-CO)beta(MnII)]2.

We have applied single-crystal X-ray diffraction methods to analyze the structure of [alpha(FeII-CO)beta(MnII)]2, a mixed-metal hybrid hemoglobin that crystallizes in the deoxyhemoglobin quaternary structure (the T-state) even though it is half liganded. This study, carried out at a resolution of 3.0 A, shows that (1) the Mn(II)-substituted beta subunits are structurally isomorphous with normal deoxy beta subunits, and (2) CO binding to the alpha subunits induces small, localized changes in the T-state that lack the main directional component of the corresponding larger structural changes in subunit tertiary structure that accompany complete ligand binding to all four subunits and the deoxy to oxy quaternary structure change. Specifically, in the T-state, CO binding to the alpha heme group draws the iron atom toward the heme plane, and this in turn pulls the last turn of the F helix (residues 85 through 89) closer to the heme group. The direction of these small movements is almost perpendicular to the axis of the F helix. In contrast, when the structures of fully liganded and deoxyhemoglobin are compared, extensive structural changes occur throughout the F helix and FG corner, and the main component of the atomic movements in the F helix (in addition to the smaller component toward the heme) is in a direction parallel to the heme plane and toward the alpha 1 beta 2 interface. These findings are discussed in terms of the current stereochemical theories of co-operative ligand binding and the Bohr effect.

Reversible and irreversible effects of alkaline pH on Photosystem II electron-transfer reactions.

Incubation of highly active, O2-evolving PS II preparations at alkaline pH inhibits donor side electron-transfer reactions in two distinct fashions, one reversible the other irreversible. In both cases, O2 evolution is inhibited, with concomitant loss of the light-induced multiline and g = 4.1 EPR signals and an increased steady-state level of EPR Signal II induced by continuous illumination. However, the inhibition that is observed between pH 7.0 and 8.0 is readily reversible by resuspension at low pH, while above pH 8.0 the effect is irreversible. In addition, under repetitive flash conditions the ms decay kinetics remains largely unchanged at pH less than or equal to 8.0 but shows about a 2-fold increase in amplitude and is slowed at pH above 8.0. The irreversible component of inhibition most likely can be attributed to the loss of Mn and the 16, 24 and 33 kDa proteins. The reversible component may be mediated by displacement of Cl- from an anion-binding site by OH- or by titration of ionizable groups on the protein(s) associated with water-splitting. We propose that the reversible inhibition blocks electron transfer between the O2-evolving complex and an intermediate which serves as the direct donor to Signal II, while the irreversible inhibition blocks the reduction of Signal II by this intermediate donor species.

The effect of mono- and divalent salts on the rise and decay kinetics of EPR signal II in Photosystem II preparations from spinach.

The rise and decay kinetics of EPR signal II have been used to probe the organization of the donor side of Photosystem II (PS II) before and after extraction of PS II preparations with high concentrations of salt. 800 mM NaCl or 500-800 mM NaBr substantially depletes the preparations of the 16 and 24 kDa proteins and decreases the steady-state rate of O2-evolution by 70-80% from control rates. These treatments do not largely alter the decay kinetics of Signal II; the rise kinetics remain in the instrument limited time range (2 microseconds or less) during the first 8-12 flashes. Treating PS II preparations with 800 mM CaCl2 removes the 16, 24 and 33 kDa proteins with at least 95% inhibition of the steady-state rates of O2 evolution. The additional removal of the 33 kDa polypeptide decreases the rates of oxidation and rereduction of Z, the species responsible for Signal II. Preparations treated with either mono- or divalent salts show a steady-state light-induced increase in Signal II similar to that seen in Tris-washed samples. Such a steady-state increase indicates that the rate of electron transport from water to Z is greatly decreased or blocked. The data are interpreted within a model in which there is an intermediate electron carrier between the O2 evolving complex and Z.

The effects of mono- and divalent salts on the O2-evolution activity and low temperature multiline EPR spectrum of Photosystem II preparations from spinach.

The ability of salts to inhibit the O2-evolution activity of PS II preparations is shown to parallel closely the Hofmeister series, suggesting that inhibition is related to the solubility of the 16, 24 and 33 kDa proteins in these salt solutions. An examination of the effect of salt inactivation on the low temperature multiline EPR signal indicates that the release of either the 16 and 24 kDa proteins, or additionally the 33 kDa protein blocks or greatly reduces the efficiency of the advancement of the water-splitting complex to the S2-state; under some conditions, this inhibition is reversible.

Carbon monoxide binding to the ferrous chains of [Mn,Fe(II)] hybrid hemoglobins: pH dependence of the chain affinity constants associated with specific hemoglobin ligation pathways.

In mixed-metal [Mn,Fe] hybrid hemoglobins (Hb), the two chains of a single type, alpha or beta, are substituted with manganese protoporphyrin IX, which does not bind CO in either the Mn(II) or Mn(III) valency states. Thus, CO binding by the two ferrous subunits of a hybrid with Mn of either valency represents a simplified two-step Hb ligation process in which ligands bind to a single-chain type. Considering the [Mn(II),Fe(II)] hybrids, which are deoxy T-state analogues, at pH 6.6 both types bind CO with low affinity (alpha-Fe, 0.38 mmHg; beta-Fe, 0.71 mmHg) and noncooperatively (Hill coefficient n = 1). At elevated pH, both exhibit an increase in affinity (Bohr effect) and strong cooperativity, with the alpha-Fe hybrid having a higher degree of cooperativity (n approximately equal to 1.6) than beta-Fe (approximately equal to 1.3) at pH 9.0. The CO association constants for the Hb ligation routes in which the first two ligands bind to the same chain type are obtained from these measurements, and their pH dependence provides estimates of the proton release at each step. Through studies of CO on- and off-rates, the [Mn(III),Fe(II)] hybrids are used to obtain the pH dependence of the association constants for binding the fourth CO to the individual Hb chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Flash photolytic studies of carbon monoxide binding to the ferrous chains of [Mn(II),Fe(II)] hybrid hemoglobins: kinetic mechanism for the early stages of hemoglobin ligation.

Flash photolysis is employed to investigate the kinetics of CO recombination to the ferrous chains of [Mn(II),Fe(II)] hemoglobin (Hb) hybrids. At low pH (6.6), Hb remains predominantly in the T quaternary state for the first two CO ligation steps, when binding to either the alpha chains or beta chains. At elevated pH, CO binding to the alpha chains produces a larger degree of T to R conversion than binding to the beta chains, in support of earlier equilibrium measurements. This study provides the full pH dependence of the CO binding rate constants for both alpha- and beta-Fe chains within the T state and at elevated values of pH gives the R-state rate constants for the monoliganded analogues. The data can be analyzed within the context of a two-state model for Hb cooperativity, but they give clear evidence for slow quaternary structure interconversion at the monoliganded level.