HYDROPEROXYNAPHTHALIMIDE DERIVATIVE-MEDIATED OXIDATION OF LYSOZYME

Abstract— Lysozyme was photoirradiated in the presence of photo-Fenton reagents (hydroperoxynaph-thalimide derivatives) at 366 nm. Enzymatic activities of photoirradiated lysozymes were lower than that of native lysozyme. Taking account of the results of amino acid analysis and of fluorescence spectra, it was probably that Trp residues in the photoirradiated lysozyme were oxidized with hydroxyl radicals. The reagents formed complexes with lysozyme as proved by the inhibitory effects of the reagents on the enzymatic activities ( K ₁= 4.7 ± 1.2 × 10⁴ M for HPO II, a hydroperoxide derivative of naphthalimide), which suggested that these reagents were bound to the active site cleft of lysozyme, and the Trp residues located in or near the active site cleft were photooxidized. Fluorescence-difference spectra of photoirradiated lysozymes showed that Trp 62 was preferentially photooxidized. Furthermore, sodium dodecyl sulfatepolyacrylamide gel electrophoresis and circular dichroism spectra showed that the photooxidation examined here induced no significant change in the molecular size but a slight change in the conformation of lysozyme, which suggests the usefulness of the reagents in the site-selective oxidation of biopolymers.     

Luminol Oxidation by Hydrogen Peroxide Catalyzed by Tobacco Anionic Peroxidase: Steady-State Luminescent and Transient Kinetic Studies

The properties of a newly isolated anionic tobacco peroxidase from transgenic tobacco plants overexpressing the enzyme have been studied with respect to the chemiluminescent reaction of luminol oxidation. These were compared to the properties of horseradish peroxidase in the cooxidation of luminol and p -iodophenol, the enhanced chemiluminescence reaction. The pH, luminol and hydrogen peroxide concentrations were optimized for maximum sensitivity using the tobacco enzyme. The detection limit for the latter under the optimal conditions (2.5 m M luminol, 2 m M hydrogen peroxide, 100 m M Naborate buffer, pH 9.3) was about 0.1 p M , which is at least five times lower than that for horseradish peroxidase in enhanced chemiluminescence with p -iodophenol. The rate constants for the elementary steps of the enzyme-catalyzed reaction have been determined: k ₁= 4.9 × 10⁶ M ⁻¹ s¹, k ₂= 7.3 × 10⁶ M ⁻¹ s⁻¹, k ³= 2.1 × 10⁶ M ⁻¹ s⁻¹ (pH 9.3). The similarity of these rate constants is unusual for plant peroxidases. The high catalytic activity of tobacco peroxidase in the luminescent reaction is explained by the high reactivity of its Compound II toward luminol and the high stability of the holoenzyme with respect to heme dissociation. This seems to be a unique property of this particular enzyme among other plant peroxidases.     

A Role for Hydrogen Peroxide in the Pro-apoptotic Effects of Photodynamic Therapy

Although the first reactive oxygen species (ROS) formed during irradiation of photosensitized cells is almost invariably singlet molecular oxygen (¹O₂), other ROS have been implicated in the phototoxic effects of photodynamic therapy (PDT). Among these are superoxide anion radical (^•O₂⁻), hydrogen peroxide (H₂O₂) and hydroxyl radical (^•OH). In this study, we investigated the role of H₂O₂ in the pro-apoptotic response to PDT in murine leukemia P388 cells. A primary route for detoxification of cellular H₂O₂ involves the peroxisomal enzyme catalase. Inhibition of catalase activity by 3-amino-1,2,4-triazole led to an increased apoptotic response. PDT-induced apoptosis was impaired by addition of an exogenous recombinant catalase analog (CAT- skl) that was specifically designed to enter cells and more efficiently localize in peroxisomes. A similar effect was observed upon addition of 2,2'-bipyridine, a reagent that can chelate Fe⁺², a co-factor in the Fenton reaction that results in the conversion of H₂O₂ to ^•OH. These results provide evidence that formation of H₂O₂ during irradiation of photosensitized cells contributes to PDT efficacy.     

FLUORESCENCE QUENCHING OF INDOLIC COMPOUNDS BY ALIPHATIC AMINO ACIDS. EVIDENCE FOR EXCITED STATE COMPLEXES

Abstract The fluorescence quenching of indole, tryptophan, tryptamine and indole-3-acetic by aliphatic amino acids was studied. The bimolecular rate constant ( k _q) for the deactivation of the excited state was determined. The k _q values were in the range 0.6 × 10⁸–1.6 × 10⁹ M ^–1 S^–1 and they increased in the order tryptophan < tryptamine < indole ≈ indole-3-acetic acid. When the rate constant was corrected for diffusion al effects a good linear correlation was found between the log ( k '_q) and the ionization equilibrium constant of the carboxylic group of the amino acid (p ^k _a1). This was interpreted as arising from a charge transfer mechanism in which the indole moiety acts as an electron donor and the carbonyl group of the amino acid as the acceptor.
The activation parameter for the quenching processes were also determined. The ΔH^≠ values were in the range —4.0 to +4.0 kcal/mol and the ΔH^≠ in the range –7 to –37 e.u. For the systems with lower values of k _q negative values for ΔH^≠ were observed. A good enthalpy-entropy compensation was found with an isokinetic temperature of 229 K. These results suggest that a common mechanism is operating for all the systems and that it involves the formation of an excited state complex between the indolic compound and the amino acid.     

HO2 ELIMINATION FROM α-HYDROXYALKYLPEROXYL RADICALS IN AQUEOUS SOLUTION

Abstract— In aqueous solutions α-hydroxyalkylperoxyl radicals undergo a spontaneous and a base catalysed HO₂ elimination. From kinetic deuterium isotope effects, temperature dependence, and the influence of solvent polarity it was concluded that the spontaneous reaction occurs via an HO₂ elimination followed by the dissociation of the latter into H⁺ and O₂^-. The rate constant of the spontaneous HO₂ elimination increases with increasing methyl substitution in α-position ( k (CH₂(OH)O₂) < 10s^-1 k (CH₃CH(OH)O₂) = 52s^-1 k ((CH₃)₂C(OH)O₂) = 665 s^-1). The OH^- catalysed reaction is somewhat below diffusion controlled. The mixture of peroxyl radicals derived from polyhydric alcohols eliminate HO₂ at two different rates. Possible reasons for this behaviour are discussed. The mixture of the six peroxyl radicals derived from d -glucose are observed to eliminate HO₂ with at least three different rates. The fastest rate is attributed to the HO₂ elimination from the peroxyl radical at C-l ( k > 7000s^-1). Because of the HO₂ eliminations the peroxyl radicals derived from d -glucose do not undergo a chain reaction in contrast to peroxyl radicals not containing an α-OH group. In competition with the first order elimination reactions the α-hydroxylalkylperoxyl radicals undergo a bimolecular decay. These reactions are briefly discussed.     

BLEACHING OF PURPLE MEMBRANE WITH O-SUBSTITUTED HYDROXYLAMINES

Abstract The retinal Schiff base of bacteriorhodopsin, in the purple membrane from Halobacterium halobium , can be cleaved by hydroxylamine in the presence of light. We have further investigated this reaction with a series of O -substituted hydroxylamines, RONH₂, where R = -H (HA), -CH₃ (MHA), -SO₃− (HAS), benzyl- (BHA), p -nitrobenzyl- (NBHA), and pentafluorobenzyl- (FBHA). All except MHA caused light-induced bleaching of the purple membrane and the chromophore could be regenerated from apomembrane and all- trans retinal. Relative bleaching rate constants were obtained from V = QI _a k ₀ X /( k _r+ k ₀ X ), where V = bleaching rate, Q = quantum yield, I a = absorbed light intensity, X = hydroxylamine concentration, k ₀= rate constant for bleaching and k _r= rate constant for return of photoexcited bacteriorhodopsin to the initial state. This equation fits the time-, concentration- and intensity-dependences of the bleaching reactions in 0.02 M phosphate, pH 7.0. The rate constants k ₀ relative to HA were: MHA: 0; HAS: 0.3; HA: 1.0; BHA: 1.8; FBHA: 10.1; NBHA: 10.8. The relative rate constants do not correlate with the basicity of the derivatives. Instead, the results suggest that the retinal Schiff base is near a non-polar cavity into which an aromatic group can be inserted.     

THE INFLUENCE OF MOLECULAR CONFORMATION ON THE STABILITY OF ULTRAVIOLET STABILIZERS TOWARD DIRECT AND DYE-SENSITIZED PHOTOIRRADIATION: THE CASE OF 2-(2'-HYDROXY-5'-METHYPHENYL)BENZOTRIAZOLE (TIN P)

Abstract— The mechanism for photodegradation of the ultraviolet photostabilizer 2-(2'-hydroxy-5'-methylphenyl)benzotriazole (TIN P) upon direct and dye-sensitized (singlet molecular oxygen [O₂(1Δ_g)]-mediated) irradiation was studied. From the experimental TIN P photodegradation rate data, and low temperature (77 K) fluorescence and phosphorescence quantum yields, one can conclude that the photodegradative process involves phosphorescent states of TIN P. The open conformer of TIN P quenches O₂(¹Δ_g) by physical scavenging with a rate constant (k_q) in dimethylsulfoxide of 2.8 times 10⁶ M ^-1 s^-1. The intramolecular hydrogen-bonded conformer does not appreciably interact with O₂(¹Δ_g). In the presence of a relatively high concentration of OH- (either 5 times 10^-2 M KOH in ethanol or water at pH 13), the ionic form of TIN P (with an ionized phenol group) physically and chemically quenches O₂(¹Δ_g). The reaction rate constant ( k _r) is 1 times 10 ⁸ M ^-1 s^-1, and the ratio k _q/ k _r is approximately three in alkaline aqueous media.     

KINETIC ANALYSIS OF THE FLUORESCENCE INDUCTION IN 3-(3,4-DICHLOROPHENYL)-1,1-DIMETHYLUREA POISONED CHLOROPLASTS

Abstract— The size of the area over the fluorescence rise curve of chloroplasts is a measure of the total number of quanta utilized in photosystem II during the fluorescence induction, while the growth of the area reflects the progress of photochemical events. In the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), the growth kinetics of the area are affected by the reoxidation of the primary acceptor Q ^- with stored oxidizing charges on the donor side of system II.
At low light intensities, a slow component of this back reaction may limit the steady state fluorescence emission. At higher intensities, however, the fluorescence rise is limited solely by photochemical events, although fast thermochemical reactions like the immediate recombination of photochemically separated charges may affect the efficiency of the photochemistry.
A kinetic analysis of the area growth at moderate light intensities revealed that it occurred in two first order phases which were described by the rate constants k _α and k _β. The biphasic nature suggested a sequential two-electron reduction of the primary acceptor Q , or the presence of two different types of photochemical centers in system II. The rate constants were light intensity dependent. They also were affected by changes in pH, by an addition of NH₂OH, or by a preillumination with short flashes prior to addition of DCMU. It is suggested that the pH of the medium, the presence of NH₂OH, and the flash induced state S_n of the water splitting enzyme, control the values of k _α and k _β by changing the rate constants of electron carrier interactions in the reaction center complex, with a resulting modification of the frequency of back reactions between the primary donor and the primary acceptor.     

EVIDENCE FOR A MAJOR STRONG BINDING SITE FOR TETRACHLOROSALICYLANILIDE ON HUMAN SERUM ALBUMIN

Abstract— Solutions of human serum albumin(HSA) monomer were irradiated with UV light(360 nm) in the presence of [₁₄C]-3,3.4'S-tetrachlorosalicylanilide([₁₄C]-T₄CS).The [₁₄C]-T₄ CS-labeiled HSA was cleaved by cyanogen bromide and separated into two fractions. These fractions were reduced carboxymethylated and separated into their seven characteristic peptides and monitored for radioactivity. Tetrachlorosalicylanilide was found to bind mainly to one region of the sequence of HSA and this covalent binding site was located in residues 124 (Cys) to 298 (Met) of the molecule. The binding of 3,5-dichlorosalicylamido-4-(2,2,6.6-tetramethylpiperidine-l-oxyl (DCS-TEMPO),a spin-label analogue of T₄CS, to HSA was studied by electron spin resonance spectroscopy. In the absence of UV light. DCS-TEMPO bound non-covalently (k = 6.1 times 10₆M₁) to one major binding site on HSA. These results are evidence for the existence of a major strong binding site for the photochemical binding of T₄CS to HSA.     

RATE PARAMETERS FOR THE QUENCHING OF SINGLET OXYGEN BY WATER-SOLUBLE AND LIPID-SOLUBLE SUBSTRATES IN AQUEOUS AND MICELLAR SYSTEMS

Abstract— The laser flash photolysis method has been used to determine the bimolecular rate constants for the reaction between O₂(¹Δ₉) and several lipid-soluble and water-soluble substrates. Values for lipid-soluble substrates have been obtained using aqueous dispersions of surfactants above the critical micelle concentration with 1,3 diphenylisobenzofuran as monitor of singlet oxygen. Under these conditions the hydrophobic substances are solubilized by the micellar phase. For substrates which are water-soluble, 9,10-anthracene dipropionic acid disodium salt was used as singlet oxygen monitor. For several substances, the values obtained are comparable to the values found in homogeneous nonaqueous solutions. In cases where significant differences have been found these have been rationalized according to the individual case. The only major unexpected result concerned β-carotene which, in micellar dispersion, failed to react at all with O₂(¹Δ₉) This may be due to multi-molecular aggregations occurring in the polar medium. The work described herein shows clearly that, under appropriate conditions, singlet oxygen kinetics can be effectively followed in aqueous solutions by time resolved methods. The indiscriminate use of β-carotene as a quencher of O₂(¹Δ₉)in mainly aqueous media is questioned.     

PULSE RADIOLYSIS STUDIES IN MODEL LIPID SYSTEMS: FORMATION AND BEHAVIOR OF PEROXY RADICALS IN FATTY ACIDS

Abstract— Radiolytic formation and peroxidation of fatty acid radicals have been investigated by pulse radiolysis techniques in oleate, linoleate, linolenate and arachidonate systems. A strong absorption band at 280 nm associated with conjugated radicals, R_conj, formed in polyunsaturated fatty acid moieties has been used as a probe for kinetic processes occurring at doubly allylic sites in the hydrocarbon chain. Formation of R_conj by O^- has been found to be more efficient than the less selective OH radical. Peroxidation of R_conj is shown to be somewhat slower, ( k _R+ O₂˜ 3 × 10⁸ M ^-1 s^-1), than O₂ reactions with radicals in oleate ( k _R+ O₂= 1 × 10⁹ M ^-1 s^-1). Peroxy radicals generated in these reactions disappear slowly by essentially second order processes (2 k RO₁˜ 10⁷ M ^-1 s^-1). The superoxide radical, O^-₂, shows little if any reactivity towards 0.01 M linolenate or arachidonate over periods of 20 s.     

A Water-Soluble Luminescence Oxygen Sensor

We developed a water-soluble luminescent probe for dissolved oxygen. This probe is based on (Ru[dpp(SO₃Na)₂]₃) Cl₂, which is a sulfonated analogue of the well-known oxygen probe (Ru[dpp]₃)Cl₂. The compound dpp is 4,7-diphenyl-1,10-phenanthroline and dpp(SO₃Na)₂ is a disulfonated derivative of the same ligand. In aqueous solution in the absence of oxygen (Ru[dpp(SO₃Na)₂]₃)Cl₂ displays a lifetime of 3.7 μs that decreases to 930 ns on equilibrium with air and 227 ns on equilibrium with 100% oxygen. The Stern–Vohner quenching constant is 11330 M⁻¹. This high oxygen-quenching constant means that the photoluminescence of Ru(dpp[SO₃Na]₂)₃Cl₂ is 10% quenched at an oxygen concentration of 8.8 x 10⁻⁶ M , or equilibration with 5.4 torr of oxygen. The oxygen probe dissolved in water displays minimal interactions with lipid vesicles composed of dipalmityl-L-α-phosphatidyl glycerol but does appear to interact with human serum albumin. The absorption maximum near 480 nm, long lifetime and large Stokes'shift allow this probe to be used with simple instrumentation based on a light-emitting diode light source, allowing low-cost oxygen sensing in aqueous solutions. To the best of our knowledge this is the first practical water-soluble oxygen sensor.     

SYNTHESES AND PHOTOREDOX PROPERTIES OF POLYELECTROLYTES FUNCTIONALIZED WITH 5-DEAZAFLAVIN

Abstract— Anionic polyelectrolytes functionalized with the 5-deazaflavin group (dFl) were synthesized. The lifetime of the triplet excited dFl in the polyelectrolytes with a 2-mol% dFl content (AdFl-2) was about 10 times longer than that of a low molecular weight analog (AdFl-M). 2-Mercaptoethanol (RSH) reduced the triplet dFl with the rate constant of k _red= 2.01 × 10⁸ M ⁻¹ s⁻¹ for AdFl-M and k _red= 4.4 × 10⁷ M ⁻¹ s⁻¹ for AdFl-2. A zwitterionic viologen (SPV) oxidized the triplet dFl with the rate constant of k _red= 3.69 × 10⁹ M ⁻¹ s⁻¹ for AdFl-M and k _ox= 7.4 × 10⁸ M⁻¹ s⁻¹ for AdFl-2. The smaller rate constants for the polymer system were discussed in terms of the hindering effect of the macromolecular microenvironment. The back electron transfer was shown to be drastically slowed in the AdFl-2-SPV system as a result of the intensive electrostatic effect of the polyelectrolytes. The buildup of the viologen radicals was studied under the steady-state illumination of the three component systems including viologen and RSH. The dFl group was demonstrated to serve as a very efficient photosensitizer in the oxidative cycle in case back electron transfer was retarded. This is the case of the AdFl-2-SPV system which gave the quantum yield of about 0.4 for the SPV⁻ buildup. By comparison, the AdFl-2-MV²+ system resulted in a much slower buildup of MV +radicals.     

ON THE REACTION OF SINGLET MOLECULAR OXYGEN WITH N-ALLYLTHIOUREA IN AQUEOUS SOLUTION

Abstract— Experiments on the photooxidation of N -allylthiourea, thiourea, and N-allylurea sensitized by the dye phenosafranine show that in N -allylthiourea the thiourea group is the site of singlet oxygen attack, while the allyl moiety neither reacts with nor quenches this metastable form of O₂ (in neutral aqueous solutions). Low concentrations of N^-₃ (a known quencher of singlet oxygen) strongly reduce the photooxidation of allylthiourea by a mechanism which apparently obeys simple competition kinetics. From these results the rate constant of the reaction between allylthiourea and singlet oxygen is obtained ( k = 4 × 10⁶ M ^-1 s^-1; pH = 7.1).     

PHOTOSENSITIZATION VIA CHARGE TRANSFER OR REVERSIBLE ELECTRON TRANSFER. OXIRANE ISOMERIZATION AND SULFUR DIOXIDE EXTRUSION

Abstract— N, N, N^' N^'-Tetramethylbenzidine (NTMB) photosensitizes the cis-trans isomerization of stilbene oxiranes (SO) and the extrusion of SO₂ from dibenzyl sulfone (DBS). In acetonitrile solution it is found that in the absence of SO or DBS, singlet NTMB undergoes three processes: intersystem crossing to triplet NTMB (φ_ISC= 0.63, k _ISC= 6.3 × 10⁷s^-1), fluorescence (φ_f= 0.30, k _f= 3 × 10⁷s^-1), and formation of a cation by electron ejection (φ_ion= 0.09). Both singlet and triplet sensitization are observed. A charge transfer or reversible electron transfer mechanism is proposed to explain the results.     

CONVENIENT AND SIMPLE METHODS FOR THE OBSERVATION OF PHOSPHORESCENCE IN FLUID SOLUTIONS. INTERNAL AND EXTERNAL HEAVY ATOM AND MICELLAR EFFECTS

Abstract— Phosphorescence of organic molecules in fluid solutions may be conveniently and readily observed under certain conditions. If k _p (radiative phosphorescence rate constant) is 10s^-1, then (in the absence of photoreaction) phosphorescence is observable upon N₂ purging. For example, nitrogen purged, acetonitrile solutions of bromo and dibromonaphthalene display readily observable phosphorescence as a result of internal heavy atom enhancement of π_S, and k _p. External heavy atom enhancement of k, (CH₂BrCH₂Br solvent) of aromatic hydrocarbons even allows observation of phosphorescence from these compounds in N₂ purged fluid solutions. Although bromonaphthalenes are not significantly phosphorescent in N₂ purged aqueous solution, phosphorescence is readily observed in N₂ purged detergent (HDTBr, HDTCl, and SDS) solutions above the critical micelle concentration. The general factors which determine whether phosphorescence is "readily" obervable in fluid solution are briefly discussed and the results are interpreted in light of these factors.     

Interaction of the excited singlet state of neutral red with aromatic amines

Quenching of neutral red (NR; neutral form of the dye) fluorescence by a number of aromatic amines has been investigated in acetonitrile solutions. The bimolecular quenching constants ( k _q) obtained from steady-state and time-resolved measurements for a particular donor–acceptor pair are seen to be the same within experimental error. Correlation of the changes in the k _q values with the oxidation potentials of the donors (amines) indicates that electron transfer (ET) is the mechanism operative in the present systems. Direct evidence for ET has been obtained from picosecond transient absorption studies on a suitable amine–NR pair. Experimentally determined k _q values are seen to correlate well with the free energy changes (Δ G ⁰) for the ET reactions, within the framework of the Marcus outer sphere ET theory. From the correlation between the experimentally determined and theoretically calculated k _q values, it appears that solvent reorganization plays a major role in governing ET dynamics in the systems investigated.     

THE HABER-WEISS CYCLE

Abstract— The Haber-Weiss cycle:
was investigated at low pH by radiolysis of oxygen or nitrogen saturated solutions of hydrogen peroxide. It was found that reaction 2 has a low rate constant: k ₂= 3.0 ± 0.6 M ^-1 s^-1 (pH 2.3, 22°C). The rate determining step of reaction 2 is most probably the transfer of an electron from a π₈* orbital of HO₂ to the empty _u* orbital of H₂O₂. Overlap between these two orbitals is hindered by the filled π₈* orbitals of H₂O₂. Fe(HI)EDTA catalyses reaction 2.     

THE PHOTOREDUCTION OF METHYLENE BLUE BY AMINES—I. A FLASH PHOTOLYSIS STUDY OF THE REACTION BETWEEN TRIPLET METHYLENE BLUE AND AMINES

Abstract— Flash photolysis was used to study the reduction of the triplet state of methylene blue by both alkyl- and aryl-amines. The extent of the formation of the semireduced form of the dye yielded rate constants of interaction between the triplet state and the amine ( k ₅). A correlation between log k ₅ and ionization potentials for alkylamines (slope = -1.75 eV^-1) was interpreted as evidence for the formation of a partial charge-transfer intermediate. The rate constants ( k ₅) calculated for aryl-amines approached the rate of diffusion in many cases. A Hammett plot for a series of N, N-dimethyl-anilines yielded a moderately large p value (– 3.28) consistent with the formation of a charge-transfer intermediate. It was concluded that reaction of amines with triplet methylene blue leads to the formation of a partial charge-transfer intermediate which may undergo complete electron transfer to yield radicals, or undergo spin inversion and return to the ground state.     

SOLVENT EFFECTS ON THE TAUTOMERIZATION OF 5'-DEOXYPYRIDOXAL. A PHOTOPHYSICAL STUDY

Abstract— Absorption and fluorescence spectra of 5'-deoxypyridoxal (DPL) in various pure solvents and mixtures were recorded both at room temperature and over the range10–65°C. The areas under the absorption bands were analyzed to obtain the mole fraction (f_N, f_z) of two tautomers (the zwitterionic, Z, and neutral, N, forms) in the ground state. The following spectral parameters were determined from the fluorescence spectra: Stokes shift (Δ v ), fluorescence quantum yield of the neutral form (Q_N), fluorescence ratio of the neutral to the zwitterionic form (ø_N/ø_Z) and the rate constant of tautomerization ( k ₁) from Z to N in the excited state. Some of these parameters (f_N, Δ v , Q_N, k ₁) were found to depend on the proton donor character of the solvent, whereas others (ø_N/ø_Z) depended on its dipole moment. Thus, the absorption and fluorescence spectra of DPL allow one to obtain information on the polarity and the concentration of –OH groups on its environment.