Edward J. Land

Bipyridylium quaternary salts and related compounds. V. Pulse radiolysis studies of the reaction of paraquat radical with oxygen. Implications for the mode of action of bipyridyl herbicides

1. Rate constants for reduction of paraquat ion (1,1′-dimethyl-4,4′-bipyridy-lium, PQ2+) to paraquat radical (PQ+·) by e−aq and CO2−· have been measured by pulse radiolysis. Reduction by e−aq is diffusion controlled (k = 8.4·1010 M−1·s−1) and reduction by CO2−· is also very fast k = 1.5·1010 M−1·s−1). 2. The reaction of paraquat radical with oxygen has been analysed to give rate constants of 7.7·108 M−1·s−1 and 6.5·108 M−1·s−1 for the reactions of paraquat radical with O2 and O2−·, respectively. The similarity in these rate constants is in marked contrast to the difference in redox potentials of O2 and O2−· (− 0.59 V and + 1.12 V, respectively). 3. These rate constants, together with that for the self-reaction of O2−·, have been used to calculate the steady-state concentration of O2−· under conditions thought to apply at the site of reduction of paraquat in the plant cell. On the basis of these calculations the decay of O2−· appears to be governed almost entirely by its self-reaction, and the concentration 5 μm away from the thylakoid is still 90% of that at the thylakoid itself. Thus, O2−· persists long enough to diffuse as far as the chloroplast envelope and tonoplast, which are the first structures to be damaged by paraquat treatment. O2−· is therefore sufficiently long-lived to be a candidate for the phytotoxic product formed by paraquat in plants.

Reactions of nitrogen dioxide in aqueous model systems: Oxidation of tyrosine units in peptides and proteins

By application of pulse radiolysis it was demonstrated that nitrogen dioxide (NO2.) oxidizes Gly-Tyr in aqueous solution with a strongly pH-dependent rate constant (k6 = 3.2 X 10(5) M-1 S-1 at pH 7.5 and k6 = 2.0 X 10(7) M-1 S-1 at pH 11.3), primarily generating phenoxyl radicals. The phenoxyl can react further with NO2. (k7 approximately 3 X 10(9) M-1 S-1) to form nitrotyrosine, which is the predominant final product in neutral solution and at low tyrosyl concentrations under gamma-radiolysis conditions. Tyrosine nitration is less efficient in acidic solution, due to the natural disproportionation of NO2., and in alkaline solutions and at high tyrosyl concentrations due to enhanced tyrosyl dimerization. Selective tyrosine nitration by interaction of NO2. with proteins (at pH 7 to 9) was demonstrated in the case of histone, lysozyme, ribonuclease A, and subtilisin Carlsberg. Nitrotyrosine developed slowly also under incubation of Gly-Tyr with nitrite at pH 4 to 5, where NO2. is formed by acid decomposition of HONO. It is recalled in this context that NO2.-induced oxidations, by regenerating NO2-, can propagate NO2./NO2- redox cycling under acidic conditions. Even faster than with tyrosine is the NO2.-induced oxidation of cysteine-thiolate (k9 = 2.4 X 10(8) M-1 S-1 at pH 9.2), involving the transient formation of cystinyl radical anions. The interaction of NO2. with Gly-Trp was comparably slow (k approximately 10(6) M-1 S-1), and no reaction was detectable by pulse radiolysis with Met-Gly and (Cys-Gly)2, or with DNA. Slow reactions of NO2. were observed with arachidonic acid (k approximately 10(6) M-1 S-1 at pH 9.0) and with linoleate (k approximately 2 X 10(5) M-1 S-1 at pH 9.4), indicating that NO2. is capable of initiating lipid peroxidation even in an aqueous environment. NO2.-Induced tyrosine nitration, using 50 microM Gly-Tyr at pH 8.2, was hardly inhibited, however, in the presence of 1 mM linoleate, and was not affected at all in the presence of 5 mM dimethylamine (a nitrosamine precursor). It is concluded that protein modifications, and particularly phenol and thiol oxidation, may be an important mechanism, as well as initiation of lipid peroxidation, of action of NO2. in biological systems.

Evidence of the Indirect Formation of the Catecholic Intermediate Substrate Responsible for the Autoactivation Kinetics of Tyrosinase

Tyrosinase (EC 1.14.18.1) exhibits unusual kinetic properties in the oxidation of monohydric phenol substrates consisting of a lag period that increases with increasing substrate concentration. The cause of this is an autocatalytic process dependent on the generation of a dihydric phenol substrate, which acts as an activator of the enzyme. Experiments with N-substituted dihydric phenol substrates (N-methyldopamine,N-acetyldopamine) demonstrate that oxygen consumption is retarded in the N-acetyl substituted material due to a diminished rate of cyclization. The oxygen uptake exhibited a similar pattern when N-acetyltyramine was oxidized, and this was reflected by a prolongation of the lag period.N,N-Dipropyldopamine was oxidized with normal kinetics but with an oxygen stoichiometry of 0.5 mol of oxygen/mol of substrate. We show that this is the result of the formation of a stable indoliumolate product with oxidation-reduction properties that prevent the formation of dopaminochrome, thus blocking further stages in the tyrosinase-catalyzed oxidation. Evidence that the indoliumolate product is formed by cyclization of theortho-quinone is presented by pulse radiolysis studies, which demonstrate the formation of the ortho-quinone (by disproportionation of the corresponding semiquinones), which cyclizes to give the indoliumolate. The rate constant for cyclization was shown to be 48 s−1 (at pH 6.0). Tyrosinase-catalyzed oxidation of the monohydric phenol analogue,N,N-dimethyltyramine, was shown to require the addition of a dihydric phenol. Oxygen utilization then exhibited a stoichiometry of 1.0, indicating that the reactions proceed only as far as the cyclization. The analogous stable cyclic indoliumolate product was shown to be formed, with UV absorption and NMR spectra closely similar to the indoliumolate derived fromN,N-dipropyldopamine. This material was methylated by catechol O-methyltransferase but was unreactive to redox reagents. The formation of the cyclic product accounts for the indefinite lag when N,N-dimethyltyramine is used as the substrate for tyrosinase in the absence of a dihydric phenol cofactor.

Free radical scavenging properties of melanin: Interaction of eu- and pheo-melanin models with reducing and oxidising radicals

The human skin and eye melanin is commonly viewed as an efficient photoprotective agent. To elucidate the molecular mechanism of the melanin-dependent photoprotection, we studied the interaction of two synthetic melanins, dopa-melanin and cysteinyldopa-melanin, with a wide range of oxidising and reducing free radicals using the pulse radiolysis technique. We have found that although both types of free radicals could efficiently interact with the synthetic melanins, their radical scavenging properties depended, in a complex way, on the redox potential, the electric charge and the lifetime of the radicals. Repetitive pulsing experiments, in which the free radicals, probing the polymer redox sites, were generated from four different viologens, indicated that the eumelanin model had more reduced than oxidised groups accessible to reaction with the radicals. Although with many radicals studied, melanin interacted via simple one-electron transfer processes, the reaction of both melanins with the strongly oxidising peroxyl radical from carbon tetrachloride, involved radical addition. Our study suggests that the free radical scavenging properties of melanin may be important in the protection of melanotic cells against free radical damage, particularly if the reactive radicals are generated in close proximity to the pigment granules.

DIRECT DETECTION OF SINGLET OXYGEN SENSITIZED BY HAEMATOPORPHYRIN AND RELATED COMPOUNDS

Abstract— Direct time‐resolved detection of the luminescence at 1270 nm from ‘singlet oxygen’ was used to estimate the quantum yield of singlet oxygen production (ΦΔ) from a series of related porphyrins in benzene and in D2O. From this and available data the fraction of oxygen quenching interactions leading to singlet oxygen production (SΔ) was derived in most cases. A marked increase in ΦΔ value was observed for di‐haematoporphyrin ester (DHE) in cetyltrimethyl ammonium bromide/D2O solution in comparison to D2O alone, this increase is attributed to a major structural alteration of DHE on introduction of the detergent.

Singlet oxygen quenching and the redox properties of hydroxycinnamic acids

The singlet oxygen quenching rate constants (kq) for a range of hydroxycinnamic acids in acetonitrile and D2O solutions were measured using time resolved near infrared phosphorescence in order to establish their antioxidant activity. The magnitude of kq observed depends on both the nature of the substituent groups and solvent polarity. The variations in kq depend on the energy of the hydroxycinnamic acid/molecular oxygen charge transfer states, (O2delta- ...HCAdelta+). In D2O the values of kq range from 4x10(7) M(-1) s(-1) to 4x10(6) M(-1) s(-1) for caffeic acid and o-coumaric acid respectively. In acetonitrile, the charge transfer energy levels are raised and this is reflected in lower singlet oxygen quenching rate constants with a kq value of 5x10(6) M(-1) s(-1) for caffeic acid. The phenoxyl radical spectra derived from the hydroxycinnamic acids were determined using pulse radiolysis of aqueous solutions and the reduction potentials were found to range from 534 to 596 mV. A linear correlation is observed between reduction potential, and hence free energy for electron transfer, and log kq. These correlations suggest a charge transfer mechanism for the quenching of singlet oxygen by the hydroxycinnamic acids.

LASER INTENSITY AND THE COMPARATIVE METHOD FOR DETERMINATION OF TRIPLET QUANTUM YIELDS

Abstract— In the comparative method of determining the triplet quantum yield øT by laser flash absorption spectroscopy, general equations are established (1) for describing the dependence of øT with laser intensity and (2) for absolute actionometry of a laser pulse. Applications to specific examples are discussed.

Interaction of radicals from water radiolysis with melanin.

Melanins are considered to be natural photoprotectors in the melanocytes and keratinocytes of the skin. These pigments have also been suggested to play an important role in protection of melanin-containing cells against ionising radiation. Various mechanisms have been proposed to explain the protective role of melanin which invoke the radical scavenging properties of the polymer. In the present work the reactions of melanins with radicals generated in aqueous media by pulse radiolysis have been studied. Time-resolved changes in absorbance of the melanin or the radical species were recorded at selected wavelengths. Experiments were carried out on synthetic dopa- and 5-S-cysteinyldopa-melanins and a natural melanin in phosphate buffer (pH 7.4). Under the conditions employed, melanin reacted predominantly with either oxidising (OH., N3.) or reducing (eaq-, CO2-) species. We were also able to monitor the interaction of melanin with superoxide radical, which was reducing in this case. Detailed analysis of transient changes in melanin absorbance, detected at different wavelengths, was demonstrated to be a convenient method for studying redox processes of this substance, as shown by model experiments using ferricyanide and dithionite as oxidising and reducing agents, respectively. Among the radicals studied, OH. exhibited the strongest reactivity with melanins. Apparent rate constants for the reactions of radicals with autoxidative dopa-melanin (1.5 X 10(9) M-1 X s-1, 2.6 X 10(8) M-1 X s-1, 1.8 X 10(8) M-1 X s-1, 5 X 10(5) M-1 X s-1, 10(6)-10(7) M-1 X s-1 for OH., eaq-, N.3. O2- and CO2-, respectively) are reported. The reactivity of melanins with radicals from water radiolysis and their effect on pigment properties are discussed in terms of the structure and possible biological role of the pigments.

Charge transfer between tryptophan and tyrosine in proteins

Abstract With numerous proteins, most of which are not involved in oxidation reduction reactions, azide radicals have been found to react with tryptophan units to give Trp. radicals. The Trp. radicals commonly transfer their electron deficiencies to tyrosine with rate constants in the region of 102-104 s-1, producing TyrO.. For β-lactoglobulin, which has been studied in most detail, the rate of transfer is independent of protein concentration, so the reaction must be intramolecular. Sodium perchlorate and SDS affect the rate, probably because of alterations in protein conformation. The activafion energy for the process is 45 kJ.mol-1, inconsistent with any mechanism involving hydrogen bounds or charge conduction throngh the chain, but consistent with tunnelling. With other proteins there is evidence of intermolecular transfer as well as intramolecular transfer, and there may be several different intramolecular steps corresponding to different tryptophan-tyrnosine pairs. Rates are not always the same as those initiated by photoionization of tryptophan, probably because different tryptophan units are involved. The possibility of transformation means that with enzymes the initial site of attnck by free radicals is not necessarily the site responsible for any consequent loss in activity. The oxidation-reduction potentials involved in the transfer are such that the process may be important in the mode of action of enzymes such as high-potential copper proteins and peroxidases, and perhaps also in the mode of action of other proteins if the environment is suitable.

PHOTOPHYSICAL PROPERTIES OF meso-TETRAPHENYLPORPHYRIN and SOME meso-TETRA(HYDROXYPHENYL)PORPHYRINS

Abstract— The o‐, m‐, and p‐isomers of 5, 10, 15, 20‐ tetra(hydroxyphenyl)‐porphyrin have been of recent interest as potential second‐generation sensitisers in tumour phototherapy. Fluorescence spectroscopy, nanosecond laser flash photolysis and pulse radiolysis have been used to characterise the singlet and triplet excited states of tetraphenylporphyrin and the o, m‐, and p‐isomers of tetra(hydroxyphenyl)porphyrin. This has included evaluation of fluorescence yields and lifetimes, triplet spectra, lifetimes, oxygen quenching rate constants, extinction coefficients, and yields and singlet oxygen yields. Whilst the fluorescence quantum yields were low, the triplet yields were all 0.7 ± 10% and the singlet oxygen yields 0.6 ± 10%: all these parameters are in the ranges shown by other efficient porphyrin photosensitisers. The similar photophysical properties found for these compounds suggest that their differing tumour sensitising efficiencies are likely to be due to other factors.