A novel and potent biological antioxidant, Kinobeon A, from cell culture of safflower

Kinobeon A was originally isolated from cultured cells of safflower (Carthamus tinctorius L., Compositae). It had never previously been directly isolated from safflower or other plants, animals or microorganisms. In this report, we demonstrate the anti-oxidative effects of kinobeon A and compare the results with those two known natural antioxidants, lignan (nordihydroguaiaretic acid) and quercetin. The NADPH-induced microsomal lipid peroxidation system was employed to assess anti-oxidative effects of kinobeon A. Addition of kinobeon A to the system significantly decreased the formation of thiobarbituric acid reactive substances (TBARS) in a dose-dependent manner with effects similar to those of lignan and quercetin. Formation of TBARS was completely inhibited at 10 microM of kinobeon A. Employing the xanthine/xanthine oxidase/nitroblue tetrazolium system and the KO2/XTT system, the superoxide anion scavenging activity of kinobeon A was greater than that of lignan or quercetin. IC50 values calculated for kinobeon A in these two systems were 1 microM and 0.8 microM, respectively. Kinobeon A exerted cytoprotective effects following oxidative treatments with hydrogen peroxide, cumene hydroperoxide, menadione and xanthine oxidase (XOD). Addition of kinobeon A to the systems markedly enhanced survival ratios of Madin-Darby bovine kidney cells, while their survival significantly decreased with the oxidative treatment alone. Kinobeon A exhibited stronger effect on the cell viability than lignan or quercetin when menadion or XOD were used as inducing reagents of oxidative stress. The present study demonstrates for the first time that kinobeon A prevents oxidative stresses and could be a useful cytoprotective reagent.     

Singlet oxygen quenching by dietary carotenoids in a model membrane environment

The ability of several dietary carotenoids to quench singlet oxygen in a model membrane system (unilamellar DPPC liposomes) has been investigated. Singlet oxygen was generated in both the aqueous and the lipid phase, with quenching by a particular carotenoid independent of the site of generation. However, singlet oxygen quenching is dependent on the carotenoid incorporated; xanthophylls exhibit a marked reduction in efficiency compared to the hydrocarbon carotenoids. Lycopene and beta-carotene exhibit the fastest singlet oxygen quenching rate constants (2.3-2.5 x 10(9)M(-1)s(-1)) with lutein the least efficient (1.1 x 10(8)M(-1)s(-1)). The other carotenoids, astaxanthin and canthaxanthin, are intermediate. Zeaxanthin exhibits anomalous behavior, and singlet oxygen quenching decreases with increasing amounts of zeaxanthin, leading to nonlinear plots for the decay of singlet oxygen with zeaxanthin concentration. Such differences are discussed in terms of carotenoid structure and their influence on the properties of the lipid membrane. The formation of aggregates by the polar carotenoids is also proposed to be of significance in their ability to quench singlet oxygen.     

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.     

Effect of vanillin on methylene blue plus light-induced single-strand breaks in plasmid pBR322 DNA

The ability of vanillin (4-hydroxy-3-methoxybenzaldehyde), a naturally occurring food flavouring agent, in inhibiting photosensitization-induced single-strand breaks (ssbs) in plasmid pBR322 DNA has been examined in an in vitro system, independent of DNA repair/replication processes. Photosensitization of DNA with methylene blue, visible light and oxygen, induced ssbs resulting in the production of open circular form (OC form) in a concentration-dependent manner. The yield of OC form induced by photosensitization was increased several-fold by deuteration of the buffer and was found to be inhibited by sodium azide, a scavenger of singlet oxygen (1O(2)). Vanillin, per se, did not induce but inhibited photosensitization-induced ssbs in plasmid DNA, at millimolar concentrations. The inhibitory effect of vanillin was both concentration- and time-dependent. On a molar basis, vanillin was, however, less effective than trolox, a water-soluble analogue of alpha-tocopherol. Photosensitization by methylene blue system generates singlet oxygen, as one of the major components of ROS. Therefore, interaction of singlet oxygen with vanillin was investigated. The rate constant of vanillin with 1O(2) was estimated to be 5.93x10(7)M(-1)s(-1) and that of sodium azide as 2. 7x10(8)M(-1)s(-1). The present investigations show that vanillin can protect against photosensitization-induced ssbs in the plasmid pBR322 DNA, and this effect may partly be due to its ability to scavenge 1O(2).     

Peridinin as the major biological carotenoid quencher of singlet oxygen in marine algae Gonyaulax polyedra

Carotenoids in light-harvesting proteins and reaction centers increase the overall efficiency of photosynthesis by transferring absorbed light energy to chlorophylls. Peridinin and beta-carotene were isolated from Gonyaulax polyedra in a one-step purification protocol using the preparative circular chromatography (Chromatotron), performed on silica gel under N(2) atmosphere and n-hexane/acetone 8:2 as mobile phase and characterized by extensive (1)H NMR, infrared, and electrospray ionization mass spectrometry analyses. The quenching of singlet molecular oxygen [O(2) ((1)Delta(g))] was evaluated by NIR-emission assays using singlet oxygen generated by sensitization of either perinaphthenone or methylene blue. The NIR-emission assay showed that peridinin quench as singlet oxygen (k(q) = 9.5 x 10(8) M(-1) s(-1)) 5-fold less efficiently than beta-carotene (52 x 10(8) M(-1) s(-1)). A method, based on the use of high-performance liquid chromatography with UV-VIS detection, was then developed for the sensitive quantification of peridinin (55% of total carotenoids) and beta-carotene (4.1% of total carotenoids). Thus, since peridinin is 10-fold more abundant than beta-carotene, it is expected to be the major protector against the deleterious effects of O(2) ((1)Delta(g)) in Gonyaulax polyedra.     

Kinetics of oxidation of serotonin by myeloperoxidase compounds I and II.

The oxidation of serotonin (5-hydroxytryptamine) by the myeloperoxidase intermediates compounds I and II was investigated by using transient-state spectral and kinetic measurements at 25.0 +/- 0.1 degrees C. Rapid scan spectra demonstrated that both compound I and compound II oxidize serotonin via one-electron processes. Rate constants for these reactions were determined using both sequential-mixing and single-mixing stopped-flow techniques. The second order rate constant obtained for the one-electron reduction of compound I to compound II by serotonin is (1.7 +/- 0.1) x 10(7) M(-1) x s(-1), and that for compound II reduction to native enzyme is (1.4 +/- 0.1) x 10(6) M(-1) x s(-1) at pH 7.0. The maximum pH of the compound I reaction with serotonin occurs in the pH range 7.0-7.5. At neutral pH, the rate constant for myeloperoxidase compound I reacting with serotonin is an order of magnitude larger than for its reaction with chloride, (2.2 +/- 0.2) x 10(6) M(-1) x s(-1). A direct competition of serotonin with chloride for myeloperoxidase compound I oxidation was observed. Our results suggest that serotonin may have a role to protect lipoproteins from oxidation and to prevent enzymes from inactivation caused by the potent oxidants HOCl and active oxygen species.     

Kinetic study of the quenching reaction of singlet oxygen by tea catechins in ethanol solution

A kinetic study of the quenching reaction of singlet oxygen ((1)O(2)) with catechins (catechin (CA), epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG)) and related compounds (5-methoxyresorcinol (MR), 4-methylcatechol (MC), and n-propyl gallate (PG)) was performed in ethanol at 35 degrees C. MR, MC, and PG are considered to be a model of resorcinol (A)-, catechol (B)-, and gallate (G)-rings in catechins, respectively. The overall rate constants, k(Q) (= k(q) + k(r), physical quenching + chemical reaction), for the reaction of catechins with (1)O(2) increased in the order of PG < MR < MC < CA < EC < EGC < ECG < EGCG. In a comparison of the rate constants, the relationship between quenching rates and chemical structures is discussed. The catechins which have lower peak oxidation potentials, E(P), show higher reactivities. It was observed that the chemical reaction (k(r)) is almost negligible in the quenching reaction of (1)O(2) by catechins. The k(Q) values of EGCG (1.47 x 10(8) M(-1) s(-1)) and ECG (7.81 x 10(7)) were found to be larger than those of lipids (1.3 x 10(5)-1.9 x 10(5) M(-1) s(-1)), amino acids (<3.7 x 10(7)), and DNA (5.1 x 10(5)). Further, these values are similar to those (1.15 x 10(8)-2.06 x 10(8) M(-1) s(-1)) of alpha- and gamma-tocopherol, ubiquinol-10, and gamma-tocopherol hydroquinone (plastoquinol model). The result suggests that catechins may contribute to the protection of oxidative damage in biological systems, by quenching (1)O(2).     

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.     

Whole animal and gill tissue oxygen uptake in the Eastern oyster, Crassostrea virginica: Effects of hypoxia, hypercapnia, air exposure, and infection with the protozoan parasite Perkinsus marinus(1)

The Eastern oyster, Crassostrea virginica, lives in shallow coastal waters and experiences many different environmental extremes including hypoxia, hypercapnia and air exposure and many oysters are infected with the protozoan parasite Perkinsus marinus. The effects of these conditions on oyster metabolism, as measured by oxygen uptake, were investigated. Mild hypercapnia had no effect on the ability of oysters to regulate oxygen uptake in hypoxic water, as measured by the B2 coefficient of oxygen regulation. The average B2 was -0.060x10(-3) (+/-0.01x10(-3) S.E.M.; n=20; low and high CO(2) treatments combined) in oysters uninfected with P. marinus and -0.056x10(-3) (+/-0.01x10(-3) S.E.M.; n=16; low and high CO(2) treatments combined) in infected oysters. There was no significant effect of light to moderate infections of P. marinus on oxygen regulation. Nor did the presence of P. marinus have an effect on the rate of oxygen uptake of whole animals in well-aerated water. In well-aerated conditions, oxygen uptake was significantly reduced by moderate hypercapnia in oysters when data from uninfected and infected oysters were combined. Mean oxygen uptake of infected oysters under hypercapnia (pCO(2)=6-8 Torr; pH 7) was 9.10 μmol O(2) g ww(-1) h(-1) +/-0.62 S.E.M. (n=9), significantly different from oxygen uptake under normocapnia (pCO(2) 相似文献   

Kinetics and mechanism of the sensitized photodegradation of lignin model compounds.

The kinetics of the sensitized photodegradation of a variety of well-defined lignin model compounds was studied to determine the mechanisms responsible for lignin's photochemically-mediated oxidation. Monomeric and dimeric models representing lignin's phenolic end groups and nonphenolic dimers representing its inner core were studied. It was determined that the rate constants for the reaction of the deprotonated phenolic models with singlet oxygen (1O2) range from 0.96 to 7.2 x 10(7) M(-1) s(-1). The models were substituted with zero, one, or two electron-donating methoxy groups on both aryl rings and, while the rate constants showed little dependence on the substitution of the nonphenolic ring, the rate constants increased dramatically with increasing methoxy substitution of the phenol. Reaction between these deprotonated models and 1O2 is thus proposed to occur at the phenolate ring. Under neutral conditions, it was observed that the phenolic models react with excited state sensitizer, with this reaction also occuring at the phenol ring. The sum of the rate constants for quenching of and reaction with excited state sensitizer by lignin model compound ranges from 5.4 to 75 x 10(7) M(-1) s(-1). This study corrects previous reports that attribute the sensitized degradation of neutral lignin model compounds to reaction with 1O2. A nonphenolic aromatic ketone inner-core model was observed to undergo direct photolysis, and its reduced analog was not degraded by direct photolysis or reaction with 1O2 or excited state sensitizer. The oxidized inner-core model was also shown to be able to act as a sensitizer for the degradation of a phenolic lignin model compound.     

Photochemically induced transformation of adrenochrome to adrenochrome-melanin

The photochemical decomposition of adrenochrome in aqueous and deuterated solutions by visible light was investigated. From the spectroscopic study the disappearance constant k = 4.8 x 10(-5) s-1 as well as quenching constant kq = 2.5 x 10(-1) [s M-1] and isotope effect kD/kH = 2 for singlet oxygen mechanism have been calculated. A possible chemical mechanism for the observed transformation of adrenochrome to the melanin polymer is discussed including the formation of the reactive intermediate species like cytotoxic quinones.     

Photodynamic action of bonellin, an integumentary chlorin of Bonellia viridis, Rolando (Echiura, Bonelliidae).

1. The photodynamic activity of bonellin, an integumentary chlorin of Bonellia viridis, is investigated. 2. 10(-6) M bonellin solutions haemolyze erythrocytes only in the presence of light. Previous illumination (tungsten lamp 2000-4000 lux for 1 hr) of the bonellin solutions does not affect the results. Under lowered oxygen tensions the bioactivity is depressed. Benzoquinone and singlet oxygen quenchers delay the photodynamic effect. 3. Bonellin (2 x 10(-6) M) destroys echinoid gamete function, depresses oxygen uptake of spermatozoa, and arrests development of echinoid and Bonellia eggs. These effects are produced only in the presence of light. 4. Copper bonellin is not photodynamically active. The role of copper may be to protect against photosensitization.     

Beta-adrenergic receptor function and oxygen radical production in bovine pulmonary alveolar macrophages

Reactive oxygen species production by bovine pulmonary alveolar macrophages was evaluated by a chemiluminescence assay utilizing luminol and opsonized zymosan. Incubation with dobutamine (5 x 10(-8) and 5 x 10(-7) M) or isoproterenol (5 x 10(-8) and 5 x 10(-7) M) prior to zymosan challenge significantly (p less than 0.05) increased the time for chemiluminescence to begin, and significantly decreased the level of maximum chemiluminescence. The agonists' inhibitory effects on maximum chemiluminescence were significantly reduced by pre-incubation with the appropriate antagonist (atenolol at 1 x 10(-6) M for dobutamine; and propranolol at 1 x 10(-6) M for isoproterenol). Salbutamol at 1 x 10(-6) M significantly reduced the level of maximum chemiluminescence only, but did not increase the time for chemiluminescence to begin. This effect was significantly reduced by the presence of the beta 2-antagonist ICI 118,551 at 1 x 10(-6) M. The results reveal the presence of beta 1- and beta 2-adrenoceptors on bovine pulmonary alveolar macrophages, and suggest that these receptors are important in the regulation of reactive oxygen species production by these cells.     

Reactivity towards singlet oxygen of propofol inside liposomes and neuronal cells

Singlet oxygen (1O2), a reactive oxygen species, has been found to be implicated in many cellular events and pathological disorders. Herein, we investigated the reactivity of 1O2 towards the anaesthetic agent propofol (PPF) encapsulated within DMPC liposomes. By time resolved luminescence, the rate constant of 1O2 quenching by PPF was evaluated, depending on the location of the sensitizer, with following values: 1.35+/-0.05x10(7) M(-1) s(-1) for deuteroporphyrin (as embedded source) and 0.8+/-0.04x10(7) M(-1) s(-1) for uroporphyrin (as external source), respectively. The nature of the oxidation product, resulting from the reaction of 1O2 with PPF, was determined using absorption and HPLC techniques. Finally, the in vitro protective effect of PPF towards the 1O2-induced neuronal cell toxicity was evaluated in terms of cell viability.     

Modulation of the phototoxic effect of hypericin in human leukemia CEM cell line by N-ethylmaleimide,amiloride and omeprazole

Hypericin is a photosensitizing plant pigment from Hypericum perforatum with multiple modes of light-induced biological activities due to production of singlet oxygen and/or excited-state proton transfer with consequent pH drop in the hypericin environment. In the present work, we studied the effects of three inhibitors of crucial mechanisms responsible for intracellular pH (pHi) regulation on hypericin phototoxicity: N-ethylmaleimide (NEM), an inhibitor of H+-ATPase, 5'-(N,N-dimethyl)-amiloride (DMA), an inhibitor of Na+/H+ exchanger, and omeprazole (OME), an inhibitor of H+K+-ATPase. Our experiments show that the effect of hypericin at 1 x 10(-5) and 1 x 10(-6) mol x l(-1) was significantly potentiated by NEM (1 x 10(-7)-1 x 10(--9) mol x l(-1)) and DMA (1 x 10(-6) and 1 x 10(-7) mol x l(-1)) in leukemic CEM cell line. On the other hand, OME had no significant effect on hypericin cytotoxicity. Our results support the hypothesis that the excited-state proton transfer and the consequent acidification of hypericin environment could play a role in the biological activity of hypericin.     

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).     

Solvent effect on the physical quenching of singlet molecular oxygen by p-quinones.

Rate constants for the interaction between singlet molecular oxygen [O2(1 delta g)] and the p-quinones 1,4-benzoquinone (BQ), duroquinone (DQ), 9,10-anthraquinone (AQ) and 1,8-dihydroxy-9,10-anthraquinone (OHAQ) are reported for several solvents at room temperature. The solvent effect on the total quenching rate constant (kt) was analysed employing the semiempirical solvatochromic equation proposed by Kamlet and Taft. The higher values of kt (2-7 x 10(7) M(-1) s(-1)) were obtained when the hydrogen-bond donor solvent ability is increased (higher alpha parameter values). The results indicate the importance of specific solvent interactions in governing the rates of the quenching.     

Spectroscopic properties and reactivity of free radical forms of A2E

A pyridinium bisretinoid (A2E) is the only identified blue-absorbing chromophore of retinal lipofuscin that has been linked to its aerobic photoreactivity and phototoxicity. Pulse radiolysis has been used to study both the one-electron oxidation and the one-electron reduction of A2E in aqueous micellar solutions. The reduction to the semireduced A2E (lambda(max) broad and between 500 and 540 nm) was achieved with formate radicals and the subsequent decay of A2E* was slow (over hundreds of milliseconds) via complex kinetics. The long lifetime of the A2E* should facilitate its reactions with other biomolecules. For example, with oxygen, the A2E* produced the superoxide radical anion with a rate constant of 3 x 10(8) M(-1) s(-1). The A2E was also reduced by the NAD radical, the corresponding rate constant being 2.3 x 10(8) M(-1) s(-1). Other experiments showed that the one-electron reduction potential of A2E lies in the range -640 to -940 mV. The semioxidized form of A2E (lambda(max) 590 nm) was formed via oxidation with the Br2*- radical and had a much shorter lifetime than the semireduced form. With strongly oxidizing peroxyl radicals (CCl3O2*) our kinetic data suggest the formation of a radical adduct followed by dissociation to the semioxidized A2E. With milder oxidizing peroxyl radicals such as that from methanol, our results were inconclusive. In benzene we observed an efficient oxidation of zeaxanthin to its radical cation by the A2E radical cation; this may be relevant to a detrimental effect of A2E in vision.     

Reactivity of 2',7'-dichlorodihydrofluorescein and dihydrorhodamine 123 and their oxidized forms toward carbonate, nitrogen dioxide, and hydroxyl radicals

The aim of this study was to investigate the oxidation of two common fluorescent probes, dichlorodihydrofluorescein (DCFH2) and dihydrorhodamine (DHR), and their oxidized forms, dichlorofluorescein and rhodamine, by the radical products of peroxynitrite chemistry, *OH, NO2*, and CO3*-. At pH 8.0-8.2, rate constants for the interaction of carbonate radical with probes were estimated to be 2.6 x 10(8) x M(-1) s(-1) for DCFH2 and 6.7 x 10(8) M(-1) s(-1) for DHR. Nitrogen dioxide interacted more slowly than carbonate radical with these probes: the rate constant for the interaction between NO2* and DCFH2 was estimated as 1.3 x 10(7) M(-1) s(-1). Oxidation of DHR by nitrogen dioxide led to the production of rhodamine, but the kinetics of these reactions were complex. Hydroxyl radical interacted with both probes with rate constants close to the diffusion-controlled limit. We also found that oxidized forms of these fluorescent probes reacted rapidly with carbonate, nitrogen dioxide, and hydroxyl radicals. These data suggest that probe oxidation may often be in competition with reaction of the radicals with cellular antioxidants.     

Photodynamic effect in lysozyme: a kinetic study in different micellar media.

The influence of medium heterogeneity on the kinetics of the photodynamic effect on native protein lysozyme (Lyso), as well as the interaction of protein and the medium, anionic (SDS) micelles, neutral (Triton X-100) micelles and reversed micelles of AOT, were investigated at pH 8. The interaction between Lyso, Triton X-100 and SDS micelles was quantified by determining the respective associations constant (K(Lyso)). Values were 37 M(-1) for Triton X-100 and 514 M(-1) for SDS, indicating that the Lyso molecule binds Triton X-100 micelles effectively and SDS micelles even more strongly. Time-resolved phosphorescence detection (TRPD) indicates that the protein interacts with O2 (1deltag), with overall rate constants of the order of 10(8) M(-1)/S in direct micelles and 10(7) M(-1)/S in reverse micelles. Apparent reactive rate constants for eosin-sensitized photo-oxidation (singlet molecular oxygen [O2 (1deltag)]-mediated) of the protein were determined through oxygen uptake experiments for the direct micelles, while the fade in the protein fluorescence spectrum upon sensitized irradiation was used in AOT. The results indicate that the O2 (1deltag) attack on the interior of Lyso on amino acid residues, was more effective in leading to a photo-oxidative reaction in SDS and in Triton X-100 at surfactant concentrations < 1 x 10(-2) M than in a homogeneous solution. However, Lyso reactivity reached a maximum when the concentration of micelles was approximately 1 x 10(-5), the same as the protein concentration In AOT reverse micelles, the quenching rate constants decreased > 75% with respect to water. This effect can be attributed to the decrease in accessibility of the amino acid residues to O2 (1deltag).