Pyrogallol red oxidation induced by superoxide radicals: application to evaluate redox cycling of nitro compounds.

The bleaching of the pyrogallol red (PGR) dye mediated by superoxide anion radicals (O(2)(-)) generated from the xanthine/xanthine oxidase system (X/XO) was studied by UV-visible spectrophotometry. The absorption band (at 540 nm) of PGR quickly decreased in the presence of X/XO, implying an efficient reaction of O(2)(-) with PGR. The process was unaffected by catalase (CAT), but completely abolished by superoxide dismutase (SOD). A mechanism of the reaction involving the consumption of one PGR molecule by two O(2)(-) to generate one molecule of H(2)O(2) is proposed. PGR was used as a probe to estimate the rate of O(2)(-) generation in redox cycling reactions of a series of nitro compounds mediated by rat liver microsomes. The consumption of PGR induced by the redox cycling of nitrofurantoin was totally eliminated by the addition of SOD but unaffected by CAT. The initial rate of consumption of PGR mediated by the redox cycling of others nitro derivatives follows the order: furazolidindione > nitrofurantoin > nifurtimox > benznidazole > chloramphenicol. We concluded that PGR can be used as a probe to estimate the release of O(2)(-) from enzymatic systems or from the redox cycling of nitro compounds.

Photobehavior of merocyanine 540 bound to human serum albumin.

The photobehavior of merocyanine 540 (MC) was studied in homogeneous media (ethanol, buffer and glycerol), and in microheterogenous systems (Triton X-100 micelles and in the presence of human serum albumin) using stationary and time-resolved techniques. Merocyanine 540 in aqueous solution mostly forms aggregates, which in the presence of Triton X-100 or HSA are disaggregated. The extent of binding to HSA and its characteristics were estimated from dye absorption and fluorescence changes following protein addition; the Trp-214 fluorescence quenching was also employed to assess the extent of dye association, and physical separation was employed to evaluate the dye's apparent association constant. These results showed that dye adsorption on HSA takes place at both main protein-binding sites (I and II). This adsorption leads to dye monomerization, changing its photobehavior remarkably, with a noticeable increase in fluorescence and triplet lifetimes. These slower decays can be ascribed to a reduction of the dye photoisomerization rate. In addition, the adsorption of the dye partially protects it from the oxygen present in solution, thus reducing the apparent dye triplet-quenching rate constant. However, singlet oxygen and MC triplet quantum yields remain very low in all the systems tested. Finally, we found that the photoconsumption of merocyanine bound to HSA takes place predominantly by a type I mechanism, being more than seven times more efficient than that taking place in ethanol.

Antioxidant capacity of human blood plasma and human urine: simultaneous evaluation of the ORAC index and ascorbic acid concentration employing pyrogallol red as probe.

The oxygen radical absorbance capacity (ORAC) methodology has been employed to estimate the antioxidant capacity of human blood plasma and human urine using pyrogallol red (ORAC-PGR) as target molecule. Uric acid, reduced glutathione, human serum albumin, and ascorbic acid (ASC) inhibited the consumption of pyrogallol red, but only ASC generated an induction time. Human blood plasma and human urine protected efficiently pyrogallol red. In these assays, both biological fluids generated neat induction times that were removed by ascorbate oxidase. From these results, ORAC-PGR method could be proposed as a simple alternative to evaluate an ORAC index and, simultaneously, to estimate the concentration of ascorbic acid in human blood plasma or human urine.

Relationship between lipid peroxidation and rigidity in L-alpha-phosphatidylcholine-DPPC vesicles.

DPPC incorporation into egg-PC unilamellar vesicles reduces their oxidation rate beyond that expected from the unsaturated lipid dilution. Addition of the unsaturated lipids produces changes in the physical properties of the inner parts of the lipid bilayer, as sensed by fluorescence anisotropy of DPH, and in the hydrophilic/hydrophobic region, as sensed by the generalized polarization of laurdan. DPPC (30 mol%) incorporation into egg-PC vesicles produces a decrease in alkyl chain mobility in the inner part of the bilayer, evaluated by the increase of DPH fluorescence anisotropy, and a rise of the generalized polarization value of laurdan in the bilayer interface. It also leads to a decrease in the rate of water efflux promoted by a hypertonic shock. Oxidation of PC LUVs, promoted by AAPH, as sensed by oxygen uptake and MDA formation, leads to qualitatively similar results than DPPC addition: rigidification at the inner part and the surface of the liposomes, and a lower rate of water permeation. It is suggested that these changes could contribute to the observed decrease in oxidation rate with conversion.

The pore forming capacity of Sticholysin I in dipalmitoyl phosphatidyl vesicles is tuned by osmotic stress.

The osmotic condition modulates the properties of liposomes, particularly those related to their stability and response to external agents such as membrane-active proteins or peptides. In a previous work, we have demonstrated that an osmotic shock can increase, per se, water influx/efflux and the exit of the fluorophore calcein entrapped in the aqueous pool of dipalmitoylphosphatidylcholine (DPPC) and DPPC:sphingomyelin (SM) large unilamellar vesicles (LUVs), suggesting a loss of integrity of the liposome bilayer. In the present work, we have extended our study in order to assess how an osmotic imbalance prior to or synchronous with the addition of a recombinant variant of the pore-forming toxin sticholysin I (rSt I) modifies its pore forming capacity in DPPC and DPPC:SM (1:1) LUVs. Our results conclusively show the capacity of hypotonic gradients to improve the pore forming capacity of rSt I molecules, even in pure DPPC liposomes, rendering pore-formation less dependent on the presence of sphyngomyelin. In fact, non-active toxins in DPPC liposomes become active by a hypotonic imbalance in a similar way to those containing SM as a second component.

Controversial alkoxyl and peroxyl radical scavenging activity of the tryptophan metabolite 3-hydroxy-anthranilic acid.

3-Hydroxy-anthranilic acid (3-OHAA), a tryptophan metabolite produced in the kynurenine pathway, is an efficient antioxidant towards peroxyl radicals (ROO) derived from the AAPH (2,2'-azobis(2-amidinopropane) dihydrochloride) thermolysis. However, self-reactions of ROO can give rise to alkoxyl radicals (RO), which could strongly affect the fate of scavenging reactions. In the present work, we studied the influence of RO in the scavenging activity of 3-OHAA in three different systems: i) Monitoring of the direct reaction between 3-OHAA and AAPH-derived free radicals (kinetic studies); ii) Evaluation of the protective effect of 3-OHAA on the AAPH-induced consumption of fluorescein; and, iii) Inhibition, given by 3-OHAA, of the AAPH-initiated lipid peroxidation of both, rat brain synaptosomes and homogenate preparations (assessed by chemiluminescence). For such purposes, the fraction of free radicals (f) trapped per 3-OHAA molecule was determined in each system. Kinetic results show that the oxidation of 3-OHAA follows a process dominated by ROO with a zero order kinetic limit in 3-OHAA, and a fraction (fri) equal to 0.88. From the induction times, elicited by 3-OHAA in the kinetic profiles of fluorescein consumption, a fraction (fT) of 0.28 was determined. 3-OHAA also generated induction times in the kinetic profiles of light emission during the AAPH-initiated lipid peroxidation of rat brain synaptosomes and homogenates. From such induction times, fractions of 0.61 and 0.63 were determined for rat brain synaptosomes (fsyn) and homogenates (fhom), respectively. These results show that during the incubation of 3-OHAA and AAPH, a low fraction of ROO self-reacts to generate RO. Nevertheless, when 3-OHAA is employed to protect particular targets, such as fluorescein, rat brain synaptosomes and homogenates, reactions of ROO and/or RO should be considered.

A novel and simple ORAC methodology based on the interaction of Pyrogallol Red with peroxyl radicals.

Oxygen radicals absorbance capacities (ORAC) indexes are frequently employed to characterize the radical trapping capacity of pure compounds and their complex mixtures. A drawback of ORAC values obtained using phycoerythrin, fluorescein (FL) or c-phycocyanin as targets, makes it possible to conclude that for very reactive compounds they are much more related to stoichiometric factors than to the reactivity of the tested compound. In the present paper, we propose a simple methodology, based on the bleaching of Pyrogallol Red (PGR) absorbance that provides ORAC indexes that are almost exclusively determined by the reactivity of the tested compounds. This difference is due to the high reactivity of PGR and the high concentrations of this compound employed in the experiments.

Structural and functional characterization of a recombinant sticholysin I (rSt I) from the sea anemone Stichodactyla helianthus.

Sticholysins I and II (Sts I and II) are two potent cytolysins from the sea anemone Stichodactyla helianthus. These isoforms present 13 substitutions, with three non-conservative located at the N-terminus. St II is considerably more hemolytic than St I in human red blood cells, a result explained by the smaller number of negatively charged groups present at St II's N-terminus. In the present work, we have obtained a recombinant St I (rSt I), differing from the wild type in a single amino acid residue (E16Q). This pseudo-wild type is structurally similar to St I and shows a similar capacity to interact with and form pores in model membranes. This was assessed by the intrinsic fluorescence increase in the presence of liposomes, their adsorption to bilayers (measured by SPR), their concentration at the air-water interface, their interaction with lipid monolayers and their capacity to promote the release of carboxyfluorescein entrapped in liposomes. In spite of these similarities, rSt I presents a larger hemolytic activity in human red blood cells than St I, being intermediate in activity between Sts I and II. The results obtained in the present work emphasize that even the change of one single E by Q at the N-terminal segment may modify the toxin HA and show that this functional property is the most sensitive to subtle changes in the protein primary structure.

Role of endogenous channels in red blood cells response to their exposure to the pore forming toxin Sticholysin II.

Sticholysin II (St II) is a highly hemolytic cytolysin isolated from the sea anemone Stichodactyla heliantus. The toxin hemolytic action takes place through the formation of channels that provoke an electrolyte unbalance leading to osmotic shock. The lytic event must involve the exchange of electrolytes and the entrance of water, leading to red blood cell disruption. These processes can occur through St II pores and/or the endogenous red blood cells transporters. In order to evaluate the contribution of these channels to water, anion and cation transport, we have measured the hemolysis and K+ efflux rates in the presence of several specific inhibitors. The results obtained in the presence of Hg, an AQP1 blocker, indicate that water transport through these channels is not essential for the occurrence of the lytic process induced by St II. The data also support a partial role of K+ and anion transporters. In particular, they are compatible with a preferential K+ efflux though the K(+)/Cl- co-transport as a response to the promoted swelling. Furthermore, they suggest that chloride influx, a process that can regulate both K+ efflux and lysis, is partially mediated by the endogenous cell transporters, in particular, band-3 anion exchange system being relevant at early stages of the lytic process.

Effect of urea on the enzymatic activity of a lipase entrapped in AOT-heptane-water reverse micellar solutions.

A study has been made of the effect of urea upon the hydrolysis of 2-naphthyl acetate (2-NA) catalyzed by lipase from Rhizopus arrhizus in AOT-heptane-water reverse micellar solutions at pH 7. The partition constants, K, of 2-NA between n-heptane and aqueous urea solutions in the absence of micelles were also determined. It was found that K decreases when the concentration of urea increases. In aqueous solution the rate of hydrolysis of 2-NA catalyzed by lipase is dependent on the concentration of urea (at a given 2-NA concentration). This result can be due to a decrease in the magnitude of the association of lipase with 2-NA and/or to changes in the reaction rate of the lipase-2-NA complex. The modifications of the enzymatic activities elicited by addition of urea show a lineal correlation with K, emphasizing the relevance of hydrophobic effects in the loss of activity. Nevertheless, the slope of the line is higher than one, suggesting that changes in the conformation of the enzyme would be also important. Addition of urea to the micellar solutions provokes a decrease of the enzyme activity. From the dependence of the reaction rate with AOT concentration, the partition constant of 2-NA between n-heptane and the micelles, K(p), was obtained. In the presence of 2 M urea a value of K(p)=0.33 M(-1) was derived. This value is lower than that measured in the absence of urea (Aguilar et al., Arch. Biochem. Biophys. 388 (2001) 231), indicating that incorporation of urea to the micellar interface produces a decrease of the association of 2-NA with the micelles. From a comparison of the results obtained in the micellar solution and in aqueous solution, it is concluded that the enzyme is more resistant to denaturation by urea in the micellar solution than in aqueous solution. Furthermore, at intermediate urea concentrations (2 M), the additive produces an increase in the Michaelis constant (K(M)) without a significant decrease (or even a small increase) in the catalytic rate constant (k(cat)).

Response of unilamellar DPPC and DPPC:SM vesicles to hypo and hyper osmotic shocks: A comparison.

DPPC and DPPC:SM large unilamellar vesicles (LUVs), prepared by extrusion, readily respond to osmotic shocks (hypo- and hyper-osmotic) by water influx/efflux (evaluated by changes in turbidity) and by entrapped calcein liberation (measured by an increase in dye fluorescence intensity). On the other hand, small unilamellar vesicles (SUVs) prepared by sonication are almost osmotically insensitive. LUVs water transport, both in hypo- and hyper-osmotic conditions, takes place faster than calcein ejection towards the external solvent. Similarly, response to a hypotonic imbalance is faster than that associated to a hypertonic stress. This difference is particularly noticeable for the increase in calcein fluorescence intensity and can be related to the large reorganization of the bilayer needed to form pores and/or to adsorb the dye to the inner leaflet of the vesicle after water efflux. Conversely, addition of SM to the vesicles barely modify the rate of calcein permeation across the bilayer.

Autocatalytic oxidation of hemoglobin by nitrite: a possible mechanism.

Oxidation of oxyhemoglobin by nitrite ions to produce methemoglobin is one of the more employed procedures to oxidize the hemoprotein. The process takes place readily after a clear induction time. This behaviour is usually explained in terms of an autocatalytic reaction mechanism. However, the generally accepted mechanism is not autocatalytic and cannot explain the main features of the process. In the present work it is proposed that the characteristics of the process require the occurrence of a fast reaction between oxyhemoglobin and nitrogen dioxide. This process acts as a branching step, leading to the observed autocatalysis.

On the use of the quenching of luminol luminescence to evaluate SOD activity.

Addition of horseradish peroxidase to a luminol solution (pH = 9.4) produces a burst of light followed by a steady luminescence that lasts for several minutes. This steady-state luminescence is readily quenched by SOD, with a Q1/2 concentration (the additive concentration needed to decrease by one-half the emitted luminescence intensity) of c.a. 4 ng/ml (14 mU/ml). The luminescence intensity decrease can then be employed to evaluate SOD activity in SOD-containing samples. However, the light intensity can also be quenched by additives, such as Trolox, that are able to trap luminol-derived intermediates. It is proposed that double quenching experiments must be performed in order to be able to relate the observed effect of an additive to its SOD-like activity.

Visible chemiluminescence from rat brain homogenates undergoing autoxidation. II. Kinetics of the luminescence decay.

The visible luminescence emitted in the autoxidation of brain homogenates is only partially quenched when antioxidants are added at concentrations such that further oxidation is prevented. From the time course of the emission after antioxidant addition, it can be estimated that nearly 50% of the light arises from an intermediate that decays with a first order kinetics and with a lifetime of ca. 40 s at 32 degrees C. The remaining light arises from the decomposition of one or several intermediates, and show a kinetics that is independent of the incubation time. From the data obtained it is concluded that bimolecular free radical processes, such as the recombination of peroxy radicals, do not significantly contribute to the observed luminescence.

Kinetics of phycocyanine bilin groups destruction by peroxyl radicals.

Bilin groups in c-phycocyanine are readily bleached by peroxyl radicals produced in the thermolysis of 2, 2'-azobis(2-amidinopropane). From an evaluation of the bilin groups destroyed per radical that interacts with the protein, it is concluded that the bilin moiety is the main target of the radicals. Kinetic expressions are derived that allows an estimation of the substrate reactivity from the analysis of the rate of bilin group modification as a function of the protein concentration. From this analysis it is concluded that micromolar concentrations of c-phycocyanine are able to reduce the steady state concentration of the peroxyl radicals by one half, indicating a high antioxidant activity for this compound. This conclusion is confirmed by measuring the capacity of the protein to protect 1-naphthol from modification by peroxyl radicals. The results obtained show that the bilin groups have, on a molar basis, an antioxidant activity similar to that of potent antioxidants such as catechin.

Evaluation of total antioxidant potential (TRAP) and total antioxidant reactivity from luminol-enhanced chemiluminescence measurements.

The effect of antioxidants and biological fluids on the intensity of luminol induced chemiluminescence by radicals derived from the thermolysis of 2,2'-azo-bis(2-amidinopropane) has been employed to monitor TRAP and TOTAL ANTIOXIDANT REACTIVITY (TAR) levels. The latter parameter, which considers not only the quantity of oxidants but also their reactivity, is considered a potentially more useful index of the antioxidant status of a biological fluid. The results obtained employing human blood plasma and human urine show that the detected antioxidant capacity of both fluids is mainly related to the uric acid concentration of the samples.

Sod and catalase inactivation by singlet oxygen and peroxyl radicals.

Both superoxide dismutase and catalase are readily deactivated by singlet oxygen and by the radicals produced in the pyrolysis of 2,2'-azo-bis-(2-amidinpropano) under aerobic conditions. The rate constant for the loss of enzymatic activity induced by singlet oxygen are 3.9 x 10(7) and 2.5 x 10(7) M-1 sec-1 for SOD and catalase, respectively. The similarity between these values implies that in systems where SOD and catalase are exposed to similar singlet oxygen concentrations, it can be expected a parallel inactivation of both enzymes. The inactivation of both enzymes by the radicals produced by 2,2'-azo-bis-(2-amidinopropane) pyrolysis under aerobic conditions follows a first-order kinetics at low enzyme concentrations and a zero-order kinetics at higher concentrations. Although at low enzymatic concentrations the rate of inactivation of both enzymes is similar, this results from a compensation of effects because there are wide differences in the reactivity of both enzymes towards peroxyalkyl radicals. Catalase is considerably more reactive, but a large number of protein/radical reactive interactions are needed to inactivate one enzyme. On the other hand, the reactivity of SOD is smaller, but the average enzyme activity decreases by nearly 20% in each SOD/radical reactive interaction.

Antioxidant capacity of diethyldithiocarbamate in a metal independent lipid peroxidative process.

2,2'-Azobis-[2-amidinopropane] initiated lipid peroxidation of egg yolk phosphatydil choline liposomes was measured by oxygen uptake and the emitted visible luminescence. Lipid peroxidation involved a chain process (kinetic chain length = 49 +/- 11) and its rate was independent of added Fe ions. Diethyldithiocarbamate (DDC) behaved as an efficient inhibitor in the microM range, being able to trap 1.05 +/- 0.25 free radicals per added molecule. The efficiency of DDC was also independent of Fe addition to the system. These results indicate that DDC is able to trap the chain carrying free radicals, showing that this compound, besides being a powerful metal chelator, is also an efficient free radical scavenger. It is proposed that the relevant step in this process involves an electron transfer from DDC to the peroxy radical LOO., LOO. + DDC- ----LOO- + DDC. followed by protonation of LOO- and dimerization of the DDC. radical.

Is spontaneous urinary visible chemiluminescence a reflection of in vivo oxidative stress?

Based on the characteristics of the visible spontaneous luminiscence of human urine, we propose that this luminescence is due to the dark decomposition of long-lived luminescent intermediates produced by oxidations at the cellular level, and that it might reflect the development of oxidative stress conditions in vivo. This hypothesis is consistent with the higher emission levels monitored in the urine of hyperthyroid patients and the correlation observed between urinary thiobarbituric acid reactive substances (TBARS) levels and urinary chemiluminescence.

Enhanced urinary spontaneous luminescence in Duchenne muscular dystrophy.

Urinary spontaneous visible luminescence is enhanced in children with Duchenne Muscular Dystrophy (DMD). This result is indicative of systemic oxidative stress in DMD patients. It is proposed that measurement of the urinary luminescence could be employed to follow the progress of the disease, as well as the response of the patients to antioxidant therapy.