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.

Inactivation of the pore-forming toxin Sticholysin I by peroxynitrite: protection by cys groups incorporated in the toxin.

Sea anemones synthesize a variety of toxic peptides and proteins of biological interest. The Caribbean Sea anemone Stichodactyla helianthus, produces two pore-forming toxins, Sticholysin I (St I) and Stichloysin II (St II), with the ability to form oligomeric pores in cell and lipid bilayers characteristically lacking cysteine in their amino acid sequences. Recently, two mutants of a recombinant variant of Sticholysin I (rSt I) have been obtained with a Cys residue in functionally relevant regions for the pore-forming activity of the toxin: r St I F15C (in the amino terminal sequence) and r St I R52C (in the binding site). Aiming at characterizing the effects of oxidants in toxins devoid (r St I) or containing -SH moieties (r St I F15C and r St I R52C), we measured their hemolytic activity and pore forming capacity prior and after their incubation with peroxynitrite (ONOO(-)). At low ONOO(-)/Toxin ratios, nearly 0.8 Trp groups are modified by each added peroxynitrite molecule, and the toxin activity is reduced in ca. 20 %. On the other hand, in -SH bearing mutants only 0.5 Trp groups are modified by each peroxynitrite molecule and the toxin activity is only reduced in 10 %. The results indicated that Cys is the initial target of the oxidative damage and that Trp residues in Cys-containing toxins were less damaged than those in r St I. This relative protection of Trp groups correlates with a smaller loss of hemolytic activity and permeabilization ability in liposomes and emphasizes the relevance of Trp groups in the pore forming capacity of the toxins.

Effect of the addition of alkanols of different topology to dipalmitoyl-phosphatidylcholine vesicles in the presence of gramicidin.

In the present work, we analyze the effect of incorporation of the nonanol family (e.g., 1-Nonanol (1-N), 5-Nonanol (5-N), and 2,6-Dimethyl-4-Heptanol (2,6-DH)) into DPPC LUVs in the presence of different gramicidin concentrations. The principal aim of this work is to study the effect of alkanols solubilization on the physicochemical properties of lipid bilayers in the presence of peptide trans-membrane channels, that is, the effects of nonanol family in the interface of lipid-peptide region, considering that the study provides the analysis of a ternary system by direct excitation as well as by Fluorescence Resonance Energy Transfer. Fluorescence measurements were carried out at 20°C after direct excitation of the extrinsic probe or by Fluorescence Resonance Energy Transfer (FRET) from the tryptophan group of gramicidin. Alkanol incorporation decreases with increasing gramicidin content and branching of the additives. 1-N generates most important changes in the inner part of the bilayer, where it produces an increase in bulk acyl chain mobility. Similarly, 1-N significantly modifies the properties of the hydrophilic-hydrophobic interface region sensed by Laurdan, increasing the polarity of the probe microenvironment and/or increasing the relaxation time of interfacial water molecules. On the other hand, 1-N produces a decrease in PDA fluorescence lifetime, a result that can be explained by a significant amount of water entrance to the inner part of the bilayer. The same behavior was observed when pseudo-first-order quenching rate constants by oxygen were measured. 1-N produces an increase in mobility/solubility of the oxygen in the lipid membrane, an effect that is more noticeable in the deep region of the bilayer sensed by PDA, in the absence and in the presence of 2 mol% of Gr. 1-N incorporation produces a greater reduction in GP value than 5-N and 2,6-DH when Laurdan was excited by FRET. These results show that 1-N has the greatest effect in the lipidic domains near the gramicidin channel. On the other hand, excimer-monomer ratios of PDA obtained by FRET show that 1-N reduces the lateral mobility of acyl chains near the lipid-gramicidin interface when gramicidin concentration in the lipid bilayer increases. This effect is more noticeable than that obtained by direct irradiation of the probe in the presence of 5-N and 2,6-DH. On the other hand, the addition of the three alkanols in the presence of Gr produces a noticeable increase in the water permeability, particularly for 1-N. In this context, we propose a scheme that represents the effect of 1-nonanol on the water outflow in DPPC LUVs in the absence and in the presence of Gr.

Evaluation of solute binding to proteins and intra-protein distances from steady state fluorescence measurements.

Steady state fluorescence measurements, due to their relative simplicity and fast and easy implementation, are one of the most employed techniques for evaluating the non-covalent binding of small molecules to proteins. In the present review we discuss the main characteristics of solute binding and the experimental procedures that can be employed for evaluating both, the efficiency of the process and the number of binding sites. It is also discussed the possibility of determining the distance between endogenous fluorophores and non-covalently bound solutes. Albumins (human serum albumin and bovine serum albumin) are considered as model proteins due to their relevance as solute carriers, the extensive available data comprising binding of a large number of solutes, and the reduced number of intrinsic fluorophores which simplifies the data treatment. It is shown that, in spite of the apparent simplicity of the systems, extreme care must be exercised in data treatment and interpretation to avoid misleading results. This applies to the evaluation of binding constants, number of binding sites, and average distance between intrinsic fluorophores and non-covalently bound solutes associated to the proteins.

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.

Interaction and reactivity of urocanic acid towards peroxyl radicals.

The capacity of urocanic acid to interact with peroxyl radicals has been evaluated in several systems: oxidation in the presence of a free radical source (2,2'-azobis(2-amidinopropane; AAPH), protection of phycocyanin bleaching elicited by peroxyl radicals, and Cu(II)- and AAPH-promoted LDL oxidation. The results indicate that both isomers (cis and trans) are mild peroxyl radical scavengers. For example, trans-urocanic acid is nearly 400 times less efficient than Trolox in the protection of the peroxyl radical promoted bleaching of phycocyanin. Regarding the removal of urocanic acid by peroxyl radicals, nearly 100 muM trans-urocanic acid is required to trap half of the produced radicals under the employed conditions (10 mM AAPH, 37 degrees C). Competitive experiments show that the cis-isomer traps peroxyl radicals 30% less efficiently than the trans-isomer. Given the high concentrations that trans-urocanic acid reaches in skin, its capacity to trap peroxyl radicals could contribute to the protection of the tissue towards ROS-mediated processes. Furthermore, both isomers, and particularly the cis-isomer, protect LDL from Cu(II)-induced oxidation.

Comparison of pore-forming ability in membranes of a native and a recombinant variant of Sticholysin II from Stichodactyla helianthus.

Sticholysin II (St II) a potent cytolysin from the sea anemone Stichodactyla helianthus was obtained by recombinant procedures exhibiting six histidine residues in its N-terminus (St IIn6H). The functional comparison between St II and St IIn6H showed a lesser pore-forming ability for the recombinant than for the native in human or rat red blood cells (RBC) and in large unilamellar vesicles (LUV) of different phospholipid composition. However, binding of St IIn6H to small unilamellar vesicles (SUV) was higher with regard to St II. The explanation to the different permeabilizing capacity of both protein variants is not clear, but a different anchoring of St IIn6H to the lipid bilayer could delay the organization of the competent pore into membrane.

Kinetics and mechanism of the reaction of a nitroxide radical (tempol) with a phenolic antioxidant.

In the absence of redox-active transition metal ions, the removal of Tempol by Trolox occurs by a simple bimolecular reaction that, most probably, involves a hydrogen transfer from phenol to nitroxide. The specific rate constant of the process is small (0.1 M(-1) s(-1)). Metals can catalyze the process, as evidenced by the decrease in rate observed in the presence of diethylenetriaminepentaacetic acid (DTPA). Furthermore, addition of Fe(II) (20 microM ferrous sulfate and 40 microM EDTA) produces a noticeable increase in the rate of Tempol consumption.

Peroxyl radicals promoted changes in water permeability through gramicidin channels in DPPC and lecithin-PC vesicles.

Gramicidin incorporation to DPPC or lecithin-PC large unilamellar vesicles (LUVs) leads to pore formation that, under hyper-osmotic conditions, produces a noticeable increase in the rate of trans-membrane water flow. This pore formation is more efficient in the more fluid lecithin-PC LUVs. Exposure of these vesicles to peroxyl radicals generated in the aerobic thermolysis of 2,2'-azo-bis(2-amidinopropane) (AAPH), changes the physical properties of the bilayer (as sensed employing fluorescent probes), modifies gramicidin molecules (as sensed by the decrease in Trp fluorescence) and notably reduces the transbilayer rate of water outflow. In order to evaluate if this reduced water-transport capacity is due to changes in the membrane due to lipid-peroxidation and/or direct damage to gramicidin channels, results obtained in the oxidable vesicles (lecithin-PC) were compared to those obtained in DPPC vesicles. The data obtained show that most of the water transport efficiency loss can be ascribed to a direct disruption of gramicidin channels by AAPH derived peroxyl radicals.

Chemiluminescence associated with amino acid oxidation mediated by hypochlorous acid.

Although most amino acids readily react with hypochlorous acid (HOCl), only the reaction involving tryptophan (Trp) produces a measurable chemiluminescence (CL). Most of this luminescence takes place after total consumption of HOCI when the process is carried out in an excess of Trp. The quantum yield of the process is relatively low (2 x 10(-8) Einstein/mol HOCl reacted). The luminescence is attributed to free radical-mediated secondary reactions of the initially produced chloramines. This is supported by experiments showing that the chloramines produced when HOCl reacts with alanine are able to induce Trp chemiluminescence, and that this luminescence is partially quenched by free radical scavengers. The spectral changes and the effect of pH upon the observed luminescence are compatible with light emission from products produced in the free radical oxidation of Trp.

Spontaneous urinary chemiluminescence and diet. An intervention study in humans.

Spontaneous urinary visible chemiluminiscence has been measured in samples from a diet intervention study in humans. For a month, two groups, each of 21 male volunteers, received either a Mediterranean-type diet or an occidental (high-fat) diet. Urinary chemiluminescence levels (in 10(3) cpm) were 8.5 +/- 3.1 and 6.0 +/- 2.0 for the high-fat and Mediterranean-type diets, respectively. The differences obtained were statistically significant. These results reflect differences in the oxidative stress associate to both diets and support the proposal that spontaneous urinary chemiluminiscence constitutes a complementary index of systemic oxidative stress.

Antioxidant properties of alpha-crystallin.

alpha-Crystallin is a major chaperone lens protein to which has been ascribed antioxidant functions. In the present work we have evaluated the antioxidant and free radical scavenging properties of bovine alpha-crystallin in a series of in vitro models: zimosan-induced, luminol-enhanced chemiluminescence response of polymorphonuclear leukocytes, the autoxidation of brain homogenate, bleaching of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)-derived radical cations, trapping of peroxyl radicals, and reactivity toward hypochloric acid. In all these systems, the reactivity of alpha-crystallin is higher than or similar to that of bovine serum albumin. It is concluded that, given the high concentrations of ol-crystallin in the lenses, its capacity to interact with free radicals and to remove hypochlorous acid could contribute to the maintenance of the lens functionality.

Kinetics of peroxidation of linoleic acid incorporated into DPPC vesicles initiated by the thermal decomposition of 2,2'-azobis(2-amidinopropane) dihydrochloride.

In a previous work [Chem. Phys. Lipids 2000 104, 49], we have derived the following rate law for the oxidation of lipids in compartmentalized systems: R(T)=(k(1)/k(t))(0.5) k(p) [In](0.5) c(0.5) [LH], where, R(T) is the total rate of oxidation, k(1) is the rate constant for the production of free radicals, k(t) and k(p) are the intra-particle rate constants for the termination and propagation sets, respectively, [In] is the concentration of a water-soluble initiator, c is the concentration of particles, and [LH] is the intra-particle concentration of oxidable lipid. In the present work, we have investigated on the applicability of the proposed kinetic rate law for a system where it takes place the oxidation of a reactive lipid incorporated into an inert matrix. With this purpose, we have measured the rate of oxidation of linoleic acid incorporated into dipalmitoylphosphatidylcholine vesicles initiated by the thermal decomposition of 2,2'-azobis(2-amidinopropane) dihydrochloride as a function of the initiator, particles, and intra-particle LH concentrations. The experimentally determined kinetic orders obtained were 0.54+/-0.02, 0.48+/-0.05 and 0.83+/-0.04 for the dependence of the oxidation rate with initiator, particles, and LH intra-particle concentrations, respectively, in agreement with those theoretically predicted. The lower value obtained for the kinetic order in LH is attributed to a change in k(t) with the increase in oxidable lipid intra-particle concentration. The main point to be emphazised from the results here obtained is that the kinetic rate law for the oxidation of lipids in compartmentalized systems can be significantly different than that observed when to the oxidation takes place in homogeneous solution.

Evaluation of total reactive antioxidant potential (TRAP) of tissue homogenates and their cytosols.

This study evaluates the possibility of obtaining total reactive antioxidant potential (TRAP) indexes in homogenates and their cytosolic fractions by a procedure based on the quenching of luminol luminescence induced by the thermolysis of 2,2'-azo-bis(2-amidinopropane). Measurements were performed in rat brain, liver, kidney, and heart homogenates. TRAP indexes can be easily determined both in homogenates and their cytosolic fractions. The results obtained indicate that heart homogenates are the least and liver homogenates the most protected of the systems considered. Glutathione is the measured antioxidant that contributes the most to TRAP values, while uric acid makes a significant contribution only in liver. A calculation of theoretical TRAP values from the measured concentrations of the main antioxidants (glutathione, uric acid, ascorbic acid, and alpha-tocopherol) for the different homogenates shows that, in most tissues (liver, brain, and kidney), nearly 50% of the experimentally determined TRAP values are not accounted for. This difference is mainly due to the contribution of proteins to the measured TRAP.

Free radical-induced protein degradation of erythrocyte membrane is influenced by the localization of radical generation.

We have investigated the role of the localization of free radical generation in erythrocyte membrane proteins degradation. The extracellular radical producer 2,2'-azobis(2-amidinopropane) hydrochloride induced a greater loss of band-3 protein in comparison with spectrin whereas the intracellular radical initiator cysteine induced the reverse effect. However, a large extent of main-chain fragmentation was observed for both proteins under the action of cysteine-derived radicals. The results show that the relative localization of the radical generation has an important influence on the degradation of specific proteins of the erythrocyte membrane.

Kinetics of the chemiluminescence associated to the reaction between peroxyl radicals and proteins.

Protein oxidation, mediated by peroxyl radicals derived from 2,2'-azobis(2-amidinopropane) dihydrochloride is sided by a significant visible chemiluminescence (CL). The light emission shows a complex dependence with the protein concentration and with the incubation time that cannot be interpreted in terms of peroxyl radicals recombination (Russell's mechanism). In all the systems studied, the chemiluminescent behavior requires to consider the participation of several oxidation products as precursors of the excited states. These compounds lead to the formation of excited states by competing radical and nonradical mediated pathways. These intermediates (most probably hydroperoxide-like compounds) would arise from the oxidation of Trp and Tyr residues. This conclusion is based on the similarity of the time profile of the chemiluminescence observed in the oxidation of the free amino acids and the proteins, both in the presence of and absence of free-radical scavengers.

Free-radical-induced inactivation of lysozyme and carbonyl residue generation in protein are not necessarily associated.

The 2,2'-azobis(2-amidinopropane) (AAPH)-induced inactivation and oxidative modification of lysozyme, as determined by the loss of tryptophan-associated fluorescence (TAF) and the increase in dinitrophenylhydrazine-reactive carbonyl groups (CO), were studied in the absence and in the presence of antioxidants. AAPH induced a progressive inactivation of the enzyme and a parallel decrease of its TAF. Both changes were closely correlated (R2 = 0.97); however, the inactivation was only partially associated with an increase in CO. The latter reached maximal values at times half those needed to attain maximal losses in both lysozyme activity and TAF. A stoichiometric comparison reveals that whereas over 74% of the enzyme molecules had lost their activity, only 5% exhibited an increment in CO. CO formation was affected differentially by boldine and trolox. Both antioxidants fully protected against the early inactivation and loss of TAF; however, the increase in CO was completely unaffected by trolox. Exposure of lysozyme to Fe3+/ascorbate induced no loss of activity or TAF, but it led to an accumulation of CO similar to that induced by AAPH. Results indicate that CO formation and lysozyme inactivation are two mechanistically dissociable events and that changes in the former parameter can perfectly occur in the absence of changes in the latter.

Kinetics and mechanism of the chemiluminescence associated with the free radical-mediated oxidation of amino acids.

When amino acids are incubated in the presence of a free radical source [2,2'-azobis(2-amidinopropane) dihydrocloride], only tyrosine (Tyr) and tryptophan (Trp) produce significant chemiluminescence. The relationship between the observed light intensity, the rate of the oxidation process and the substrate concentration is complex and can not be explained in terms of the formation of excited states in termination processes involving two peroxyl radicals (Russell's mechanism). The observed increase in light emission with the incubation time, for both Trp and Tyr, would indicate the participation of more than one reaction product as intermediates in the pathways leading to the production of excited molecules. However, the fact that after product accumulation a high proportion of the observed luminescence is quenched by Trolox addition, implies that the main chemiluminescent process must involve the interaction of product(s) and free radicals. From the effect of added Ebselen, it is proposed that hydroperoxides and peroxides, formed along the reaction path, are the intermediates whose accumulation leads to the observed increase in chemiluminescence with elapsed time. The observed time profiles and the proposed mechanism strongly resemble those associated with the oxidation of complex biological systems, suggesting that protein oxidation could be one of the main sources of chemiluminescence in biological oxidations.

Acid phosphatase reaction with peroxyl radicals: inactivation mechanism and behavior of the partially modified ensemble.

Acid phosphatase (AP) is readily inactivated when exposed to the free radicals generated in the pyrolysis of 2, 2'-azobis(2-amidinopropane) hydrochloride (AAPH) under aerobic conditions. On average, a large number of tryptophan groups are modified by each protein molecule that loses its catalytic activity. In spite of this, the enzyme inactivation takes place without induction times, a result that indicates either that damage is progressive or that damage of a critical target is needed to inactivate the enzyme (all-or-nothing mechanism). A Lineweaver-Burk plot of the enzyme activity measured at pH 4.8 is not compatible with an all-or-nothing mechanism, showing that after exposure of the native protein ensemble to the free radical source there are partially damaged molecules whose affinity for the substrate is widely different from that of the native molecules. On the other hand, the partially damaged ensemble shows a normal Michaelis-Menten behavior when the activity is measured at pH 7.0, with only a reduced value of V(M), relative to that of the unmodified ensemble. These results show that the native protein and modified proteins that remain active constitute different populations, with different responses to pH changes. Comparative heat denaturation studies of the native and pretreated proteins support this proposal.

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.