Kinetic and structural assessment of the reduction of human 2-Cys peroxiredoxins by thioredoxins.

We have studied the reduction reactions of two cytosolic human peroxiredoxins (Prx) in their disulfide form by three thioredoxins (Trx; two human and one bacterial), with the aim of better understanding the rate and mechanism of those reactions, and their relevance in the context of the catalytic cycle of Prx. We have developed a new methodology based on stopped-flow and intrinsic fluorescence to study the bimolecular reactions, and found rate constants in the range of 105 -106 m-1 s-1 in all cases, showing that there is no marked kinetic preference for the expected Trx partner. By combining experimental findings and molecular dynamics studies, we found that the reactivity of the nucleophilic cysteine (CN ) in the Trx is greatly affected by the formation of the Prx-Trx complex. The protein-protein interaction forces the CN thiolate into an unfavorable hydrophobic microenvironment that reduces its hydration and results in a remarkable acceleration of the thiol-disulfide exchange reactions by more than three orders of magnitude and also produces a measurable shift in the pKa of the CN . This mechanism of activation of the thiol disulfide exchange may help understand the reduction of Prx by alternative reductants involved in redox signaling.

Biophysical tools to study the oligomerization dynamics of Prx1-class peroxiredoxins.

Peroxiredoxins (Prx) are ubiquitous, highly conserved peroxidases whose activity depends on catalytic cysteine residues. The Prx1-class of the peroxiredoxin family, also called typical 2-Cys Prx, organize as head-to-tail homodimers containing two active sites. The peroxidatic cysteine CP of one monomer reacts with the peroxide substrate to form sulfenic acid that reacts with the resolving cysteine (CR) of the adjacent subunit to form an intermolecular disulfide, that is reduced back by the thioredoxin/thioredoxin reductase/NADPH system. Although the minimal catalytic unit is the dimer, these Prx oligomerize into (do)decamers. In addition, these ring-shaped decamers can pile-up into high molecular weight structures. Prx not only display peroxidase activity reducing H2O2, peroxynitrous acid and lipid hydroperoxides (antioxidant enzymes), but also exhibit holdase activity protecting other proteins from unfolding (molecular chaperones). Highly relevant is their participation in redox cellular signaling that is currently under active investigation. The different activities attributed to Prx are strongly ligated to their quaternary structure. In this review, we will describe different biophysical approaches used to characterize the oligomerization dynamics of Prx that include the classical size-exclusion chromatography, analytical ultracentrifugation, calorimetry, and also fluorescence anisotropy and lifetime measurements, as well as mass photometry.

Fluorescent detection of hydrogen sulfide (H2S) through the formation of pyrene excimers enhances H2S quantification in biochemical systems.

Hydrogen sulfide (H2S) is produced endogenously by several enzymatic pathways and modulates physiological functions in mammals. Quantification of H2S in biochemical systems remains challenging because of the presence of interferents with similar reactivity, particularly thiols. Herein, we present a new quantification method based on the formation of pyrene excimers in solution. We synthesized the probe 2-(maleimido)ethyl 4-pyrenylbutanoate (MEPB) and determined that MEPB reacted with H2S in a two-step reaction to yield the thioether-linked dimer (MEPB)2S, which formed excimers upon excitation, with a broad peak of fluorescence emission centered at 480 nm. In contrast, we found that the products formed with thiols showed peaks at 378 and 398 nm. The difference in emission between the products prevented the interference. Furthermore, we showed that the excimer fluorescence signal yielded a linear response to H2S, with a limit of detection of 54 nM in a fluorometer. Our quantification method with MEPB was successfully applied to follow the reaction of H2S with glutathione disulfide and to quantify the production of H2S from cysteine by Escherichia coli. In conclusion, this method represents an addition to the toolkit of biochemists to quantify H2S specifically and sensitively in biochemical systems.

Fluorescence Lifetime Phasor Analysis of the Decamer-Dimer Equilibrium of Human Peroxiredoxin 1.

Protein self-assembly is a common feature in biology and is often required for a myriad of fundamental processes, such as enzyme activity, signal transduction, and transport of solutes across membranes, among others. There are several techniques to find and assess homo-oligomer formation in proteins. Naturally, all these methods have their limitations, meaning that at least two or more different approaches are needed to characterize a case study. Herein, we present a new method to study protein associations using intrinsic fluorescence lifetime with phasors. In this case, the method is applied to determine the equilibrium dissociation constant (KD) of human peroxiredoxin 1 (hPrx1), an efficient cysteine-dependent peroxidase, that has a quaternary structure comprised of five head-to-tail homodimers non-covalently arranged in a decamer. The hPrx1 oligomeric state not only affects its activity but also its association with other proteins. The excited state lifetime of hPrx1 has distinct values at high and low concentrations, suggesting the presence of two different species. Phasor analysis of hPrx1 emission lifetime allowed for the identification and quantification of hPrx1 decamers, dimers, and their mixture at diverse protein concentrations. Using phasor algebra, we calculated the fraction of hPrx1 decamers at different concentrations and obtained KD (1.1 × 10-24 M4) and C0.5 (1.36 µM) values for the decamer-dimer equilibrium. The results were validated and compared with size exclusion chromatography. In addition, spectral phasors provided similar results despite the small differences in emission spectra as a function of hPrx1 concentration. The phasor approach was shown to be a highly sensitive and quantitative method to assess protein oligomerization and an attractive addition to the biophysicist's toolkit.

Nitro-fatty acids as activators of hSIRT6 deacetylase activity.

Sirtuin 6, SIRT6, is critical for both glucose and lipid homeostasis and is involved in maintaining genomic stability under conditions of oxidative DNA damage such as those observed in age-related diseases. There is an intense search for modulators of SIRT6 activity, however, not many specific activators have been reported. Long acyl-chain fatty acids have been shown to increase the weak in vitro deacetylase activity of SIRT6 but this effect is modest at best. Herein we report that electrophilic nitro-fatty acids (nitro-oleic acid and nitro-conjugated linoleic acid) potently activate SIRT6. Binding of the nitro-fatty acid to the hydrophobic crevice of the SIRT6 active site exerted a moderate activation (2-fold at 20 µm), similar to that previously reported for non-nitrated fatty acids. However, covalent Michael adduct formation with Cys-18, a residue present at the N terminus of SIRT6 but absent from other isoforms, induced a conformational change that resulted in a much stronger activation (40-fold at 20 µm). Molecular modeling of the resulting Michael adduct suggested stabilization of the co-substrate and acyl-binding loops as a possible additional mechanism of SIRT6 activation by the nitro-fatty acid. Importantly, treatment of cells with nitro-oleic acid promoted H3K9 deacetylation, whereas oleic acid had no effect. Altogether, our results show that nitrated fatty acids can be considered a valuable tool for specific SIRT6 activation, and that SIRT6 should be considered as a molecular target for in vivo actions of these anti-inflammatory nitro-lipids.

Catalysis of Peroxide Reduction by Fast Reacting Protein Thiols.

Life on Earth evolved in the presence of hydrogen peroxide, and other peroxides also emerged before and with the rise of aerobic metabolism. They were considered only as toxic byproducts for many years. Nowadays, peroxides are also regarded as metabolic products that play essential physiological cellular roles. Organisms have developed efficient mechanisms to metabolize peroxides, mostly based on two kinds of redox chemistry, catalases/peroxidases that depend on the heme prosthetic group to afford peroxide reduction and thiol-based peroxidases that support their redox activities on specialized fast reacting cysteine/selenocysteine (Cys/Sec) residues. Among the last group, glutathione peroxidases (GPxs) and peroxiredoxins (Prxs) are the most widespread and abundant families, and they are the leitmotif of this review. After presenting the properties and roles of different peroxides in biology, we discuss the chemical mechanisms of peroxide reduction by low molecular weight thiols, Prxs, GPxs, and other thiol-based peroxidases. Special attention is paid to the catalytic properties of Prxs and also to the importance and comparative outlook of the properties of Sec and its role in GPxs. To finish, we describe and discuss the current views on the activities of thiol-based peroxidases in peroxide-mediated redox signaling processes.

Unraveling the effects of peroxiredoxin 2 nitration; role of C-terminal tyrosine 193.

Peroxiredoxins (Prx) are enzymes that efficiently reduce hydroperoxides through active participation of cysteine residues (CP, CR). The first step in catalysis, the reduction of peroxide substrate, is fast, 107 - 108 M-1s-1 for human Prx2. In addition, the high intracellular concentration of Prx positions them not only as good antioxidants but also as central players in redox signaling pathways. These biological functions can be affected by post-translational modifications that could alter the peroxidase activity and/or interaction with other proteins. In particular, inactivation by hyperoxidation of CP, which occurs when a second molecule of peroxide reacts with the CP in the sulfenic acid form, modulates their participation in redox signaling pathways. The higher sensitivity to hyperoxidation of some Prx has been related to the presence of structural motifs that disfavor disulfide formation at the active site, making the CP sulfenic acid more available for hyperoxidation or interaction with a redox protein target. We previously reported that treatment of human Prx2 with peroxynitrite results in tyrosine nitration, a post-translational modification on non-catalytic residues, yielding a more active peroxidase with higher resistance to hyperoxidation. In this work, studies on various mutants of hPrx2 confirm that the presence of the tyrosyl side-chain of Y193, belonging to the C-terminal YF motif of eukaryotic Prx, is necessary to observe the increase in Prx2 resistance to hyperoxidation. Moreover, our results underline the critical role of this structural motif on the rate of disulfide formation that determines the differential participation of Prx in redox signaling pathways.

Differential parameters between cytosolic 2-Cys peroxiredoxins, PRDX1 and PRDX2.

Peroxiredoxins are thiol-dependent peroxidases that function in peroxide detoxification and H2 O2 induced signaling. Among the six isoforms expressed in humans, PRDX1 and PRDX2 share 97% sequence similarity, 77% sequence identity including the active site, subcellular localization (cytosolic) but they hold different biological functions albeit associated with their peroxidase activity. Using recombinant human PRDX1 and PRDX2, the kinetics of oxidation and hyperoxidation with H2 O2 and peroxynitrite were followed by intrinsic fluorescence. At pH 7.4, the peroxidatic cysteine of both isoforms reacts nearly tenfold faster with H2 O2 than with peroxynitrite, and both reactions are orders of magnitude faster than with most protein thiols. For both isoforms, the sulfenic acids formed are in turn oxidized by H2 O2 with rate constants of ca 2 × 103 M-1 s-1 and by peroxynitrous acid significantly faster. As previously observed, a crucial difference between PRDX1 and PRDX2 is on the resolution step of the catalytic cycle, the rate of disulfide formation (11 s-1 for PRDX1, 0.2 s-1 for PRDX2, independent of the oxidant) which correlates with their different sensitivity to hyperoxidation. This kinetic pause opens different pathways on redox signaling for these isoforms. The longer lifetime of PRDX2 sulfenic acid allows it to react with other protein thiols to translate the signal via an intermediate mixed disulfide (involving its peroxidatic cysteine), whereas PRDX1 continues the cycle forming disulfide involving its resolving cysteine to function as a redox relay. In addition, the presence of C83 on PRDX1 imparts a difference on peroxidase activity upon peroxynitrite exposure that needs further study.

Differential Kinetics of Two-Cysteine Peroxiredoxin Disulfide Formation Reveal a Novel Model for Peroxide Sensing.

Two-cysteine peroxiredoxins (Prx) have a three-step catalytic cycle consisting of (1) reduction of peroxide and formation of sulfenic acid on the enzyme, (2) condensation of the sulfenic acid with a thiol to form disulfide, also known as resolution, and (3) reduction of the disulfide by a reductant protein. By following changes in protein fluorescence, we have studied the pH dependence of reaction 2 in human peroxiredoxins 1, 2, and 5 and in Salmonella typhimurium AhpC and obtained rate constants for the reaction and p Ka values of the thiol and sulfenic acid involved for each system. The observed reaction 2 rate constant spans 2 orders of magnitude, but in all cases, reaction 2 appears to be slow compared to the same reaction in small-molecule systems, making clear the rates are limited by conformational features of the proteins. For each Prx, reaction 2 will become rate-limiting at some critical steady-state concentration of H2O2 producing the accumulation of Prx as sulfenic acid. When this happens, an alternative and faster-resolving Prx (or other peroxidase) may take over the antioxidant role. The accumulation of sulfenic acid Prx at distinct concentrations of H2O2 is embedded in the kinetic limitations of the catalytic cycle and may constitute the basis of a H2O2-mediated redox signal transduction pathway requiring neither inactivation nor posttranslational modification. The differences in the rate constants of resolution among Prx coexisting in the same compartment may partially explain their complementation in antioxidant function and stepwise sensing of H2O2 concentration.

N-acetylcysteine improves the quality of red blood cells stored for transfusion.

Storage inflicts a series of changes on red blood cells (RBC) that compromise the cell survival and functionality; largely these alterations (storage lesions) are due to oxidative modifications. The possibility of improving the quality of packed RBC stored for transfusion including N-acetylcysteine (NAC) in the preservation solution was explored. Relatively high concentrations of NAC (20-25 mM) were necessary to prevent the progressive leakage of hemoglobin, while lower concentrations (≥2.5 mM) were enough to prevent the loss of reduced glutathione during the first 21 days of storage. Peroxiredoxin-2 was also affected during storage, with a progressive accumulation of disulfide-linked dimers and hetero-protein complexes in the cytosol and also in the membrane of stored RBC. Although the presence of NAC in the storage solution was unable to avoid the formation of thiol-mediated protein complexes, it partially restored the capacity of the cell to metabolize H2O2, indicating the potential use of NAC as an additive in the preservation solution to improve RBC performance after transfusion.

Potential Modulation of Sirtuins by Oxidative Stress.

Sirtuins are a conserved family of NAD-dependent protein deacylases. Initially proposed as histone deacetylases, it is now known that they act on a variety of proteins including transcription factors and metabolic enzymes, having a key role in the regulation of cellular homeostasis. Seven isoforms are identified in mammals (SIRT1-7), all of them sharing a conserved catalytic core and showing differential subcellular localization and activities. Oxidative stress can affect the activity of sirtuins at different levels: expression, posttranslational modifications, protein-protein interactions, and NAD levels. Mild oxidative stress induces the expression of sirtuins as a compensatory mechanism, while harsh or prolonged oxidant conditions result in dysfunctional modified sirtuins more prone to degradation by the proteasome. Oxidative posttranslational modifications have been identified in vitro and in vivo, in particular cysteine oxidation and tyrosine nitration. In addition, oxidative stress can alter the interaction with other proteins, like SIRT1 with its protein inhibitor DBC1 resulting in a net increase of deacetylase activity. In the same way, manipulation of cellular NAD levels by pharmacological inhibition of other NAD-consuming enzymes results in activation of SIRT1 and protection against obesity-related pathologies. Nevertheless, further research is needed to establish the molecular mechanisms of redox regulation of sirtuins to further design adequate pharmacological interventions.

Structural changes upon peroxynitrite-mediated nitration of peroxiredoxin 2; nitrated Prx2 resembles its disulfide-oxidized form.

Peroxiredoxins are cys-based peroxidases that function in peroxide detoxification and H2O2-induced signaling. Human Prx2 is a typical 2-Cys Prx arranged as pentamers of head-to-tail homodimers. During the catalytic mechanism, the active-site cysteine (CP) cycles between reduced, sulfenic and disulfide state involving conformational as well as oligomeric changes. Several post-translational modifications were shown to affect Prx activity, in particular CP overoxidation which leads to inactivation. We have recently reported that nitration of Prx2, a post-translational modification on non-catalytic tyrosines, unexpectedly increases its peroxidase activity and resistance to overoxidation. To elucidate the cross-talk between this post-translational modification and the enzyme catalysis, we investigated the structural changes of Prx2 after nitration. Analytical ultracentrifugation, UV absorption, circular dichroism, steady-state and time-resolved fluorescence were used to connect catalytically relevant redox changes with tyrosine nitration. Our results show that the reduced nitrated Prx2 structurally resembles the disulfide-oxidized native form of the enzyme favoring a locally unfolded conformation that facilitates disulfide formation. These results provide structural basis for the kinetic analysis previously reported, the observed increase in activity and the resistance to overoxidation of the peroxynitrite-treated enzyme.

Purification of a recombinant glutathione transferase from the causative agent of hydatidosis, Echinococcus granulosus.

This practical class activity was designed to introduce students to recombinant protein expression and purification. The principal goal is to shed light on basic aspects concerning recombinant protein production, in particular protein expression, chromatography methods for protein purification, and enzyme activity as a tool to evaluate purity and conformation of the recombinant product. Herein, we describe the purification of a glutathione transferase from the human parasite Echinococcus granulosus (EgGST1), the causative agent of hydatidosis. EgGST1 is expressed fused to a histidine tag and is purified by immobilized metal affinity chromatography. Protein quantification based on direct (UV absorbance) and indirect (colorimetric) methods are used and discussed. A simple colorimetric assay is used to measure GST activity and special emphasis is put on how to use these measurements to follow protein purification yields, its enrichment and its correct folding along the purification process. EgGST1 is easily expressed with high yields, purified in absence of protease inhibitors and proved to be robust concerning enzyme activity and protein integrity on a 1 week practical activity.

Kinetics of the Reaction of Pyrogallol Red, a Polyphenolic Dye, with Nitrous Acid: Role of ŸNO and ŸNO2.

In the present work we studied the reaction under gastric conditions of pyrogallol red (PGR), a polyphenolic dye, with nitrous acid (HONO). PGR has been used as a model polyphenol due to its strong UV-visible absorption and its high reactivity towards reactive species (radicals and non-radicals, RS). The reaction was followed by UV-visible spectroscopy and high performance liquid chromatography (HPLC). A clear decrease of the PGR absorbance at 465 nm was observed, evidencing an efficient bleaching of PGR by HONO. In the initial stages of the reaction, each HONO molecule nearly consumed 2.6 PGR molecules while, at long reaction times, ca. 7.0 dye molecules were consumed per each reacted HONO. This result is interpreted in terms of HONO recycling. During the PGR-HONO reaction, nitric oxide was generated in the micromolar range. In addition, the rate of PGR consumption induced by HONO was almost totally abated by argon bubbling, emphasising the role that critical volatile intermediates, such as ŸNO and/or nitrogen dioxide (ŸNO2), play in the bleaching of this phenolic compound.

Nitration transforms a sensitive peroxiredoxin 2 into a more active and robust peroxidase.

Peroxiredoxins (Prx) are efficient thiol-dependent peroxidases and key players in the mechanism of H2O2-induced redox signaling. Any structural change that could affect their redox state, oligomeric structure, and/or interaction with other proteins could have a significant impact on the cascade of signaling events. Several post-translational modifications have been reported to modulate Prx activity. One of these, overoxidation of the peroxidatic cysteine to the sulfinic derivative, inactivates the enzyme and has been proposed as a mechanism of H2O2 accumulation in redox signaling (the floodgate hypothesis). Nitration of Prx has been reported in vitro as well as in vivo; in particular, nitrated Prx2 was identified in brains of Alzheimer disease patients. In this work we characterize Prx2 tyrosine nitration, a post-translational modification on a noncatalytic residue that increases its peroxidase activity and its resistance to overoxidation. Mass spectrometry analysis revealed that treatment of disulfide-oxidized Prx2 with excess peroxynitrite renders mainly mononitrated and dinitrated species. Tyrosine 193 of the YF motif at the C terminus, associated with the susceptibility toward overoxidation of eukaryotic Prx, was identified as nitrated and is most likely responsible for the protection of the peroxidatic cysteine against oxidative inactivation. Kinetic analyses suggest that tyrosine nitration facilitates the intermolecular disulfide formation, transforming a sensitive Prx into a robust one. Thus, tyrosine nitration appears as another mechanism to modulate these enzymes in the complex network of redox signaling.

Peroxiredoxins as preferential targets in H2O2-induced signaling.

Evidence has accumulated showing that hydrogen peroxide (H2O2) acts as a signaling molecule via oxidation of critical cysteine residues on target proteins. The reaction of H2O2 with thiols is thermodynamically favored, but its selectivity is imposed by differences in reaction kinetics. Previously proposed signal relaying mechanisms, such as the floodgate hypothesis and widespread protein sulfenylation, appear inconsistent with kinetic and diffusion considerations. Among all cellular thiols, the peroxidatic cysteines of peroxiredoxins (Prxs) represent preferential targets considering their high rate constants and their cellular abundance that place them as the first step in the H2O2-induced signaling pathways. The oxidized Prxs could transfer the signal either via thiol-disulfide redox reactions or through nonredox protein-protein interactions. Recent studies evidence Prxs interactions with protein tyrosine kinases and phosphatases, indicating a potential connection between redox and phosphorylation signaling pathways that does not need the direct reaction of H2O2 with phosphatase or kinase critical cysteines. Posttranslational modifications of Prxs have been observed in vivo (mainly overoxidation of cysteines and phosphorylation of threonines) that affect their peroxidase activity, redox state, and/or oligomeric structure, and likely impact on H2O2 signaling. More focus on kinetic data and redox-sensitive protein-protein interactions are needed to unravel the molecular mechanisms of H2O2 signaling.

Inhibition of Mycobacterium tuberculosis PknG by non-catalytic rubredoxin domain specific modification: reaction of an electrophilic nitro-fatty acid with the Fe-S center.

PknG from Mycobacterium tuberculosis is a Ser/Thr protein kinase that regulates key metabolic processes within the bacterial cell as well as signaling pathways from the infected host cell. This multidomain protein has a conserved canonical kinase domain with N- and C-terminal flanking regions of unclear functional roles. The N-terminus harbors a rubredoxin-like domain (Rbx), a bacterial protein module characterized by an iron ion coordinated by four cysteine residues. Disruption of the Rbx-metal binding site by simultaneous mutations of all the key cysteine residues significantly impairs PknG activity. This encouraged us to evaluate the effect of a nitro-fatty acid (9- and 10-nitro-octadeca-9-cis-enoic acid; OA-NO2) on PknG activity. Fatty acid nitroalkenes are electrophilic species produced during inflammation and metabolism that react with nucleophilic residues of target proteins (i.e., Cys and His), modulating protein function and subcellular distribution in a reversible manner. Here, we show that OA-NO2 inhibits kinase activity by covalently adducting PknG remote from the catalytic domain. Mass spectrometry-based analysis established that cysteines located at Rbx are the specific targets of the nitroalkene. Cys-nitroalkylation is a Michael addition reaction typically reverted by thiols. However, the reversible OA-NO2-mediated nitroalkylation of the kinase results in an irreversible inhibition of PknG. Cys adduction by OA-NO2 induced iron release from the Rbx domain, revealing a new strategy for the specific inhibition of PknG. These results affirm the relevance of the Rbx domain as a target for PknG inhibition and support that electrophilic lipid reactions of Rbx-Cys may represent a new drug strategy for specific PknG inhibition.

Evaluating the antioxidant capacity of natural products: a review on chemical and cellular-based assays.

Oxidative stress is associated with several pathologies like cardiovascular, neurodegenerative, cancer and even aging. It has been suggested that a diet rich in antioxidants would be beneficial to human health and a lot of interest is focused on the determination of antioxidant capacity of natural products. Different chemical methods have been developed including the popular ORAC that evaluates the potential of a sample as inhibitor of a target molecule oxidation. Chemical-based methods are useful for screening, they are low cost, high-throughput and yield an index value (expressed as equivalents of Trolox) that allows comparing and ordering different products. More recently, nanoparticles-based assays have been developed to sense the antioxidant power of natural products. However, the antioxidant capacity indexes obtained by chemical assays cannot extrapolate the performance of the sample in vivo. Considering that antioxidant action is not limited to scavenging free radicals but includes upregulation of antioxidant and detoxifying enzymes, modulation of redox cell signaling and gene expression, it is necessary to move to cellular assays in order to evaluate the potential antioxidant activity of a compound or extract. Animal models and human studies are more appropriate but also more expensive and time-consuming, making the cell culture assays very attractive as intermediate testing methods. Cellular antioxidant activity (CAA) assays, activation of redox transcription factors, inhibition of oxidases or activation of antioxidant enzymes are reviewed and compared with the classical in vitro chemical-based assays for evaluation of antioxidant capacity of natural products.

Tools to evaluate the conformation of protein products.

Production of recombinant proteins is a process intensively used in the research laboratory. In addition, the main biotechnology market products are recombinant proteins and monoclonal antibodies. The biological (and clinical) properties of the protein product strongly depend on the conformation of the polypeptide. Therefore, assessment of the correct conformation of the produced protein is crucial. There is no single method to assess every aspect of protein structure or function. Depending on the protein, the methods of choice vary. There are general methods to evaluate not only mass and primary sequence of the protein, but also higher-order structure. This review outlines the principal techniques for determining the conformation of a protein from structural (biophysical methods) to functional (in vitro binding assays) analyses.

Antioxidant activity of uruguayan propolis. In vitro and cellular assays.

The antioxidant capacity of propolis from the southern region of Uruguay was evaluated using in vitro as well as cellular assays. Free radical scavenging capacity was assessed by ORAC, obtaining values significantly higher than those of other natural products (8000 µmol Trolox equiv/g propolis). ORAC values correlated well with total polyphenol content (determined by Folin-Ciocalteu method) and UV absorption. Total polyphenol content (150 mg gallic acid equiv/g propolis) and flavonoids (45 mg quercetin equiv/g propolis) were similar to values reported for southern Brazilian (group 3) and Argentinean propolis. Flavonoid composition determined by RP-HPLC indicates a strong poplar-tree origin. Samples high in polyphenols efficiently inhibit low-density lipoprotein lipoperoxidation and tyrosine nitration. In addition, Uruguayan propolis was found to induce the expression of endothelial nitric oxide synthase and inhibit endothelial NADPH oxidase, suggesting a potential cardiovascular benefit by increasing nitric oxide bioavailability in the endothelium.