Photoproperties of favipiravir and its 6-substituted analogues: fluorescence controlled through halogen substitution and tautomerism.

Herein, we have showed the photophysical properties of favipiravir and its 6-substituted analogues. Also, we interpreted the origin of fluorescence of favipiravir and its 6-substituted analogues as a function of tautomerism modulation in ground and excited states. Favipiravir, the 6-fluorine derivative, showed the best photophysical profile, exhibiting a dominant emission wavelength of 430 nm, a high quantum yield (Q.Y.) of 1.0 and a long-lived state (10 ns). Its analogues also showed a maximum emission at 430 nm, but their Q.Y. values were 5-fold lower than that found for favipiravir, decreasing as a function of 6-substitution as follows: F > Cl > Br > I > H. Pyrazines bearing the least electronegative 6-substituent (X = Br, I, H) showed an extra lifetime, which was shorter (0.2-0.3 ns) and less abundant (>15%) than the main lifetime (10 ns, 85%). Further 2D excitation-emission matrix and solvent studies supported that these 3-hydroxy-2-pyrazinecarboxamides present two emissive states. The first of them (λem = 430 nm), which was the most abundant, most fluorescent and long-lived state, was characterized as "locally excited" (LE). Its fluorescence was favored with an increase of the hydrogen-donor nature of the solvent and for pyrazines having a high enolic characteristic. Thus, the high LE-fluorescence of these types of pyrazines depends on the keto-tautomerization of the ground state using a protic solvent and its feasible enol-tautomerization upon excitation. Finally, the second excited state (λem = 536 nm) was suggested as an excited-state intramolecular proton-transfer (ESIPT), and it was observed only, although discretely, for pyrazines bearing the least electronegative 6-substituent.

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.

Oxidative Stress in Healthy and Pathological Red Blood Cells.

Red cell diseases encompass a group of inherited or acquired erythrocyte disorders that affect the structure, function, or production of red blood cells (RBCs). These disorders can lead to various clinical manifestations, including anemia, hemolysis, inflammation, and impaired oxygen-carrying capacity. Oxidative stress, characterized by an imbalance between the production of reactive oxygen species (ROS) and the antioxidant defense mechanisms, plays a significant role in the pathophysiology of red cell diseases. In this review, we discuss the most relevant oxidant species involved in RBC damage, the enzymatic and low molecular weight antioxidant systems that protect RBCs against oxidative injury, and finally, the role of oxidative stress in different red cell diseases, including sickle cell disease, glucose 6-phosphate dehydrogenase deficiency, and pyruvate kinase deficiency, highlighting the underlying mechanisms leading to pathological RBC phenotypes.

Disulfides form persulfides at alkaline pH leading to potential overestimations in the cold cyanolysis method.

It is well established that proteins and peptides can release sulfur under alkaline treatment, mainly through the ß-elimination of disulfides with the concomitant formation of persulfides and dehydroalanine derivatives. In this study, we evaluated the formation of glutathione persulfide (GSSH/GSS-) by exposure of glutathione disulfide (GSSG) to alkaline conditions. The kinetics of the reaction between GSSG and HO- was investigated by UV-Vis absorbance, reaction with 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB), and cold cyanolysis, obtaining an apparent second-order rate constant of ∼10-3 M-1 s-1 at 25 °C. The formation of GSSH and the dehydroalanine derivative was confirmed by HPLC and/or mass spectrometry. However, the mixtures did not equilibrate in a timescale of hours, and additional species, including thiol and diverse sulfane sulfur compounds were also formed, probably through further reactions of the persulfide. Cold cyanolysis is frequently used to quantify persulfides, since it measures sulfane sulfur. This method involves a step in which the sample to be analyzed is incubated with cyanide at alkaline pH. When cold cyanolysis was applied to samples containing GSSG, sulfane sulfur products that were not present in the original sample were measured. Thus, our results reveal the risk of overestimating the amount of sulfane sulfur compounds in samples that contain disulfides due to their decay to persulfides and other sulfane sulfur compounds at alkaline pH. Overall, our study highlights that the ß-elimination of disulfides is a potential source of persulfides, although we do not recommend the preparation of GSSH from incubation of GSSG in alkali. Our study also highlights the importance of being cautious when doing and interpreting cold cyanolysis experiments.

Oxidants and Antioxidants in the Redox Biochemistry of Human Red Blood Cells.

Red blood cells (RBCs) are exposed to both external and internal sources of oxidants that challenge their integrity and compromise their physiological function and supply of oxygen to tissues. Autoxidation of oxyhemoglobin is the main source of endogenous RBC oxidant production, yielding superoxide radical and then hydrogen peroxide. In addition, potent oxidants from other blood cells and the surrounding endothelium can reach the RBCs. Abundant and efficient enzymatic systems and low molecular weight antioxidants prevent most of the damage to the RBCs and also position the RBCs as a sink of vascular oxidants that allow the body to maintain a healthy circulatory system. Among the antioxidant enzymes, the thiol-dependent peroxidase peroxiredoxin 2, highly abundant in RBCs, is essential to keep the redox balance. A great part of the RBC antioxidant activity is supported by an active glucose metabolism that provides reducing power in the form of NADPH via the pentose phosphate pathway. There are several RBC defects and situations that generate oxidative stress conditions where the defense mechanisms are overwhelmed, and these include glucose-6-phosphate dehydrogenase deficiencies (favism), hemoglobinopathies like sickle cell disease and thalassemia, as well as packed RBCs for transfusion that suffer from storage lesions. These oxidative stress-associated pathologies of the RBCs underline the relevance of redox balance in these anucleated cells that lack a mechanism of DNA-inducible antioxidant response and rely on a complex and robust network of antioxidant systems.

Possible molecular basis of the biochemical effects of cysteine-derived persulfides.

Persulfides (RSSH/RSS-) are species closely related to thiols (RSH/RS-) and hydrogen sulfide (H2S/HS-), and can be formed in biological systems in both low and high molecular weight cysteine-containing compounds. They are key intermediates in catabolic and biosynthetic processes, and have been proposed to participate in the transduction of hydrogen sulfide effects. Persulfides are acidic, more acidic than thiols, and the persulfide anions are expected to be the predominant species at neutral pH. The persulfide anion has high nucleophilicity, due in part to the alpha effect, i.e., the increased reactivity of a nucleophile when the neighboring atom has high electron density. In addition, persulfides have electrophilic character, a property that is absent in both thiols and hydrogen sulfide. In this article, the biochemistry of persulfides is described, and the possible ways in which the formation of a persulfide could impact on the properties of the biomolecule involved are discussed.

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.

The permeability of human red blood cell membranes to hydrogen peroxide is independent of aquaporins.

Hydrogen peroxide (H2O2) not only is an oxidant but also is an important signaling molecule in vascular biology, mediating several physiological functions. Red blood cells (RBCs) have been proposed to be the primary sink of H2O2 in the vasculature because they are the main cellular component of blood with a robust antioxidant defense and a high membrane permeability. However, the exact permeability of human RBC to H2O2 is neither known nor is it known if the mechanism of permeation involves the lipid fraction or protein channels. To gain insight into the permeability process, we measured the partition constant of H2O2 between water and octanol or hexadecane using a novel double-partition method. Our results indicated that there is a large thermodynamic barrier to H2O2 permeation. The permeability coefficient of H2O2 through phospholipid membranes containing cholesterol with saturated or unsaturated acyl chains was determined to be 4 × 10-4 and 5 × 10-3 cm s-1, respectively, at 37 °C. The permeability coefficient of human RBC membranes to H2O2 at 37 °C, on the other hand, was 1.6 × 10-3 cm s-1. Different aquaporin-1 and aquaporin-3 inhibitors proved to have no effect on the permeation of H2O2. Moreover, human RBCs devoid of either aquaporin-1 or aquaporin-3 were equally permeable to H2O2 as normal human RBCs. Therefore, these results indicate that H2O2 does not diffuse into RBCs through aquaporins but rather through the lipid fraction or a still unidentified membrane protein.

Photo-Induced Partially Aromatized Intramolecular Charge Transfer.

Diverse models of intramolecular charge transfer (ICT) have been proposed for interpreting the origin of the charge-transfer (CT) state in donor-acceptor (D-A) dyes. However, a large variety of fused-heterocyclic dyes containing a pseudo-aromatic ring in the rigid structure have shown to be incompatible with them. To approximate a solution within the ICT concept, we reported a novel ICT model called partially aromatized intramolecular charge transfer (PAICT). PAICT involves the generation of a CT state from an ICT that occurred within a pre-excited D-A fused-heterocyclic structure possessing a pseudo-aromatic or unstable aromatic ring as the acceptor moiety. The model was proposed from the multiple-emissive mesomeric D-A N1-aryl-2-(trifluoromethyl)benzo[b][1,8]naphthyridin-4(1H)-one, whose excited mesomeric states, which are defined by the aromatic and pseudo-aromatic forms of the pyrindin-4(1H)-one ring, led to a common partial aromatized CT state upon excitation via PAICT. The latter was supported through theoretical calculations on the excited mesomeric states, one-dimensional (1D) and two-dimensional (2D) excitation-emission measurements in different solvents, and the detection of three excited states by lifetime measurements upon 370 nm excitation. The existence of mesomerism was supposed from: (i) two overlapping bands at 370-390 (or 400-420 nm) in UV-vis spectra, (ii) the direct interaction between the pyridinic nitrogen of one molecule and the carbonylic oxygen of the other found in the solid state and, (iii) the detection of three excited states by lifetime measurements. The PAICT opens new perspectives for interpreting the charge-transfer phenomenon in fused-heterocyclic dyes, in particular, those containing a pseudo-aromatic or unstable aromatic ring as an acceptor moiety.

Acidity and nucleophilic reactivity of glutathione persulfide.

Persulfides (RSSH/RSS-) participate in sulfur trafficking and metabolic processes, and are proposed to mediate the signaling effects of hydrogen sulfide (H2S). Despite their growing relevance, their chemical properties are poorly understood. Herein, we studied experimentally and computationally the formation, acidity, and nucleophilicity of glutathione persulfide (GSSH/GSS-), the derivative of the abundant cellular thiol glutathione (GSH). We characterized the kinetics and equilibrium of GSSH formation from glutathione disulfide and H2S. A pKa of 5.45 for GSSH was determined, which is 3.49 units below that of GSH. The reactions of GSSH with the physiologically relevant electrophiles peroxynitrite and hydrogen peroxide, and with the probe monobromobimane, were studied and compared with those of thiols. These reactions occurred through SN2 mechanisms. At neutral pH, GSSH reacted faster than GSH because of increased availability of the anion and, depending on the electrophile, increased reactivity. In addition, GSS- presented higher nucleophilicity with respect to a thiolate with similar basicity. This can be interpreted in terms of the so-called α effect, i.e. the increased reactivity of a nucleophile when the atom adjacent to the nucleophilic atom has high electron density. The magnitude of the α effect correlated with the Brønsted nucleophilic factor, ßnuc, for the reactions with thiolates and with the ability of the leaving group. Our study constitutes the first determination of the pKa of a biological persulfide and the first examination of the α effect in sulfur nucleophiles, and sheds light on the chemical basis of the biological properties of persulfides.

Human papillomavirus type 18 E5 oncoprotein cooperates with E6 and E7 in promoting cell viability and invasion and in modulating the cellular redox state.

BACKGROUND: High-risk human papillomaviruses (HR-HPVs) are the etiological agents of cervical cancer. Among them, types 16 and 18 are the most prevalent worldwide. The HPV genome encodes three oncoproteins (E5, E6, and E7) that possess a high transformation potential in culture cells when transduced simultaneously. In the present study, we analysed how these oncoproteins cooperate to boost key cancer cell features such as uncontrolled cell proliferation, invasion potential, and cellular redox state imbalance. Oxidative stress is known to contribute to the carcinogenic process, as reactive oxygen species (ROS) constitute a potentially harmful by-product of many cellular reactions, and an efficient clearance mechanism is therefore required. Cells infected with HR-HPVs can adapt to oxidative stress conditions by upregulating the formation of endogenous antioxidants such as catalase, glutathione (GSH), and peroxiredoxin (PRX). OBJECTIVES: The primary aim of this work was to study how these oncoproteins cooperate to promote the development of certain cancer cell features such as uncontrolled cell proliferation, invasion potential, and oxidative stress that are known to aid in the carcinogenic process. METHODS: To perform this study, we generated three different HaCaT cell lines using retroviral transduction that stably expressed combinations of HPV-18 oncogenes that included HaCaT E5-18, HaCaT E6/E7-18, and HaCaT E5/E6/E7-18. FINDINGS: Our results revealed a statistically significant increment in cell viability as measured by MTT assay, cell proliferation, and invasion assays in the cell line containing the three viral oncogenes. Additionally, we observed that cells expressing HPV-18 E5/E6/E7 exhibited a decrease in catalase activity and a significant augmentation of GSH and PRX1 levels relative to those of E5, E6/E7, and HaCaT cells. MAIN CONCLUSIONS: This study demonstrates for the first time that HPV-18 E5, E6, and E7 oncoproteins can cooperate to enhance malignant transformation.

Persulfides, at the crossroads between hydrogen sulfide and thiols.

Persulfides (RSSH/RSS-) can be formed in protein and non-protein thiols (RSH) through several different pathways, some of which are dependent on hydrogen sulfide (H2S/HS-). In addition to their roles in biosynthetic processes, persulfides are possible transducers of physiological effects of H2S through the modification of critical cysteines. Persulfides have a very rich biological chemistry that is currently under investigation. They are more nucleophilic and acidic than thiols and, unlike thiols, they can also be electrophilic. They are especially good one-electron reductants. Methods to detect their formation are under continuous development. In this minireview we describe the pathways of formation of persulfides, their biochemical properties and the techniques available for their detection, and we discuss the possible implications of their formation in biological systems.

Detection and quantification of nitric oxide-derived oxidants in biological systems.

The free radical nitric oxide (NO•) exerts biological effects through the direct and reversible interaction with specific targets (e.g. soluble guanylate cyclase) or through the generation of secondary species, many of which can oxidize, nitrosate or nitrate biomolecules. The NO•-derived reactive species are typically short-lived, and their preferential fates depend on kinetic and compartmentalization aspects. Their detection and quantification are technically challenging. In general, the strategies employed are based either on the detection of relatively stable end products or on the use of synthetic probes, and they are not always selective for a particular species. In this study, we describe the biologically relevant characteristics of the reactive species formed downstream from NO•, and we discuss the approaches currently available for the analysis of NO•, nitrogen dioxide (NO2•), dinitrogen trioxide (N2O3), nitroxyl (HNO), and peroxynitrite (ONOO-/ONOOH), as well as peroxynitrite-derived hydroxyl (HO•) and carbonate anion (CO3•-) radicals. We also discuss the biological origins of and analytical tools for detecting nitrite (NO2-), nitrate (NO3-), nitrosyl-metal complexes, S-nitrosothiols, and 3-nitrotyrosine. Moreover, we highlight state-of-the-art methods, alert readers to caveats of widely used techniques, and encourage retirement of approaches that have been supplanted by more reliable and selective tools for detecting and measuring NO•-derived oxidants. We emphasize that the use of appropriate analytical methods needs to be strongly grounded in a chemical and biochemical understanding of the species and mechanistic pathways involved.

Diffusion and Transport of Reactive Species Across Cell Membranes.

This chapter includes an overview of the structure of cell membranes and a review of the permeability of membranes to biologically relevant oxygen and nitrogen reactive species, namely oxygen, singlet oxygen, superoxide, hydrogen peroxide, hydroxyl radical, nitric oxide, nitrogen dioxide, peroxynitrite and also hydrogen sulfide. Physical interactions of these species with cellular membranes are discussed extensively, but also their relevance to chemical reactions such as lipid peroxidation. Most of these species are involved in different cellular redox processes ranging from physiological pathways to damaging reactions against biomolecules. Cell membranes separate and compartmentalize different processes, inside or outside cells, and in different organelles within cells. The permeability of these membranes to reactive species varies according to the physicochemical properties of each molecule. Some of them, such as nitric oxide and oxygen, are small and hydrophobic and can traverse cellular membranes virtually unhindered. Nitrogen dioxide and hydrogen sulfide find a slightly higher barrier to permeation, but still their diffusion is largely unimpeded by cellular membranes. In contrast, the permeability of cellular membranes to the more polar hydrogen peroxide, is up to five orders of magnitude lower, allowing the formation of concentration gradients, directionality and effective compartmentalization of its actions which can be further regulated by specific aquaporins that facilitate its diffusion through membranes. The compartmentalizing effect on anionic species such as superoxide and peroxynitrite is even more accentuated because of the large energetic barrier that the hydrophobic interior of membranes presents to ions that may be overcome by protonation or the use of anion channels. The large difference in cell membrane permeability for different reactive species indicates that compartmentalization is possible for some but not all of them.

Hydrogen Sulfide and Persulfides Oxidation by Biologically Relevant Oxidizing Species.

Hydrogen sulfide (H2S/HS⁻) can be formed in mammalian tissues and exert physiological effects. It can react with metal centers and oxidized thiol products such as disulfides (RSSR) and sulfenic acids (RSOH). Reactions with oxidized thiol products form persulfides (RSSH/RSS⁻). Persulfides have been proposed to transduce the signaling effects of H2S through the modification of critical cysteines. They are more nucleophilic and acidic than thiols and, contrary to thiols, also possess electrophilic character. In this review, we summarize the biochemistry of hydrogen sulfide and persulfides, focusing on redox aspects. We describe biologically relevant one- and two-electron oxidants and their reactions with H2S and persulfides, as well as the fates of the oxidation products. The biological implications are discussed.

Acceleration of the autoxidation of nitric oxide by proteins.

Lipoproteins and lipid membranes accelerate •NO autoxidation by increasing local concentration of •NO and O2. Although the idea that proteins could also accelerate this reaction was presented some time ago, it was largely criticized and dismissed. Herein the effect of proteins on •NO autoxidation rates was studied following •NO disappearance with a selective electrode. It was found that human serum albumin (HSA) accelerated •NO autoxidation by a factor of 9 per g/mL of protein, much less than previously suggested. The acceleration by HSA was sensitive to pH and significantly decreased at pH lower than 4.5 coincident with the acid structure transition of HSA to a partially unfolded and rigid conformation. Other proteins with different surface hydrophobicity also accelerated •NO autoxidation and it was found to depend mostly on the protein size and dynamics. Mathematical simulations were performed to assess the physiological importance of this acceleration. It was calculated that in plasma the autoxidation of •NO is accelerated 1.38 times by HSA relative to water alone, but this becomes of little relevance when whole blood is simulated because of the rapid rate of •NO consumption by red blood cells.

Kinetic studies reveal a key role of a redox-active glutaredoxin in the evolution of the thiol-redox metabolism of trypanosomatid parasites.

Trypanosomes are flagellated protozoan parasites (kinetoplastids) that have a unique redox metabolism based on the small dithiol trypanothione (T(SH)2). Although GSH may still play a biological role in trypanosomatid parasites beyond being a building block of T(SH)2, most of its functions are replaced by T(SH)2 in these organisms. Consequently, trypanosomes have several enzymes adapted to using T(SH)2 instead of GSH, including the glutaredoxins (Grxs). However, the mechanistic basis of Grx specificity for T(SH)2 is unknown. Here, we combined fast-kinetic and biophysical approaches, including NMR, MS, and fluorescent tagging, to study the redox function of Grx1, the only cytosolic redox-active Grx in trypanosomes. We observed that Grx1 reduces GSH-containing disulfides (including oxidized trypanothione) in very fast reactions (k > 5 × 105 m-1 s-1). We also noted that disulfides without a GSH are much slower oxidants, suggesting a strongly selective binding of the GSH molecule. Not surprisingly, oxidized Grx1 was also reduced very fast by T(SH)2 (4.8 × 106 m-1 s-1); however, GSH-mediated reduction was extremely slow (39 m-1 s-1). This kinetic selectivity in the reduction step of the catalytic cycle suggests that Grx1 uses preferentially a dithiol mechanism, forming a disulfide on the active site during the oxidative half of the catalytic cycle and then being rapidly reduced by T(SH)2 in the reductive half. Thus, the reduction of glutathionylated substrates avoids GSSG accumulation in an organism lacking GSH reductase. These findings suggest that Grx1 has played an important adaptive role during the rewiring of the thiol-redox metabolism of kinetoplastids.

Kinetic and stoichiometric constraints determine the pathway of H2O2 consumption by red blood cells.

Red blood cells (RBC) are considered as a circulating sink of H2O2, but a significant debate remains over the role of the different intraerythocyte peroxidases. Herein we examined the kinetic of decomposition of exogenous H2O2 by human RBC at different cell densities, using fluorescent and oxymetric methods, contrasting the results against a mathematical model. Fluorescent measurements as well as oxygen production experiments showed that catalase was responsible for most of the decomposition of H2O2 at cell densities suitable for both experimental settings (0.1-10â€¯× 1010 cell L-1), since sodium azide but not N-ethylmaleimide (NEM) inhibited H2O2 consumption. Oxygen production decreased at high cell densities until none was detected above 1.1 × 1012 cell L-1, being recovered after inhibition of the thiol dependent systems by NEM. This result underlined that the consumption of H2O2 by catalase prevail at RBC densities regularly used for research, while the thiol dependent systems predominate when the cell density increases, approaching the normal number in blood (5â€¯× 1012 cell L-1). The mathematical model successfully reproduced experimental results and at low cell number it showed a time sequence involving Prx as the first line of defense, followed by catalase, with a minor role by Gpx. The turning points were given by the total consumption of reduced Prx in first place and reduced GSH after that. However, Prx alone was able to account for the added H2O2 (50 µM) at physiological RBC density, calling attention to the importance of cell density in defining the pathway of H2O2 consumption and offering an explanation to current apparently conflicting results in the literature.