ES2333391A1 - In vitro method of selection of compounds with antioxidant activity and uses of the selected compounds. (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

In vitro method of selection of compounds with antioxidant activity and uses of the selected compounds. The present invention relates to a method (hereinafter method of the invention) for testing or detecting, in vitro, the ability of compounds or molecules to inhibit or stimulate oxidative/nitrative/nitrosative damage caused by ros and/or rns in systems biological, comprising the inclusion in the reaction medium of said reactive species together with the compound or molecule to be tested, using the recombinant sod enzyme, characterized by seq id no: 1, as a positive control of the activity of said ros and/or rns. (Machine-translation by Google Translate, not legally binding)

Description

In vitro method of selecting compounds with antioxidant activity and uses of the selected compounds.

Field of the Invention

The present invention can be encompassed general, within the field of biology and can be applied in wide variety of life sciences sectors as per example: biotechnology, biochemistry, medicine, pharmacy and chemistry.

State of the art

In the presence of oxygen (O2), they are produced in organisms, reactive oxygen species (ROS) and / or nitrogen (RNS) which, due to its high reactivity, they have a very short half-life and exercise important actions deleterious or harmful on essential biological components, causing numerous pathologies. Therefore the identification, establishment and quantification of compounds and / or molecules antioxidants capable of eliminating, or preventing the formation, of said ROS and / or RNS, within which the peroxynitrite (ONOO -) due to its high harmful capacity of biological systems, acquires in our times a special and relevant interest

DNA is one of the cellular components most susceptible to attack by ROS and / or RNS. Such reactive species can alter or mutate the DNA causing the emergence of various types of diseases. Such reactive species have been associated with all kinds of degenerative diseases, arthritis, cancer, Alzheimer's disease and Parkinson's disease. Peroxynitrite (ONOO -), on the other hand, can be directly or indirectly responsible for various diseases and, in particular, is known to be an important mediator in kidney diseases (MacMillan-Crow et al ., 1996).

Peroxynitrite (ONOO -) is a highly oxidizing reactive species associated with nitrosative stress, capable of oxidizing biological components and causing cytotoxicity (MacMillan-Crow et al ., 2001). It is generated from nitric oxide (NO) together with the superoxide anion (O2 -) (Feelisch et al ., 1999), said superoxide anion being one of radicals considered more dangerous. Peroxynitrite (ONOO -) can be produced in vitro by molecules such as SIN-1, which simultaneously generates equimolar amounts of nitric oxide (NO) and superoxide anion (O2 -) (Hogg et al ., 1992).

Nitric oxide (NO) and its derivatives, such as peroxynitrite (ONOO -), are of great importance in biological systems, since they can also mediate in different processes such as homeostasis of Fe in plants, the metabolic regulation of genes, trigger the defense against pathogens or mechanisms inflammation and participate in various diseases in animals (Ridnour et al . , 2004; Radi et al . , 2001, Feelisch et al . , 1999; Graziano and Lamattina, 2005). Although the metabolism of peroxynitrite (OONO -) has been studied in mammalian cells, the main studies conducted refer to the synthesis of its precursor, nitric oxide (NO) (Gardner et al ., 2001).

One of the most important antioxidant enzymes It is superoxide dismutase (SOD). SOD is truly the master cell defense mechanism to trap free radicals and prevent diseases due to them (Orr and, Sohal).

On the other hand, hemoglobins are proteins capable of reversibly binding oxygen (O2). At least three different lineages of hemoglobins are known, which are grouped into two structural classes, called 2/2 globins and 3/3 globins within archeobacteria, eubacteria and metazoans. The main function of 2/2 globins as well as bacterial flavo-hemoglobins is the detoxification or elimination of nitric oxide (NO). Some hemoglobins have been proposed as oxygen sensors (O2), while many metazoan hemoglobins, as well as symbiotic hemoglobins, which include plant leghemoglobins, are oxygen transporters (O2) without catalytic activity known (Vinogradov et al ., 2005). In contrast, hexacoordinated plant hemoglobins, like other hexacoordinated animal hemoglobins (neuroglobin, cytoglobin), appear to be involved in the cellular response during hypoxia conditions (Dordas et al ., 2003a; Dordas et al ., 2003b; Kundu et al ., 2003; Sun et al ., 2001). Class I (hexacoordinated) hemoglobins, such as rice hemoglobin I (Hbt), are a heterogeneous group of hemoglobins and, as they are hemoglobins, bind oxygen (O2) reversibly, and also do so with very high activity . It has also been shown, using transgenic plants, that hexacoordinated vegetable hemoglobins participate in the elimination of nitric oxide (NO) (Dordas et al ., 2003b; Igamberdiev et al ., 2007). This same function of eliminating nitric oxide (NO) seems to be essential in the hemoglobins of other organisms such as bacteria (Gardner et al ., 2001), although it is considered that the catalytic functions of hemoglobins are not clearly characterized yet (Vinogradov et al . , 2005).

On the other hand, paradoxically, pentacoordinated hemoglobins, such as human hemoglobin or myoglobin, or leguminous leghemoglobins, exhibit an important tendency to spontaneously oxidize, producing superoxide radical (Ec .: a.1; Misra and Fridovich 1972 ; Puppo et al ., 1981). The superoxide radical produced is capable, in turn, of producing new oxidations in hemoglobin that end with its total inactivation.

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Class I hemoglobins are present in animals, and also in humans, where a neuroglobin has been described (Kundu et al , 2003). Although it is unknown what exactly the physiological function of class I hemoglobins is, it has been proposed that they are part of a respiratory system that, residually, has been maintained until now in eukaryotic organisms and that would have special relevance in anoxia conditions (Igamberdiev et al . 2007).

There are numerous bioassays to estimate oxidative damage on biological components produced by free radicals such as the hydroxyl radical, or even to quantify lipid peroxidation (Halliwell and Gutteridge, 2007). From these bioassays in which they are used as deoxyribose target molecules or fatty acids (linolenic acid), respectively, it has been possible to evaluate the ability of numerous molecules, potentially antioxidants, to reduce or sometimes stimulate the oxidative effect on mentioned target molecules (Moran et al ., 1997).

On the other hand, in biological systems, the nitration of the amino acid tyrosine (Tyr) residues of proteins is produced specifically by said anion (OONO -) (Radi, 2001). The peroxynitrite attack (ONOO -) is capable of causing oxidation and nitration of proteins such as bovine albumin (BSA) and superoxide dismutase (SOD). At present, very few valid methods are known for detecting reactive nitrogen species such as peroxynitrite (ONOO -) or its precursor nitric oxide (NO). The average life time for nitric oxide (NO) is 2 ms in erythrocytes (Thomas et al ., 2001), so it is currently dependent on indirect measurements to identify and measure their levels. The nitration of tyrosines in proteins causes the modification of Tyr residues to 3-nitroTyr. One system to detect such modification is the use of polyclonal or monoclonal antibodies to detect 3-nitroTyr (Radi, 2001). Among the proteins that can be nitrated in Tyr residues causing their inactivation are Mn-superoxide dismutase (MnSOD) and Fe-superoxide dismutase (FeSOD) (Ischiropoulos, 1992a). The first has been associated with renal degenerative processes (MacMillan-Crow et al ., 1996) and with model systems of activated macrophages of rat alveolus (Ischiropoulos, 1992b). It has also been shown that Cu, ZnSOD, another SOD, is nitrated by peroxynitrite in the Tyr near its active center (Ischiropoulos, 1992a), which also causes its inactivation.

The detection of SOD activities has already been measured by spectrophotometric tests (McCord and Fridovich, 1969) or by gel electrophoresis assays (Beauchamp and Fridovich, 1971). SOD spectrophotometric detection is based in the ability of SOD to inhibit the reduction of cytochrome C by the superoxide radical (O 2 -) in a reaction medium containing xanthine and xanthine oxidase as generators of said radical. However, it had never been used, until the patent ES200702035, this technique to quantify quantities of peroxynitrite (ONOO -) using as a target a recombinant SOD Easy to purify and produce in large quantities.

The American patent application US2006 / 0211079A1 discloses a method to diagnose and predict asthma or other diseases whose etiology is related to existence of a high level of oxidative / nitrosative stress. Bliss US patent application cites several reactive species of oxygen / nitrogen, among which is peroxynitrite (ONOO -), capable of generating oxidatively SOD enzymes modified. This US patent application exposes two possibilities to carry out this method: on the one hand performs an estimate of the reduction of endogenous SOD activity total and on the other hand an appreciation of the high levels of any of the three endogenous SOD isoforms, in their oxidatively modified state, due to the exposure of said endogenous SOD isoforms to reactive species of oxygen / nitrogen, such as peroxynitrite (ONOO -). Be describes in said patent that the total SOD activity levels are due to the sum of activities of three endogenous SOD isoforms concrete: EC-SOD, Cu ZnSOD and MnSOD. This application US patent discloses the nitrifying activity that lead to out the reactive oxygen / nitrogen species, such as peroxynitrite (ONOO -), at the level of the Tyr residue of the SOD. Fruit of that nitrifying activity, the Tyr becomes 3-nitroTyr against which, due to the large affinity that antibodies have, antibodies are directed monoclonal (anti-nitroTyr antibodies) as a form of identifying said increase in oxidatively SOD levels modified.

In addition, there are others in the state of the art documents related to the information shown in the paragraph previous as they are:

"Evidence of peroxynitrite formation in vivo : Detection of nitrated proteins with an antinitrotyrosine antibody" Crow JP; Ye YZ; Royall J; Kooy N; Beckman JS Univ. Ala. Birmingham, Birmingham, AL 35233-1924, USA Portland Press Proceedings; The biology of nitric oxide, Vol. 4. 1994,

"Tyrosine nitration by superoxide and nitric oxide fluxes in biological systems: Modeling the impact of superoxide dismutase and nitric oxide diffusion" Quijano Celia; Romero Natalia; Radi Rafael Univ Republ, Dept Bioquim, Fac Med, Avda Gral Flores 2125, Montevideo 1800, Uruguay; rradi@fmed.edu.uy PUB - Free Radical Biology & Medicine SEP 15 2005 IRN-ISSN 0891-5849 VOL-39 NR-6 PG-728-741 and

"The impact of metal catalysis on protein tyrosine nitration by peroxynitrite " Daiber A; Bachschmid M; Beckman J S; Munzel T; Ullrich V Biochemical  and Biophysical Research Communications 07 MAY 2004 United States IRN-ISSN 0006-291X VOL-317 NR-3 PG-873-881.

In all of them the use of peroxynitrite (ONOO -) as promoter of nitration of Tyr residue of SOD and the use of antibodies to detect SOD a once exposed to peroxynitrite, due to the great affinity they have antibodies by oxidatively modified forms of SOD

In the American patent application US2006 / 0211079A1, and in the rest of the documents present in the state of the art, the total endogenous SOD activity is evaluated (EC-SOD, Cu ZnSOD and MnSOD) and / or the level increase of any of said endogenous isoforms in their oxidative state modified.

In contrast, in patent application ES200702035 the method of evaluating the capacity of a cell, group of cells, tissue or organ of any living organism to stimulate the production of reactive species of Oxygen and / or Nitrogen, and the estimation of the The concentration of said reactive species, which involves the quantification of oxidative and / or nitrative and / or nitrosative damage or stress, was performed in vitro by titration, gel or by a spectrophotometric test or other technique that serves the same purpose, decrease in activity of a recombinant SOD enzyme, such as the recombinant SOD enzyme characterized by SEQ ID NO: 1, and / or increase in the level of said recombinant enzyme when it is oxidatively modified. The chosen recombinant enzyme (FeSOD) is a recombinant enzyme that contains Fe as a cofactor at its catalytic center and differs from the SOD enzymes used in US2006 / 02 1 1 079A1. In order to carry out the method set forth in patent application ES200702035, the choice of an enzyme that is not present in any animal is relevant, as is the case of the recombinant FeSOD enzyme characterized by SEQ ID NO: 1. The use of an SOD Recombinant allowed the use of the exogenous enzyme as a target molecule in an in vitro assay since the recombinant enzyme can be purified simply in relatively large quantities as described (Moran et al ., 2003; Muñoz et al ., 2005). Thus, the enzyme used was distinguishable from the intrinsic SOD enzymes of animals, used in the state of the art.

On the other hand, and unlike all the information set forth above, the present invention relates to a method, in vitro , (High-throughput screening) for testing or detecting the ability of compounds or molecules (such as hemoglobin) to inhibit or stimulating the oxidative / nitrative / nitrosative damage caused by ROS and / or RNS (such as peroxynitrite) in biological systems, which includes the inclusion in the reaction medium of said reactive species together with the compound or molecule to be tested (as per example hemoglobin), using the recombinant SOD enzyme characterized by SEQ ID NO: 1 as a positive control of the activity of said ROS and / or RNS.

Although it would foreseeably be thought that the SOD enzyme, which eliminates superoxide anion (O2 {-}), can prevent the formation and oxidative action of peroxynitrite (ONOO-), the truth is that the present invention has evidenced that SOD is readily inactivated in in vitro assays in which peroxynitrite (ONOO -), nitric oxide (NO) and superoxide anion (O 2 -) are produced. Therefore, said recombinant SOD enzyme was used in the present invention to carry out positive control of the activity of ROS and / or RNS, since it was found that when ROS and / or RNS (such as peroxynitrite) were introduced into the test medium the recombinant SOD enzyme was inactivated. In addition, surprisingly and unexpectedly, it was shown that in the presence of some of the molecules tested (such as hemoglobin) there was no inactivation mediated by ROS and / or RNS (such as peroxynitrite) on the recombinant SOD enzyme and, for therefore, in the present invention it was concluded that hemoglobins have antioxidant activity against peroxynitrite (ONOO -). Thus, the use of hemoglobin is proposed for the preparation of a pharmaceutical or nutraceutical composition intended for the treatment and / or prevention of diseases whose etiology is related to the oxidative / nitrosative / nitrative stress caused by ROS and / or RNS, as per example peroxynitrite (ONOO -). In addition, an important part of this antioxidant capacity is due to the hexacoordinated hemoglobin rHbI to its SOD activity discovered in this invention. In addition, it was concluded that an important part of said antioxidant capacity is due to the SOD activity of hemoglobin I which was surprisingly evidenced in the present invention.

Therefore the technical problem solved by the The present invention relates to a method for testing or detecting the ability of compounds or molecules to inhibit or stimulate damage oxidative / nitrative / nitrosative caused by ROS and / or RNS (as per peroxynitrite example) in biological systems, and the elaboration of a pharmaceutical or nutraceutical composition, based on tested compounds that have a positive result, intended for treatment and / or prevention of diseases whose etiology is based on the existence of oxidative / nitrosative / nitrative stress caused by ROS and / or RNS such as peroxynitrite (ONOO -).

It is considered that this technical problem does not could be resolved, obviously, by an average expert in the matter, in the light of the documentation existing in the state of the technique because, on the one hand, in this state of the art is not yet have described molecules that prevent formation, or eliminate, peroxynitrite (ONOO -) in biological systems (even when this is one of the biggest responsible for the death of cells by oxidative stress and, therefore, a wide range of diseases), nor have the properties that it should have been determined a molecule to prevent its formation. On the other hand, to Although it is known that hemoglobins can participate in different physiological processes, such as homeostasis and in the elimination of nitric oxide (NO), however, in no case, its SOD activity has been described avoiding the formation of other ROS and / or RNS, such as peroxynitrite (ONOO -), and preventing its attack on biological components.

Description of the invention Brief Description of the Invention

As mentioned above, the present invention relates to a method (hereinafter method of the invention) for testing or detecting, in vitro , the ability of compounds or molecules (such as hemoglobin) to inhibit or stimulate damage oxidative / nitrative / nitrosative caused by ROS and / or RNS (such as peroxynitrite) in biological systems, which includes the inclusion, in the reaction medium, of said reactive species, together with the compound or molecule to be tested (such as hemoglobin), using an SOD enzyme, in particular the recombinant SOD characterized by SEQ ID NO: 1, as a positive control of the activity of said ROS and / or RNS.

In the present invention it has been shown that recombinant SOD, characterized by SEQ ID NO: 1, is inactivated in in vitro assays in which peroxynitrite (ONOO -) -, nitric oxide (NO) and superoxide anion are produced (O 2 -), as seen in examples 1 and 2. Therefore, said recombinant SOD enzyme was used in the present invention to perform positive control of the activity of ROS and / or RNS , since it was found that when ROS and / or RNS (such as peroxynitrite) were introduced into the medium, the recombinant SOD enzyme was inactivated. Furthermore, surprisingly and unexpectedly, it was evidenced that in the presence of some of the molecules tested (such as hemoglobin), inactivation mediated by ROS and / or RNS (such as peroxynitrite) did not occur on the recombinant SOD enzyme and Therefore, in the present invention it was concluded that hemoglobins have SOD activity. Thus, the use of hemoglobin is proposed for the preparation of a pharmaceutical or nutraceutical composition intended for the treatment or prevention of diseases whose etiology is related to the oxidative / nitrosative / nitrative stress caused by ROS and / or RNS, such as peroxynitrite (ONOO -).

As cited in the present invention, it is shown as an example of a recombinant SOD enzyme to the SOD enzyme of the cowpea legume plant, Vigna unguiculata , characterized by SEQ ID NO: 1, whose gene has been donated in an expression vector bacterial and has been expressed in bacteria recombinantly purifying homogeneity.

As cited in the present invention, the oxidative and / or nitrative and / or nitrosative damage or stress, refers to the presence of high concentrations of any of the ROS and / or RNS included in the group: superoxide anion (O2 -), nitric oxide (NO), hyponitrous acid (HNO), nitrosyl anion (NO -), nitrite (NO 2 -), trioxodinitrate (N 2 O 3), nitronium cation (NO 2 +), radical hydroxyl (.OH), radical (hydro) peroxyl (.COO), radical alkoxy (.CO.CO), carbonate radical (CO.sub.2), singlet of oxygen (1 O 2), hydrogen peroxide (H 2 O 2), ozone (O 3), ONOO -, hypochlorous acid (HClO), acid hypobromous (HBrO), selenite (Na2 SeO3), oxygen (O2), or the precursors thereof, as well as catalytic metals that they lead to the formation of reactive species such as Fe or Cu; in a cell, group of cells, tissue or organ of any organism alive.

The group of diseases treated with the composition of the invention, are all those whose etiology is related to the existence of oxidative and / or nitrative damage or stress and / or nitrosative. Understand in a non-limiting way: neuropathies, lung diseases, heart disease, asthma, diseases renal diseases caused by inflammation, mycoplasmosis, Alzheimer's, Parkinson's disease, acute multiple sclerosis, gliomas and adenocarcinomas.

As cited in the present invention the expression "biological system" includes not limiting: cells, tissues, organs or systems of any living organism

Description of the figures

Figure 1. Estimation of the SOD capacity of the hemoglobin I (HbI) and leghemoglobin (Lba) and / or capacity to inhibit the inactivation of the recombinant SOD enzyme (FeSOD), characterized by SEQ ID NO: 1, in reaction mixtures with SIN-1, by determining SOD activity in gel.

(A) SOD activity test in gel. They were added 2 µg of each of the proteins used as indicates:

1. FeSOD.

2. FeSOD + SIN-1 (1 mM).

3. FeSOD + Lba + SIN-1 (1 mM).

4. FeSOD + Hb1 + SIN-1 (1 mM).

(B) Replica of the gel set forth in Figure 1.A, but in this case the bright blue staining of Coomassie and all proteins were stained (regardless of its enzymatic activity).

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Figure 2. Determination of SOD activity by the spectrophotometric method.

The activity of the control samples was taken as 100%, corresponding to 99.9 units of SOD activity. A unit of SOD activity is the amount of enzyme that is capable of 50% inhibit superoxide anion dependent reduction (O 2-) of ferrocytochrome C. KCN and H 2 O 2, to induce the oxidation of SOD, where indicates:

B. White: 100% reduction of ferricitochrome c;

Street 1: FeSOD.

Street 2: FeSOD + KCN (10 µM).

Street 3: FeSOD + KCN (3 mM).

Street 4: FeSOD + H_ {2} O_ {2} (10 µM).

Street 5: HbI.

Street 6: HbI + KCN (10 µM).

Street 7: HbI + KCN 3 mM.

Street 8: HbI + H 2 O 2 (10 µM).

Street 9: Lba.

Street 10: Lba + KCN (10 µM).

Street 11: Lba + KCN (3 mM).

Street 12: Lba + H_ {2} O_ {2} (10 µM).

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The following proteins were used and concentrations: FeSOD (SEQ ID NO: 1) (0.9 µg / µl), HbI (5.5 ug / ul); Lba (6.5 \ mug / \ mul). Enzymatic activities are express in the graph regarding protein micrograms employee.

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Figure 3. Determination of SOD activity of gel hemoglobins by staining with NBT .

(A) Gel SOD activity test.

Street 1: FeSOD (SEQ ID NO: 1)

Street 2: Hb1

Street 3: Lba

Street 4: Hb1 + rFLbR2 (Reductase of recombinant leghemoglobin 2)

Street 5: Hb1 + NADH (2 mM)

Street 6: Hb1 + NADH (2 mM) + rFLbR2

Street 7: Hb1. + SIN-1 (1 mM)

(B) Replica of the gel stained with bright blue Coomassie 2 µg of protein was loaded on each street.

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Detailed description of the invention

In the present invention it was shown that the inclusion of one or more compounds to be measured in the test medium with the recombinant SOD enzyme, characterized by SEQ ID NO: 1 as a target, it allowed its standardization as a "high system troughput screening "to measure / detect compounds that can avoid the formation, or eliminate, various ROS and / or RNS, such as example, peroxynitrite (ONOO -).

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Surprisingly and unexpectedly, through the implementation of the method of the invention, it was concluded that hemoglobins have an essential SOD activity, anti-ROS and anti-RNS, showing different chemical reaction mechanism depending on the type of hemoglobin tested.

After the evaluation of the results obtained Once the method of the invention was carried out, it was concluded that these hexacoordinated hemoglobins I show, surprising and unexpectedly, SOD enzymatic activity and, therefore, an effect  antioxidant against peroxynitrite (ONOO -) and other RNS and / or ROS becoming the first known enzyme that protects the biological system against the activity of peroxynitrite (ONOO -). The fact that it presents this enzymatic activity means that hemoglobins I can bind nitric oxide (NO), superoxide radical (O 2 -), and peroxynitrite (ONOO -) (molecules that occur in numerous pathological situations in human), so they can be used as sensors of said ROS and / or RNS.

On the other hand, in the present invention evidenced that leghemoglobin of soybean nodules is capable of prevent the formation or performance of peroxynitrite (ONOO -).  This assumes that the anti-peroxynitrite (ONOO -) capacity of the hemoglobins extends, not only to hemoglobins hexacoordiandas but also to pentacoordinated hemoglobins of the legume leghemoglobin type, or myoglobin or animal hemoglobin.

Therefore, the present invention has two surprising and unexpected conclusions: on the one hand the use of a recombinant SOD enzyme, characterized by SEQ ID NO: 1, as a target for positive activity control of ROS and / or RNS, since it was found that when ROS and / or RNS (as for example peroxynitrite) were introduced into the test medium the recombinant SOD enzyme was inactivated and, on the other hand, in addition it was evidenced that in the presence of some of the molecules tested (specifically in the presence of hemoglobin) the inactivation mediated by ROS and / or RNS (specifically the peroxynitrite-mediated inactivation) on the SOD enzyme recombinant and, therefore, in the present invention first identified that hemoglobins have activity SOD Thus, the use of hemoglobin for the preparation of  a pharmaceutical or nutraceutical composition intended for treatment and / or prevention of diseases whose etiology is related to oxidative / nitrosative / nitrative stress caused for ROS and / or RNS, such as peroxynitrite (ONOO -).

The present invention is susceptible to apply in various fields related to the sciences of life, specifically those related to study and characterization of chemical and biological compounds such as antioxidants, within the field of pharmacology, and their use for disease therapies in which they participate ROS and RNS species such as peroxynitrite (ONOO -). Examples of such diseases are those that occur at the renal level, in which it is known that peroxynitrite nitration increases (ONOO -), or other diseases such as ischemia in which produce increases in nitric oxide (NO).

Therefore, the first aspect of the present invention relates to a method, in vitro , for testing or detecting the ability of compounds or molecules to inhibit or stimulate oxidative and / or nitrative and / or nitrosative damage caused by reactive species of oxygen (ROS) and / or nitrogen (RNS) on biological systems, which includes the inclusion in the reaction medium of said reactive species together with the compound or molecule of which said capacity is to be tested or detected, characterized by using an SOD recombinant as a positive control of the activity of said reactive species. In a preferred embodiment of the invention the recombinant SOD enzyme used is characterized by SEQ ID NO: 1, the molecule tested is hemoglobin which can be hexacoordinated (rice hemoglobin I) or pentacoordinated (leghemoglobin or myoglobin) and ROS and / or RNS are selected from the group comprising in a non-limiting manner: superoxide anion (O 2 -), nitric oxide (NO), hyponitrous acid (HNO), nitrosyl anion (NO -), nitrite (NO 2 -), trioxodinitrate (N 2 O 3), nitronium cation (NO 2 +), hydroxyl radical (. OH), radical (hydro) peroxyl (.COO), alkoxy radical (.CO), carbonate radical (CO 2), oxygen singlet (1 O 2), hydrogen peroxide (H 2 O 2), ozone (O 3), ONOO -, hypochlorous acid (HClO), hypobromous acid (HBrO), selenite (Na 2 SeO 3), oxygen (O2), or the precursors thereof, as well as catalytic metals that lead to the formation of reactive species such as Fe or Cu.

The second aspect of the present invention is refers to the use of hemoglobin for the preparation of a pharmaceutical and / or nutraceutical composition intended to eliminate, or avoid the formation of ROS and / or RNS in a cell group of cells, tissue or organ of any living organism and, by consequently, destined to the treatment of diseases whose etiology is related to the existence of oxidative stress and / or  nitrosative and / or nitrative, except diseases whose etiology is related to stress caused by nitric oxide (NO); to level cellular, tissue or organ. In particular embodiments of the invention ROS and / or RNS are selected from the group comprising non-limiting form: superoxide anion (O2 -), acid hyponitrous (HNO), nitrosyl anion (NO -), nitrite (NO 2 -), trioxodinitrate (N 2 O 3), nitronium cation (NO2 +), hydroxyl radical (.OH), radical (hydro) peroxy ((.COO), alkoxy ((.CO)) radical, radical carbonate (CO 2), oxygen singlet (1 O 2), hydrogen peroxide (H 2 O 2), ozone (O 3), ONOO -, hypochlorous acid (HClO), hypobromous acid (HBrO), selenite (Na2 SeO3), oxygen (O2), or the precursors thereof, as well as catalytic metals that lead to the formation of reactive species such as Fe or Cu, or the precursors of the same as well as catalytic metals that lead to formation  of reactive species such as Fe or Cu; diseases whose etiology is related to the existence of stress oxidative / nitrosative / nitrative are selected from the group that includes but is not limited to: neuropathies, lung diseases, heart disease, asthma, kidney disease, disease caused by inflammation, mycoplasmosis, Alzheimer's disease Parkinson's, acute multiple sclerosis, gliomas and adenocarcinomas; Y hemoglobin can be hexacoordinated (rice hemoglobin I) or pentacoordinated (leghemoglobin or myoglobin).

The third aspect of the present invention relates to the use of hemoglobin, due to its SOD activity, as a sensor of the presence or activity of ROS and / or RNS, except for nitric oxide (NO) and oxygen (O2) ). In preferred embodiments of the invention, ROS and / or RNS are selected from the group comprising but not limited to: superoxide anion (O 2 -), hyponitrous acid (HNO), nitrosyl anion (NO - ), nitrite (NO2 -), trioxodinitrate (N2O3), nitronium cation (NO2 +), hydroxyl radical (<2> OH), (hydro) peroxyl radical (.COO), alkoxy radical (.CO), carbonate radical (CO 2 .-),
oxygen singlet (1 O 2), hydrogen peroxide (H 2 O 2), ozone (O 3), ONOO -, hypochlorous acid (HClO), hypobromous acid (HBrO), selenite (Na2 SeO3), or the precursors thereof, as well as catalytic metals that lead to the formation of reactive species such as Fe or Cu; and hemoglobin can be hexacoordinated (rice hemoglobin I) or pentacoordinated (leghemoglobin or myoglobin).

The fourth aspect of the present invention is refers to a nutraceutical composition intended for prevention and / or treatment of diseases whose etiology is related to the existence of oxidative and / or nitrosative and / or nitrative stress at cellular, tissue or organ level, except those diseases whose etiology is related to stress caused by rust nitric (NO), characterized by understanding hemoglobin as active principle. In preferred embodiments of the invention the diseases whose etiology is related to the existence of oxidative and / or nitrosative and / or nitrative stress is selected from group comprised of: neuropathies, lung diseases, heart disease, asthma, kidney disease, disease caused by inflammation, mycoplasmosis, Alzheimer's disease Parkinson's, acute multiple sclerosis, gliomas and adenocarcinomas, degenerative diseases, arthritis or cancer; and hemoglobin it can be hexacoordinated (rice hemoglobin I) or pentacoordinated (leghemoglobin or myoglobin).

The above mentioned compositions may understand, combined with hemoglobin, other active ingredients and carry used excipients as stabilizers or vehicles for the same, facilitating its absorption by the human body. The type of excipient was selected based on the route of administration (oral, intravenous, intradermal, etc.) chosen and, in general, they can be: ties, fillers, disintegrators, lubricants, coaters, sweeteners, flavor enhancers or dyes

The last aspect of the present invention is refers to a method of disease prevention whose etiology It is related to the existence of oxidative stress and / or nitrosative and / or nitrative at the cellular, tissue or organ level, except for those diseases whose etiology is related with stress caused by nitric oxide (NO), characterized by understand the administration of a quantity pharmaceutically acceptable of a pharmaceutical and / or nutraceutical composition that Understand hemoglobin.

The following are examples of embodiment, which aims to illustrate the invention if limiting the same.

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Examples of the invention Example 1 The amount of nitration of the recombinant SOD produced by the SIN-1 can be estimated by the loss of activity enzymatic determined by the recombinant SOD activity assay in gel

Recombinant SOD was incubated for 120 min at 37 ° C with increasing amounts of SIN-1. Be they used the following concentrations of SIN-1: 0.2, 0.4, 0.6, 0.8 and 1 mM. SOD gel activity was visualized after protein separation in 15% native PAGE (w / v). To estimate the loss of enzyme activity in the assay gel, densitometry analyzes were performed using the Quant 1 software on the GelDoc 2000 (BioRad). It was considered that control samples incubated in the absence of the nitrating agent They showed 100% activity.

Enzyme inactivation occurred in a dose-dependent manner, as observed by the gradation of residual SOD activity in the gel, which indicated that the loss of activity in the gel test could be utility for estimating the nitration of recombinant SOD, when peroxynitrite is formed from SIN-1. The Loss of enzyme activity was most evident in the higher concentrations of SIN-1, and began to decrease as the concentration of SIN-1 employed. Quantification with densitometry of the gel SOD activity showed that about 57% was lost of the activity with the 1 mM treatment in three repetitions different. Deferroxyamine did not completely suppress the loss of activity but reduced the effect of SIN-1, as it had been observed in the detection of Tyr nitration.

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Example 2 The amount of nitration of the recombinant SOD produced by the SIN-1 can be quantified by the loss of Enzymatic activity by spectrophotometric assay

Total SOD activity was analyzed by a spectrophotometric method based on the ability of SODs to inhibit the reduction of ferric cytochrome C, by the system xanthine-xanthine oxidase.

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The cytochrome C reduction was quantified to 25 ° C following the increase in absorbance at 550 nm for 2 min. The reaction cocktail pre-incubated at 25 ° C and kept in Darkness contained 50 mM phosphate buffer with 0.1 Na 2 EDTA mM (pH 7.8), 1 mM xanthine, and 1 mM horse heart cytochrome c (Sigma-Aldrich, St. Louis, MO, USA). The reaction started with the addition of 20 µl of diluted xanthine oxidase to Starting from the commercial solution. This dilution of xanthine oxidase is optimized in order to reach 0.1 units of variation of absorbance from the sample taken as blank. In the present invention, the dilution of xanthine oxidase was prepared diluting 10 times the milk xanthine oxidase of Sigma-Aldrich in 50 mM phosphate buffer with Na2EDTA 0.1 mM (pH 7.8). Sample volumes for control and for Recombinant SOD treated with SIN-1 is also optimized in order to obtain an approximate 50% reduction in the activity rate, since higher values of SOD activity could lead to nonlinear kinetics or saturation of the enzymatic activity. Finally 5 µl of Sample volume as appropriate for our conditions.

In order to obtain one more estimate requires the nitration of the protein by the SIN-1, the spectrophotometric test is also used to detect the inactivation of recombinant SOD. Saying assay confirmed that enzyme inactivation occurred gradually, in a dose-dependent manner. The Recombinant SOD was increasingly inactivated with the concentration of SIN-1 For the concentration of SIN-1 (0.2 mM) inactivation was 17.8% and for the sample of SIN-1 (1 mM) was inactivated by 61.6%, which which confirmed the result obtained in the gel test of the example 1. Using this calibration with increasing concentrations of SIN-1 shown, and assuming a relationship equimolar production of peroxynitrite (ONOO -), you can calculate from the loss of activity of recombinant SOD the initial peroxynitrite concentration in any solution trouble.

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Example 3 Use, in an in vitro bioassay, of recombinant SOD (SEQ ID NO: 1) to assess the antioxidant capacity of molecules, such as hemoglobins

The reaction media comprised the agent. SIN-1 nitrant and the compound to be tested: in this example hemoglobin I (HbI) or leghemoglobin (Lba). One time performed the incubation the proteins were separated in electrophoresis native and gels were analyzed for SOD activity. The present Example is illustrated in Figure 1 where it is shown:

Figure 1A: SOD activity of the tested mixtures.

Figure 1B: Coomasie staining of mixtures equivalent to those used in Figure 1A, in which all proteins present are stained blue regardless of its enzymatic activity.

As you can see, the mixture of reaction containing only FeSOD gives positive reaction in the assay of enzymatic activity SOD in gel (lane 1; Figure 1A). Further, as set forth in examples 1 and 2, when the compound SIN-1 was largely inactivated the Enzymatic activity of the FeSOD enzyme (lane 2; Figure 1A). By Therefore, this essay offers the possibility of including compounds or macromolecules and test their ability to prevent or eliminate the nitrating and inactivating effect of SIN-1 Specifically, in the present essay tested two macromolecules: soy leghemoglobin (lane 3), and Hemoglobin I of rice (lane 4). Although in Figure 1A it does not SOD activity of leghemoglobin was observed, in lane 3 evidenced that the presence of said reactive leghemoglobin or protects the SOD activity of the FeSOD by preventing its inactivation by the SIN-1 In the present invention, leghemoglobin protected the FeSOD and it maintained 100% of the initial activity in the concentrations of SIN-1, FeSOD and leghemoglobin tested. Similarly, it was observed that the rice hemoglobin I was able to maintain 100% activity of FeSOD in the test conditions (lane 4). However, the hemoglobin I, in addition to protecting the SOD activity of FeSOD, surprisingly and unexpectedly, as shown on 4th street of Figure 1A, showed a new positive band, with lower relative mobility that the FeSOD, which evidenced the SOD activity of hemoglobin I, above the FeSOD activity line.

The preventive effects on the inactivation of FeSOD may be due to two causes:

I.

nitric oxide eliminating effect (NO) by hemoglobins (which is known in the state of the art) or

II.

superoxide anion eliminating effect (O2 {-}) that was evidenced for the first time here invention for hemoglobin I (Figure 1, lane 4).

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Since SIN-1 produces so much nitric oxide (NO) as superoxide anion (O2 -) in equimolar amounts, the two previous possibilities are valid, and converts hexacoordinated hemoglobins into enzymes that prevent the formation of peroxynitrite (ONOO -) due to its double antioxidant capacity against nitric oxide (NO) and anion superoxide (O 2 -). In addition, since it was not possible to discard the production of a certain amount of peroxynitrite (ONOO -) to from SIN-1, that FeSOD maintained 100% of residual activity and that the reversal of nitration over amino acids of the active center of FeSOD was not reversible, in The present invention concluded that the hemoglobin I was able to eliminate at least part of the peroxynitrite (ONOO -) formed.

On the other hand, only hemoglobins with Faith Hemic in reduced form (Fe 2+) are able to bind oxygen. However, hemoglobins were included in this trial in their oxidized form (Fe 3+), so it was shown that they exert their effect regardless of its functionality to bind oxygen. Be considers that given the similarity between O 2 and O 2 - and the high electronegativity of O2 and the negative charge of O2 -, both molecules are capable of interacting with the Fe molecule in the active center of Hemoglobin I.

To confirm that rice hemoglobin I showed SOD activity similar to that of recombinant SOD (SEQ ID NO: 1), gel SOD activity tests were performed (Figure 3) as described in Beauchamp and Fridovich (1971). These essays are based on SOD inhibition of the reduction of nitro tetrazolium blue (NBT) by superoxide radicals generated photochemically SOD hemoglobin I samples recombinant (SEQ ID NO: 1) and leghemoglobin (2 µg / lane) were ran on a polyacrylamide gel (PAGE) -native at 15% (w / v) at 200 V for approximately 2 hours, that is, he was running 1 more hour after bromophenol blue I reached the end of the gel. This allowed for better mobility. of SODs in gels.

After electrophoresis, the gels were incubated in reaction buffer (phosphate buffer 50 mM sodium, pH 7.8) for 30 minutes. Second, they were transferred to reaction buffer containing blue of 0.5 mM nitro-tetrazolium (NBT) and incubated Again, this time for 20 minutes.

Finally, they were incubated in buffer reaction supplemented with 0.03 mM riboflavin and 0.2% TEMED (v / v) for another 20 minutes. All incubations were performed in darkness. For the visualization of the SOD activity, the gels were exposed to white light for 2-5 minutes, as described in Beauchamp and Fridovich (1971).

As shown in Figure 3A, the Rice hemoglobin I has SOD activity, in a very similar to recombinant SOD (SEQ ID NO: 1). Hemoglobin I presented less mobility than recombinant SOD (SEQ ID NO: one). In the panel of Fig. 1B it was shown by Coomasie staining the amount of total protein loaded in each assay (2 µg). He delayed mobility of SOD activity for hemoglobin I coincided with the delay of hemoglobin I protein in staining with Coomasie dye. Also, on street 2 of Figure 3 only hemoglobin I was added and therefore the SOD activity cannot correspond to another enzyme. The fact that there is none in the two tests another electron giver that the superoxide anion (O 2) - and that the activity values shown in the Figures 1 and 3 are similar to those obtained for SOD recombinant (SEQ ID NO: 1), indicated that it is an activity SOD, and not of pseudoperoxidase activity.

The use of two methods of determination of activity provided independent information and showed convergent way that hemoglobin I has SOD activity.

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Example 4 SOD activity by hemoglobin I test rice cytochrome c reduction

Rice hemoglobin I also presented superoxide dismutase activity in the test of cytochrome-c, similar to the SOD enzyme recombinant characterized by SEQ ID NO: 1 (Figure 2).

The SOD activity of rice hemoglobin I was examined using the spectrophotometric method based on the ability of superoxide dismutases to inhibit the reduction of ferricitochrome c by the superoxide radical producing system (O2 -) "xanthine- xanthine oxidase "(McCord and Fridovich, 1969), with the specifications indicated in Moran et al . (2003). The reduction of ferricitochrome-c causes a change in the absorption spectrum of cytochrome-c causing an increase in absorption at 550 nm. Therefore, this reduction was monitored following the increase in absorbance at 550 nm, in a spectrophotometer with thermostat at 25 ° C, for two minutes. Three proteins were tested under the conditions indicated: hemoglobin I, FeSOD (recombinant SOD characterized by SEQ ID NO: 1), and soy leghemoglobin. The reaction mixture contained reaction buffer (50 mM phosphate buffer, 0.1 mM Na2 EDTA, pH 7.8), 1 mM xanthine, and 1 mM horse heart cytochrome-c (Sigma-Aldrich, St Louis, MO, USA). The reaction began with the addition of 20 µl of diluted xanthine oxidase from the commercial solution. This dilution of xanthine oxidase was optimized in order to reach 0.1 units of absorbance variation from the sample taken as blank. In the present example, the dilution of xanthine oxidase was prepared by diluting the Sigma-Aldrich milk xanthine oxidase 10 times in 50 mM phosphate buffer with 0.1 mM Na 2 EDTA (pH 7.8). Finally, 10 µl of each protein volume was established as appropriate for our conditions. Specific inhibitors were used in the in vitro assays at the final concentrations indicated in Figure 1, and were prepared using concentrated solutions of 10 mM and 300 mM KCN and 100 mM H2O2, both prepared in deionized water . The amounts of FeSOD used were calculated using the Bradford method (BioRad protein assay, Bio-Rad), while the amounts of rHb and Lba were estimated using the pyridine method (Jun et al ., 1994).

Figure 2 shows the SOD activity obtained from the incubation of FeSOD, rHb1 and Lba in the assay described above. The superoxide dismutase activity inhibits the reduction by the superoxide radical of cytochrome c to its ferrous form cytochrome c2 +, which is quantifiable by spectrophotometry at 550 nm. It was observed how FeSOD and rHbI show specific activities within the same range with respect to the micrograms of protein used (trials 1 and 5). VuFeSOD was not inhibited by CN - either at low or high concentration (10 µM and 3 mM), nor at low concentration of hydrogen peroxide (assays 2, 3, and 4), which is typical of the FeSODs (Moran et al ., 2003). In contrast, the SOD activity in the rHbI enzyme is inhibited by both CN - and hydrogen peroxide, with inhibition with CN - being more efficient than with peroxide, which is consistent with the greater susceptibility of other hemoproteins such as cytochrome c oxidase by CN - with respect to peroxide.

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Example 5 Confirmation of the SOD activity of hemoglobin I

The SOD activity of hemoglobin is independent of the presence of reducers or catalysts of the reduction of said hemoglobin, so it is considered that the reaction is a dismutation. In Figure 3 it was shown that Rice hemoglobin I has catalytic SOD activity. For it hemoglobin was included in the gel test described above I of rice in the absence of another protein (Lane 2, Figure 3) and it compared its activity against FeSOD (SEQ ID NO: 1) (Street 1, Figure 3) and leghemoglobin (Street 3, Figure 3). Again it was observed that the rice hemoglobin I, in the absence of another compound, shows SOD activity, in a manner very similar to FeSOD (Fig 3). In This test also tested compounds and catalysts of the reduction of the heme group of hemoglobin I and its effect was studied on the redox state of hemoglobins and SOD activity. The rFLbR2 is an enzyme that catalyzes the reduction of hemoglobins in presence of a reducer like the NADH. As we mentioned only reduced hemoglobins (Hb2 +) are capable of binding oxygen, although they are capable of interacting with O_ {2} -.

The addition of reducing agents such as NADH and catalysts such as rFLbR2 showed no effect on the SOD activity of hemoglobin I. This is in accordance with an enzymatic dismutation reaction in which it does not occur a net reduction reaction but two reactions: one of reduction initial hemoglobin by the superoxide radical (since the hemoglobin is added in oxidized form), and a subsequent reaction of oxidation of a second superoxide radical with production concomitant of H 2 O 2. Because the H 2 O 2 is a ROS, in the present invention the possibility of use agents that eliminate H 2 O 2 in combination with the hemoglobin I which, as demonstrated in the present invention, It has SOD activity.

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References

1. Arredondo-Peter , R., Hargrove , MS, Moran , JF, Sarath , G., and Klucas , RV ( 1998 ). Plant hemoglobins Plant Physiol 118, 1121-1125.

2. Beauchamp , CO, and Fridovich , I. ( 1971 ). Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem 44, 276-287.

3. Dordas , C., Rivoal , J., and Hill RD ( 2003a ) Plant haemoglobins, nitric oxide and hypoxic stress. Annals of Botany 91, 173-178.

4. Dordas , C., Hasinoff , BB, Igamberdiev , AU, Manac'h , N., Rivoal , J., Hill , RD ( 2003b ) Expression of a stress-induced hemoglobin affects NO levels produced by alfalfa roots culture under hypoxic stress Plant J. 35: 763-770

5. Feelisch , M., Kotsonis , P., Siebe , J., Clement , B., and Schmidt HH ( 1999 ). The soluble guanylyl cyclase inhibitor 1H- [1,2,4] oxadiazolo [4,3, -a] quinoxalin-1-one is a nonselective heme protein inhibitor of nitric oxide synthase and other cytochrome P-450 enzymes involved in nitric oxide donor bioactivation Mol. Pharmacol 56, 243-253.

6. Gardner , PR, Martin , LA, Hall , D., and Gardner , AM ( 2001 ) Dioxygen-dependent metabolism of nitric oxide in mammalian cells. Free Radic Biol Med . 31, 191-204.

7. Graziano, M., and Lamattina, L. (2005). Nitric oxide and iron in plants: an emerging and converging story. Trends Plant Sci . 10, 4-8. (Review). Halliwell and Gutteridge, 2007.

8. Igamberdiev , I., Hill , RD, ( 2007 ) Nitric oxide as alternative electron carrier during oxygen deprivation. In Lamattina L, Polacco JC, Eds. Nitric oxide in plant growth, development and stress physiology. Springer-Verlag Berlin Heidelberg.

9. Ischiropoulos , H., Zhu , L., Chen , J., Tsai , M., Martin , JC, Smith , CD, and Beckman , JS ( 1992a ). Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase. Arch. Biochem. Biophys 298 , 431-437.

10. Ischiropoulos , H., Zhu , L., and Beckman , JS ( 1992b ). Peroxynitrite formation from macrophage-derived nitric oxide. Arch. Biochem. Biophys 298 , 446-451.

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11. Jun , HK, Sarath , G., Moran , JF, Becana , M., Klucas , RV, Wagner , FW ( 1994 ) Characteristics of modified leghemoglobins isolated from soybean (Glycine max Men.) Root nodules. Plant Physiol 104: 1231-1236

12. Kundu , S., Trent III, JT, and Hargrove , MS ( 2003 ). Plants, humans and hemoglobins. Trends in Plant Science . 8, 387-393.

13. MacMillan-Crow , LA, Crow , JP, Kerby , JD, Beckman , JS, and Thompson , JA ( 1996 ). Nitration and inactivation of manganese superoxide dismutase in chronic rejection of human renal allografts. Proc. Natl Acad. Sci. USA 93, 11853-11858.

14. MacMillan-Crow LA, Cruthirds DL ( 2001 ) Invited review: manganese superoxide dismutase in disease. Free Radic Res . 34, 325-36. Review

15. McCord , JM, and Fridovich , I. ( 1969 ). Superoxide dismutase: an enzymic function for erythrocuprein (hemocuprein). J. Biol. Chem . 244, 6049-6055.

16. Moran , JF, Klucas , RV, Grayer , R., Abian , J., and Becana , M. ( 1997 ) Complexes of iron with phenolic compounds from soybean nodules and other legume tissues: prooxidant and antioxidant properties. Free Radicals in Biology and Medicine 22, 861-870.

17. Moran , JF, Rubio , MC, James , EK, Sarath , G., Klucas , RV, and Becana , M. ( 2003 ). Functional characterization and expression of a cytosolic iron-superoxide dismutase from cowpea (Vigna unguiculata) root nodules. Plant Physiology 133, 1-10.

18. Munoz, I., Moran, JF, Becana, M., Montoya, G. (2005). The crystal structure of the eukaryotic iron superoxide dismutase suggests intersubunit cooperation during catalysis. Protein Science 14, 387-394.

19. On , WC, Sohal , RS ( 1994 ). Extension of life-span by overexpression of superoxide dismutase and catalase in Drosophila melanogaster. Science 263: 1128-30

20. Radi , R., Peluffo , G., Alvarez , MN, Naviliat , M., and Cayota , A. ( 2001 ). Unraveling peroxynitrite formation in biological systems. Free Radic Biol. Med . 30, 463-488.

21. Ridnour , LA, Thomas , DD, Mancardi , D., Espey , MG, Miranda , KM, Paolocci , N., Feelisch , M., Fukuto J., and Wink , DA ( 2004 ). The chemistry of nitrosative stress induced by nitric oxide and reactive nitrogen oxide species. Putting perspective on stressful biological situations. Biol. Chem . 385, 1-10.

22. Sun , Y., Jin , K., Mao , XO, Zhu , Y., and Greenberg , DA ( 2001 ). Neuroglobin is up-regulated by and protects neurons from hypoxic-ischemic injury. Proc. Natl Acad. Sci. USA . 98, 15306-15311.

23. Thomas , DD, Xiaoping L., Kantrow SP, Lancaster JR Jr ( 2001 ). The biological lifetime of nitric oxide: Implications for the perivascular dynamics of NO and O_ {2}. Proc. Natl Acad. Sci. USA 98 , 355-360.

24. Vinogradov , SN, Hoogewijs , D., Bailly , X., Arredondo-Peter , R., Guertin , M., Gough , J., Dewilde , S., Moens , L., and Vanfleteren , JR ( 2005 ) . Three globin lineages belonging to two structural classes in genomes from the three kingdoms of life. Proc. Natl Acad. Sci. USA . 102, 11385-11389.

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one

Claims (8)

1. Method, in vitro , to select compounds or molecules capable of inhibiting or stimulating oxidative and / or nitrative and / or nitrosative stress caused by reactive oxygen (ROS) and / or nitrogen (RNS) species on biological systems, which comprises including in the reaction medium, of said reactive species with the compound or molecule which is to be measured and / or detect such capacity, characterized by using a SOD enzyme as positive control for the activity of these reactive species. 2. Method according to claim 1, wherein the SOD enzyme is characterized by being recombinant and represented by SEQ ID NO: 1. 3. Method according to claim 1, characterized in that the molecule tested is hemoglobin. 4. Method according to claim 3, characterized in that the hemoglobin is hexacoordinated. 5. Method according to claim 4, characterized in that the hemoglobin is rice hemoglobin I. 6. Method according to claim 3, characterized in that the hemoglobin is pentacoordinated. 7. Method according to claim 6, characterized in that the hemoglobin is leghemoglobin or myoglobin. Method according to claim 1, characterized in that the ROS and / or RNS are selected from the group: superoxide anion (O2 -), nitric oxide (NO), hyponitrous acid (HNO), nitrosyl anion ( NO -), nitrite (NO 2 -), trioxodinitrate (N 2 O 3), nitronium cation (NO 2 +), hydroxyl radical (^. OH), radical (hydro) peroxyl (.COO), alkoxy radical (.CO), carbonate radical (CO 2), oxygen singlet (1 O_ {2}, hydrogen peroxide (H 2 O 2), ozone (O 3), peroxynitrite (ONOO -), hypochlorous acid (HClO), hypobromous acid (HBrO), selenite ( Na 2 SeO 3), oxygen (O 2), or the precursors thereof, as well as catalytic metals that lead to the formation of reactive species such as Fe or Cu.