Correlations between hemes I and IV, II and III, II and IV; and histidine 67 and hemes I and III; are mainly a result of indirect interactions mediated by other redox/protonable sites. Thus, they are expected to vary in an unpredictable manner within charge set and simulated E values. This expectation is corroborated by our results. From Figure 3.15 it is indeed difficult to establish a rational cause-effect relation for most cases. However, Figure 3.15a presents a striking example of how an unlikely positive cooperativity can be established by indirect interactions. Correlations of any kind between hemes II and IV are usually not noticeable in most studies [21, 29, 30]. Yet, because these two heme groups are positively correlated with the protonable site histidine 67, they are prone to exist in the same redox state. In other words, given the fact that the correlation values at E ' −300 mV for heme II and histidine 67, and heme IV and histidine 67 are positive with similar magnitudes, these heme groups are very often in the same redox state, thus, showing a strong positive correlation between them. On a different note, it is relevant to mention that the correlations obtained with this work are, in absolute terms, higher than any of those previously published [21, 30]. A direct comparison of our results with the correlations obtained by Machuqueiro and Baptista for simulations at ε = 2  (unpublished data), shows that both sets of results differ qualitatively (a sign that they are electrostatically different), but assume similar magnitudes (a consequence of using the same dielectric constant for the protein).
by cyclic and square-wave voltametries. This drug was irreversibly reduced on glassy carbon electrode and the peak potential values are pH independent, however the biggest value of current peak was observed at pH 6.0. The voltammetric behavior of artemisinin was significantly changed in the hemegroup presence, provoking an anticipation of about 600 mV on cathodic peak. By square-wave voltammetry it was observed that this new peak was sensitive to the hemin concentration, reaching a value around 10 times larger regarding the original cathodic peak of artemisinin, being the concentration of 20 μmol/L for the former and 50 μmol/L for the latter. In addition, results indicated that this electro-catalytic process depends on the Fe(II)-hemin formation on the electrode surface, indicating the possible electro-polymerization of hemin on the glassy carbon electrode. This adsorptive effect was evaluated from the superficial concentration (Γ) estimation of the hemin on the working electrode at pH 6.0. The modification of the glassy carbon electrode using hemin showed that the interaction between artemisinin and the hemegroup predominantly occurs on the electrode surface and not in solution. Therefore, clarifying artemisinin mechanism of action is important in order to contribute for the design and development of new antimalarial agents.
Enterococcus faecalis is one of the few species of lactic acid bacteria that exhibit catalase activity, but only when grown in the presence of heme . E. faecalis catalase (KatA) belongs to the group of heme-containing mono-functional catalases (EC 188.8.131.52). It is a homo-tetrameric protein containing one hemegroup (protoheme IX) per subunit and its crystal structure has been determined . Catalase protects E. faecalis against hydrogen peroxide stress but is not the only enzyme with this function. The bacterium can produce a NADH peroxidase (Npr) that degrades hydrogen peroxide to water and which seems sufficient for protection against high concentrations of hydrogen peroxide when heme is not available . E. faecalis cells cannot synthesize heme and thus have to take it up from the environment in order to form active catalase. Despite the fact that catalase has been studied intensively for many years biogenesis of this enzyme is not understood. The chronological order of assembly events such as KatA polypeptide folding, tetramer formation and heme insertion into the apopro- tein is not known. The complex structure of the catalase tetramer as well as the hydrophobic nature of heme and its potential in generating reactive oxygen species indicate the need for tightly controlled and assisted assembly of catalase. No catalase biogenesis factors have been identified except in the bacterium Campylobacter jejuni which was recently reported to contain a protein (Cj1386)
Clinical and experimental evidences have reported that intestinal ischemia and reperfusion (I/R-intestinal) induces acute lung injury (ALI), which in severe cases can progress to acute respiratory distress syndrome (ARDS). The ALI is characterized by the release of a broad spectrum of inflammatory mediators, neutrophil infiltration and increased vascular permeability. It is known that inflammatory mediators generated at the site of I/R-intestinal are transported by the mesenteric lymphatic system and, on reaching the lung, contribute to the ALI. The enzyme heme oxygenase 1 (HO-1) plays an important role in cellular homeostasis, due to its catabolic action on hemegroup of hemoproteins, forming as by-products such as iron, biliverdin and carbon monoxide. It is known that these by-products have anti-inflammatory, antioxidant and antiapoptotic actions. However, the role of HO-1 in the control of ALI caused by I/R-intestinal is not yet fully understood. In the present study we investigated the expression of HO-1 and the effects of its induction on pulmonary complications resulting from I/R-intestinal. So, male Wistar rats (220-250 g) were subjected to 45 min of intestinal ischemia by occlusion of the superior mesenteric artery and 2 h of reperfusion. The control group consisted of animals falsely operated (Sham). Still, the induction of HO-1 was performed by treating animals with the compound Hemin (10 mg/kg) 48 and 24 h before the induction of I/R- intestinal. The I/R-intestinal increased the pulmonary activity of the myeloperoxidase (MPO) and the extravasation of Evans blue dye (EB) in the lung. Levels of IL-1 β increased in lung explant (24 h) while the IL-10 were reduced after I/R- intestinal. Further, the I/R-intestinal reduces pulmonary expression of SOD-1 and promoted the increase of iNOS expression. The results indicate that the I/R-intestinal alone did not induce gene expression of HO-1 enzyme, but the treatment of animals with Hemin increased its expression which was accompanied by reduction of pulmonary activity of MPO and extravasation of the dye EB. The high pulmonary levels of IL-1 were reduced by treatment of animals with Hemin and there was an increase of IL-10 and VEGF in the same tissue. The Induction of HO-1 prevented the increased levels of IL- β 1 and IL-10 and promoted increasing of the VEGF levels in the animals’ lymph. With respect to the antioxidant system, our data indicate that induction of HO-1, seems to be related to the elevation of expression of SOD-1, SOD-2 and reduction of iNOS expression. In conclusion, our data may suggest that prior induction of HO-1 expression controls the magnitude of lung injury caused by I/R-intestinal by mechanisms involving increased activity of a portion of the antioxidant system and regulation of the balance between generation anti-inflammatory cytokines and pro-inflammatory in the lung.
Pathologies related with myoglobin can range from membrane lipids peroxidation to serious hepatopathologies. Some pathologies are related with the capability of myoglobin to acquired oxidase activity after an interaction with exogenous agents as alkyl halides, or endogenous agents exemplified by hydrogen peroxide which is normally produced by cellular metabolism. Previous studies showed that it was necessarie a specific tyrosine residue presence to covalently bind hemegroup at mioglobin proteic part to acquires oxidase activity after being transformed using hydrogen peroxide; distal histidine e histidine residue 97 are recquired for oxidase activity if alkys halide are used.
Indeed, non-covalently bound heme proteins lose their hemegroup upon denaturation with a concomitant and significant shift of the Soret peak maximum to ~ 365 nm , which is not observed in this case. It was also noticed that both thermal and chemical unfolding are reversible, as the far-UV CD and visible absorption fingerprints corresponding to the native conformers are restored upon removal of the destabilizing condition. This allows a thermodynamic analysis of the reaction, as the system reacts under equilibrium conditions: this is not always the case among metallo proteins which frequently unfold irreversibility either due to loss or deficient reintegration of the metal cofactor upon refolding [205-209], although other cytochromes also undergo reversible unfolding . Overall, the spectroscopic analysis of the PAS-like sensor native and unfolded conformers shows that i) heme and secondary structure changes can be independently monitored as conformational probes and ii) the reversibility of the unfolding reaction allows a quantitative thermodynamic analysis of the folding properties of both proteins. The latter is determinant for understanding mechanistic differences between the different heme sensor domains.
With the above understanding, it is possible to analyze these three spectra simultaneously using one set of parameters. To reduce the number of variable parameters, the following rational assumptions were made: (1) the total absorption intensity (oxidized plus reduced) for each one of the four Fe sites is 1/4 of the total Fe absorption, (2) the line widths of a quadrupole doublet arising from a hemegroup are the same, and (3) for each sample, the [oxidized]/[reduced] ratio of heme b and heme c is the same. (This assumption is based on the observed similar midpoint redox potentials for heme b and heme c; see above.) During the analysis, we also realized that the electronic relaxation rate of ferric Fe B is not much faster than the 57 Fe precession rate,
No fígado (Tabela 5), um compartimento de reserva corporal de ferro, não houve diferença entre todos os grupos experimentais quanto ao peso do órgão, ferro total, ferro heme e ferro não- -heme (p>0,05). Todavia, houve uma clara ten- dência a menor concentração de ferro heme [10,5 (2,5) µg/g] e ferro não-heme [28,6(22,3)µg/g] no fígado dos leitões que receberam rações forti- ficadas com Fe 0 . Para o grupo Ferlim estas concen-
O ferro heme apresenta-se na forma iônica reduzida Fe +2 (ferroso), representando 40%-55% do ferro presente na carne bovina, aves ou peixes, formando parte das moléculas de hemoglobina e mioglobina, sendo altamente disponível. Esse tipo de ferro entra diretamente nas células da mucosa na forma de complexo ferro-porfirina, e sua absorção é determinada principalmente pelo nível de ferro corporal e, em pequena parte, por fatores dietéticos, possivelmente o cálcio. O ferro não-heme, predominante na forma iônica oxidada Fe +3 (férrico), é menos solúvel no organismo; encontra-se nos cereais, leguminosas, verduras. A biodisponibilidade de ferro não-heme é menor em relação ao ferro heme, sendo influenciada por fatores dietéticos, além do nível do ferro corpóreo. O ferro não-heme, em meio ácido, é transportado quelado, aumentando a absorção na membrana do duodeno, facilitando a transferência do ferro através das microvilosidades da membrana (Hallberg et al., 1992; House & Welch, 1987; Schricker et al., 1982; FAO/OMS, 1988).
As porfirias agudas são de herança autossômica dominante, com exceção da PALAD, que possui herança autossômica recessiva (Tabela I). A expressividade das porfirias agudas é variável, sendo que os pacientes podem permanecer assintomáticos durante anos. A expressão clínica da doença geralmente está ligada a fatores ambientais ou adquiridos que provocam os ataques agudos por aumentar a demanda de heme, estimulando a síntese da enzima ALA-S (Moore, 1980; Kappas et al., 1995). O fator predisponente mais importante é a ingestão de medi- camentos que sofrem metabolização pelo sistema citocromo P 450 (dependente de heme), induzindo a sínte- se deste complexo enzimático com conseqüente consumo de heme (Moore et al., 1983; De Matteis, 1988).
CHASIN, A.; CHASIN, M.; SALVADORI, M. C. Validação de métodos cromatográficos em análises toxicológicas. Rev. Farm. Bioquím. Univ. S. Paulo, v.30, n.2, p.49-53, 1994. DANIELL, W. E.; STOCKBRIDGE, H. L.; LABBE, R. F.; WOODS, J. S.; ANDERSON, K. E.; BISSELL, M. D.; BLOOMER, J. R.; ELLEFSON, R. D.; MOORE, R. M.; PIERACH, C. A.; SCHREIBER, W. E.; TEFFERI, A.; FRANKLIN, G. M. Chemical exposures and disturbances of heme synthesis. Environ. Health Perspect., v.105, n.1, p.37-53, 1997.
Quanto maior a perda de água no corte, menos concentrados ficam os teores de ferro, visto que pode haver perda do ferro junto com a água. Como a mioglobina e outras proteínas heme são proteínas sarcoplasmáticas e estão localizadas dentro das células, são solúveis em água. Sendo assim, estas proteínas podem, em parte, ser perdidas com a ocorrência de exsudação, ou seja, durante a perda de umidade ou suco da carne (SHIMOKOMAKI et al., 2006). Esse aumento na perda de água foi observado antes da análise de ferro heme, em que foi feita a análise de umidade dos cortes. Os antibióticos estudados também se mostraram influentes, a norfloxacina apresentou valores superiores ao ciprofloxacina, mostrando que antibióticos influenciam de forma diferente na qualidade da carne pois são diferentes. A ciprofloxacina possui um grupo ciclopropil na posição N-1, o que melhora sua atividade antimicrobiana e também altera o seu comportamento em relação à norfloxacina (MARTINEZ; MCDERMOTT; WALKER, 2006; ROYBAL et al. 2002).
Os estados oligoméricos da DevS foram detectados através de cromatografia de exclusão por tamanho (SEC) usando uma coluna TSKgel GFC-300 (7,8 mm x 150 mm, TOSOH) acoplada a uma HPLC usando um detector de arranjo de diodo. A fase móvel usada para todos os experimentos consistiu de um tampão Tris-HCl 50 mM a pH 7,4 contendo cloreto de sódio 50 mM, a não ser que seja indicado diferentemente. Os espectros eletrônicos foram coletados para cada pico durante a corrida de filtração em gel, permitindo-nos confirmar o estado do grupo heme, se ligado ao oxigênio (oxi-DevS), monóxido de carbono (carboxi-DevS) ou reduzido em condições anaeróbicas (deoxi-DevS) ou oxidado (met-DevS). Esta proteína foi originalmente isolada e armazenada no estado DevS reduzido ligada a O 2 . A forma férrica de DevS (met) foi obtida por incubação da amostra de proteínas
The activation of NF-KB involves a major signal transduction pathway, which regulates the expression of genes related to inflammation. NF- KB regulates the expression of many cytokines, such as TNF-α and IL-1β, related to inflammatory responses, with TNF-α playing a major role (Schreiber et al. 1999, He et al. 2007). A previous study showed that losartan decreased TNF-α and IL-1β levels (Imai et al. 2005, Yuksel et al. 2015). Research also demonstrated that ALIS treatment reduced levels of TNF-α and IL-1β in acute lung injury (Akpinar et al. 2014). In accordance with these results, we showed that ALIS treatment decreased TNF-α and IL-1β mRNA expression in CAR-induced lung injury, with the strongest effect apparent in the 200 mg/kg treatment group.
(ligação peróxido e carbonila) interage por meio de uma interação polar com um dos grupos propionato do heme (parte polar) (Figura 7B). Esta diferença nos resultados está relacionada com a metodologia computacional adotada por aqueles autores, em que o anel porfirínico do heme permaneceu fixo, enquanto que os grupos metil e vinil, um dos grupos propionato do heme, bem como a artemisinina, permaneceram totalmente livres durante a otimização de geometria em nível de mecânica molecular. Além disto, os gru- pos propionato estavam na forma neutra e a histidina ligada ao fer- ro foi incluída nos cálculos. No presente trabalho, devido os grupos propionato na forma aniônica estarem fixos durante os cálculos, não se observaram interações efetivas entre os átomos de oxigênio destes grupos e os átomos de hidrogênio da artemisinina.
Estudos sobre o efeito da irradiação e do armazenamento em carnes de frango foram realizados para se conhecer melhor sua influência nos teores de ferro heme, não-heme, cor e pigmentos totais. Foram estudados coxa e filé de peito de frango. Estes foram irradiados a 0, 1 e 2 kGy e armazenados por 14 dias a 4 °C em câmara refrigerada. A determinação do conteúdo de heme e não-heme de carnes foi realizada através do método colorimétrico, empregando-se o reagente Ferrozine. Os valores de ferro heme foram influenciados tanto pela irradiação quanto pelo armazenamento, diminuindo seus teores com o passar do tempo. O ferro não-heme também foi influenciado tanto pelas doses empregadas quanto pelo tempo de estocagem, porém aumentou seus valores com o passar do tempo, devido à conversão do heme em não- heme. A cor não se mostrou influenciada pelas doses estudadas, somente pela estocagem, e os pigmentos totais foram afetados tanto pela irradiação quanto pelo tempo, diminuindo seus valores com o aumento do tempo de armazenamento. A irradiação se mostrou um bom método para conservação do ferro, visto que aumentou os teores de acordo com o aumento das doses.
Putative mechanism of action of free heme on neutrophils. Resting mature neutrophils express high levels of pro-apoptotic Bcl-2 protein Bad while very low or undetectable amounts of the anti-apoptotic protein Bcl-X L , facilitating Bax insertion into the mitochondria and consequent launch of the apoptotic cascade. When neutrophil interact with free heme, the activation of multiple signaling pathways occurs, which can be triggered or not by NADPH oxidase-dependent reactive oxygen species (ROS) production. This activation ultimately results in degradation of Bad and de novo synthesis of Bcl-X L , corroborating for the maintenance of mitochondria stability, as well as up- regulates the expression of proinflammatory and anti-apoptotic genes.
The exposure of animals to threatening situations of innate or learned nature results in exhibition of a repertoire of species-specific defensive behaviors, autonomic alterations and pain inhibition. This group of reactions has high relevance for the survival of species. In this context, an important component of the response of the organism in the emergency situations is the reduction of the capacity to perceive pain. The processing of nociceptive stimulus can be modulated in forebrain, in spinal, and in midbrain, for mechanisms involving different neurotransmitters and neuromodulators. Recently, evidence has demonstrated that carbon monoxide gas (CO), produced from the enzyme heme oxygenase (HO), stimulate the formation of 3', 5' - cyclic guanosine monophosphate (cGMP), and this molecule has participated as neuromodulator in some physiological processes. In this way, it has shown that the HO-CO-cGMP pathway is involved in the peripheral and spinal modulation of inflammatory pain, as well as in the modulation of the stress. However, the involvement of this pathway in the modulation of acute painful stimulus, as well as in the antinociception induced by stress isn´t clarified. Thus, this study evaluated the involvement of the HO-CO- cGMP pathway in nociception and in antinociception induced by acute stress in rats, by means the of analgesia index in the tail flick test (AITF). Our results demonstrated that the activation of the HO-CO-cGMP pathway by means of heme-lysinate ICV administration has antinociceptive effect. Again, the increase of the AITF was dependent of the cGMP activity, since that the pretreatment with inhibitor of soluble guanylase cyclase (sGC), ODQ, blocked the antinociceptive effect. This modulation occurs in fasic and not tonic manner, because per se ICV treatment with inhibitor of the HO, ZnDBPG or with inhibitor of the sGC, ODQ did not modify the AITF. The antinociception induced by acute stress (physical restriction during 120 min) is not dependent of the HO-CO-cGMP pathway, since that neither the treatment with the ZnDBPG, nor with the heme-lysinate had modified the AITF. However, this antinociception is dependent of the activity of the cGMP, because the pretreatment with ODQ blocked the increase of the AITF induced by acute stress.