RESUMO
The presence of hemoglobin A-S (HbAS) in erythrocytes has been related to the high production of reactive oxygen species (ROS) and an increased in intracellular oxidative stress that affects the progress of Plasmodium erythrocytic cycle life and attenuates its serious clinical symptoms. Nevertheless, oxidative effects on P. falciparum proteome across the intraerythrocytic cycle in the presence of HbAS traits have not been described yet. Here, an immune dot-blot assay was used to quantify the carbonyl index (C.I) on P. falciparum 3D7 proteome at the different asexual erythrocytic stages. Protein carbonylation on parasites cultivated in erythrocytes from two donors with HbAS increased 5.34 ± 1.42 folds at the ring stage compared to control grown in hemoglobin A-A (HbAA) red blood cells. Whereas at trophozoites and schizonts stages were augmented 2.80 ± 0.52 and 3.05 ± 0.75 folds, respectively. Besides proteins involved in processes of the stress response, recognition and invasion were identified from schizonts carbonylated bands by combining SDS-PAGE with MALDI-TOF-TOF analysis. Our results reinforce the hypothesis that such oxidative modifications do not appear to happen randomly, and the sickle cell trait affects mainly a small fraction of parasite proteins particularly sensitive to ROS.
Assuntos
Eritrócitos/metabolismo , Plasmodium falciparum/crescimento & desenvolvimento , Proteoma/análise , Traço Falciforme/patologia , Eletroforese em Gel de Poliacrilamida , Eritrócitos/parasitologia , Hemoglobina A/química , Hemoglobina A/metabolismo , Hemoglobina Falciforme/química , Hemoglobina Falciforme/metabolismo , Humanos , Estágios do Ciclo de Vida , Estresse Oxidativo , Plasmodium falciparum/metabolismo , Plasmodium falciparum/patogenicidade , Carbonilação Proteica , Proteoma/metabolismo , Proteínas de Protozoários/análise , Proteínas de Protozoários/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Human hemoglobin (Hb) is a benchmark protein of structural biology that shaped our view of allosterism over 60 years ago, with the introduction of the MWC model based on Perutz structures of the oxy(R) and deoxy(T) states and the more recent Tertiary Two-State model that proposed the existence of individual subunit states -"r" and "t"-, whose structure is yet unknown. Cooperative oxygen binding is essential for Hb function, and despite decades of research there are still open questions related to how tertiary and quaternary changes regulate oxygen affinity. In the present work, we have determined the free energy profiles of oxygen migration and for HisE7 gate opening, with QM/MM calculations of the oxygen binding energy in order to address the influence of tertiary differences in the control of oxygen affinity. Our results show that in the α subunit the low to high affinity transition is achieved by a proximal effect that mostly affects oxygen dissociation and is the driving force of the allosteric transition, while in the ß subunit the affinity change results from a complex interplay of proximal and distal effects, including an increase in the HE7 gate opening, that as shown by free energy profiles promotes oxygen uptake.
Assuntos
Hemoglobina A/química , Hemoglobina A/metabolismo , Oxigênio/metabolismo , Humanos , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de ProteínaRESUMO
Raman spectroscopy has been proposed as a tool for diagnosis of human blood diseases aiming a quick and accurate diagnosis. Sickle cell disease arises in infancy and causes a severe anemia; thus, an early diagnosis may avoid pathological complications such as vasoocclusion, hemolytic anemia, retinopathy, cardiovascular disease, and infections. This work evaluated spectral differences between hemoglobin S (HbS) and hemoglobin A (HbA) to be used in a diagnostic model based on principal components analysis. Blood samples of patients with a previous diagnosis of sickle cell disease were hemolyzed with water, centrifuged, and the pellet was collected with a pipette. Near-infrared Raman spectra (830 nm, 200 mW) were obtained from these samples, and a model based on principal components analysis and Mahalanobis distance were used to discriminate HbA from HbS. Differences were found in the spectra of HbS and HbA, mainly in the 882 and 1,373 cm(-1) (valine, HbA) and 1,547 and 1,622 cm(-1) (glutamic acid, HbS). The spectral model could correctly discriminate 100% of the samples in the correspondent groups. Raman spectroscopy was able to detect the subtle changes in the polypeptide chain (valine and glutamic acid substitution) due to the sickle cell disease and could be used to discriminate blood samples with HbS from HbA with minimum sample preparations (hemolysis with water and centrifugation).
Assuntos
Anemia Falciforme/sangue , Anemia Falciforme/diagnóstico , Hemoglobina A/química , Hemoglobina Falciforme/química , Análise Espectral Raman , Adulto , Interpretação Estatística de Dados , Feminino , Hemólise , Humanos , Masculino , Análise de Componente Principal , Reprodutibilidade dos Testes , Adulto JovemRESUMO
The role of chloride in the stabilization of the deoxy conformation of hemoglobin (Hb), the low oxygen affinity state, has been studied in order to identify the nature of this binding. Previous studies have shown that arginines 141α could be involved in the binding of this ion to the protein. Thus, des-Arg Hb, human hemoglobin modified by removal of the α-chain C-terminal residue Arg141α, is a possible model for studies of these interactions. The loss of Arg141α and all the salt bridges in which it participates is associated with subtle structural perturbations of the α-chains, which include an increase in the conformational flexibility and further shift to the oxy state, increasing oxygen affinity. Thus, this Hb has been the target of many studies of structural and functional behavior along with medical applications. In the present study, we describe the biochemical characterization of des-Arg Hb by electrophoresis, high-performance liquid chromatography and mass spectroscopy. The effects of chloride binding on the oxygen affinity and on the cooperativity to des-Arg Hb and to native human hemoglobin, HbA, were measured and compared. We confirm that des-Arg Hb presents high oxygen affinity and low cooperativity in the presence of bound chloride and show that the binding of chloride to des-Arg does not change its functional characteristics as observed with HbA. These results indicate that Arg141α may be involved in the chloride effect on Hb oxygenation. Moreover, they show that these residues contribute to lower Hb oxygen affinity to a level compatible with its biological function.
Assuntos
Humanos , Masculino , Cloretos/sangue , Hemoglobina A/química , Oxigênio/metabolismo , Cromatografia Líquida de Alta Pressão , Eletroforese em Acetato de Celulose , Hemoglobina A/metabolismo , Espectrometria de Massas , Ligação ProteicaRESUMO
The role of chloride in the stabilization of the deoxy conformation of hemoglobin (Hb), the low oxygen affinity state, has been studied in order to identify the nature of this binding. Previous studies have shown that arginines 141alpha could be involved in the binding of this ion to the protein. Thus, des-Arg Hb, human hemoglobin modified by removal of the alpha-chain C-terminal residue Arg141alpha, is a possible model for studies of these interactions. The loss of Arg141alpha and all the salt bridges in which it participates is associated with subtle structural perturbations of the alpha-chains, which include an increase in the conformational flexibility and further shift to the oxy state, increasing oxygen affinity. Thus, this Hb has been the target of many studies of structural and functional behavior along with medical applications. In the present study, we describe the biochemical characterization of des-Arg Hb by electrophoresis, high-performance liquid chromatography and mass spectroscopy. The effects of chloride binding on the oxygen affinity and on the cooperativity to des-Arg Hb and to native human hemoglobin, HbA, were measured and compared. We confirm that des-Arg Hb presents high oxygen affinity and low cooperativity in the presence of bound chloride and show that the binding of chloride to des-Arg does not change its functional characteristics as observed with HbA. These results indicate that Arg141alpha may be involved in the chloride effect on Hb oxygenation. Moreover, they show that these residues contribute to lower Hb oxygen affinity to a level compatible with its biological function.
Assuntos
Cloretos/sangue , Hemoglobina A/química , Oxigênio/metabolismo , Cromatografia Líquida de Alta Pressão , Eletroforese em Acetato de Celulose , Hemoglobina A/metabolismo , Humanos , Masculino , Espectrometria de Massas , Ligação ProteicaRESUMO
Hemoglobin is known to be a source of peptides involved in several functions. The peptide FLSFPTTKTYFPHFDLSHGSAQVKGHGAK (Hb33-61) is a proteolytic product of the bovine hemoglobin alpha-chain found in the gut content of the cattle tick, Boophilus microplus, and it possesses antimicrobial activity. Since in the past we showed that the amidated form of Hb33-61, Hb33-61a, is active against a few Gram-positive bacteria and fungi strains at micromolar concentration [Fogaca et al. (1999) J. Biol. Chem. 274, 25330-25334], we have been prompted to shed more light on its functional and structural features. Here we show that the peptide is able to disrupt the bacterial membrane ofMicrococcus luteus A270. As for its structure, it has a random conformation in water, and it does not interact with zwitterionic micelles. On the other hand, it binds to negatively charged micelles acquiring a finite structural organization. The 3D structure of Hb33-61a bound to SDS micelles exhibits a nonconventional conformation for an antimicrobial peptide. The backbone is characterized by the presence of a beta-turn in the N-terminus and by a beta-turn followed by a alpha-helical stretch in the C-terminus. A hinge, whose spatial organization is stabilized by side-chain-side-chain interactions, joins these two regions. Interestingly, it preserves structural features present in the corresponding segment of the bovine hemoglobin alpha-chain. Hb33-61a does not possess a well-defined amphipathic nature, and H/D exchange experiments show that while the C-terminal region is embedded in the SDS micelle, one face of the N-terminal half is partly exposed to the solvent.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Hemoglobina A/química , Ixodidae/química , Micelas , Fragmentos de Peptídeos/química , Subunidades Proteicas/química , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/farmacologia , Sítios de Ligação , Bovinos , Dicroísmo Circular/métodos , Medição da Troca de Deutério , Hemoglobina A/isolamento & purificação , Hemoglobina A/farmacologia , Espectroscopia de Ressonância Magnética/métodos , Micrococcus luteus/efeitos dos fármacos , Micrococcus luteus/crescimento & desenvolvimento , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/farmacologia , Estrutura Secundária de Proteína , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/farmacologia , Relação Estrutura-AtividadeRESUMO
Interrelationships between Erythrocyte Protoporphyrin (EP), dietary Iron/Protein ratio (Fe/Prot) and Fe liver content (Feh) were studied during nutritional recovery in an experimental model: weanling female Wistar rats (To) were depleted with a protein-free diet (LP), losing 20% of their initial body weight. Then they were recovered until 45 days of age (T45) with diets containing: casein: 20 g/100 g; Fe (ammonium Fe citrate) (ppm.): 0, 75 or 100 (groups A1, A2 and A3, respectively). Hematocrit, Hemoglobin (Hb) (g/dL). Erythrocyte Protoporphyrin (EP) (microgram/dL Red Blood Cells) and Feh (microgram) were determined at To, LP and T45. Results were compared with control rats (C) fed with 20% of casein and Fe, 50 ppm. EP: a) decreased in C from To to T45 (99 +/- 24; 36 +/- 9; p < 0.01); b) increased in A1 and A2 at T45 (123 +/- 21; 93 +/- 29, respectively, p < 0.01) while A3 did not show significant difference (45 +/- 7) regarding to C: c) correlated inversely with Feh. According to the inverse correlation between EP and Fe/Prot (r = -0.99), we found that 92 ppm was an adequate Fe amount to prevent EP increase. These results confirm that during recovery from undernutrition EP depends on iron liver content, being an adequate indicator of iron nutritional status; therefore, EP would be useful as a predictor of the optimum Fe/Prot ratio for nutritional recovery.
Assuntos
Eritrócitos/química , Ferro da Dieta/sangue , Distúrbios Nutricionais/sangue , Estado Nutricional , Protoporfirinas/sangue , Fatores Etários , Animais , Peso Corporal , Feminino , Hematócrito , Hemoglobina A/química , Avaliação Nutricional , Distúrbios Nutricionais/dietoterapia , Ratos , Ratos WistarRESUMO
Time courses of total (GSH-t), disulfide (GSSG), and mixed disulfide (PSSG) forms of glutathione were studied in chicken blood submitted to oxidative stress induced by diamide or by the reactive oxygen species (ROS)-producing system xanthine/xanthine oxidase (X/XO). Diamide-treated blood induced an immediate increase in GSSG and PSSG, while X/XO produced a slow and sustained stress with increased values of GSSG and PSSG only after 30 and/or 60 min of incubation. Both total protein S-thiolation (mixed disulfide with glutathione) and dethiolation and hemoglobin A S-thiolation and dethiolation were clearly observed. Hemoglobin A (Hb A) was the major S-thiolated protein. We further characterized chicken Hb S-thiolation through the reaction of Hb with GSSG or the GSH/GSSG redox couple. Methemoglobin levels did not change with diamide or with X/XO treatment. Present results suggest that the most reactive cysteine pair of Hb A, the major chicken Hb, might function as an antioxidant under in vivo oxidative stress conditions.