Oxidized dimeric Scapharca inaequivalvis. Co-driven perturbation of the redox equilibrium.

The dimeric hemoglobin isolated from Scapharca inaequivalvis, HbI, is notable for its highly cooperative oxygen binding and for the unusual proximity of its heme groups. We now report that the oxidized protein, an equilibrium mixture of a dimeric high spin aquomet form and a monomeric low spin hemichrome, binds ferrocyanide tightly which allows for internal electron transfer with the heme iron. Surprisingly, when ferricyanide-oxidized HbI is exposed to CO, its spectrum shifts to that of the ferrous CO derivative. Gasometric removal of CO leads to the oxidized species rather than to ferrous deoxy-HbI. At equilibrium, CO binds with an apparent affinity (p50) of about 10-25 mm of Hg and no cooperativity (20 degrees C, 10-50 mM buffers at pH 6.1). The kinetics of CO binding under pseudo-first order conditions are biphasic (t1/2 of 15-50 s at pH 6.1). The rates depend on protein, but not on CO concentration. The nitrite-oxidized protein is not reduced readily in the presence of CO unless one equivalent of ferrocyanide, but not of ferricyanide, is added. We infer that ferrocyanide, produced in the oxidation reaction, is tightly bound to the protein forming a redox couple with the heme iron. CO shifts the redox equilibrium by acting as a trap for the reduced heme. The equilibrium and kinetic aspects of the process have been accounted for in a reaction scheme where the internal electron transfer reaction is the rate-limiting step

Effect of heme and non-heme ligands on subunit dissociation of normal and carboxypeptidase-digested hemoglobin. Gel filtration and flash photolysis studies.

The dissociation of normal and carboxypeptidase-digested human hemoglobin has been studied by gel filtration under several experimental conditions. These include (a) different derivatives, notably deoxy-, oxy-, and CO-hemoglobin, (b) changes in solvent composition and in pH, and (c) addition of inositol hexaphosphate. In normal hemoglobin, in agreement with previous results, the deoxygenated derivative is much less dissociated than the oxy or CO ones. This difference is observed also in some of the digested hemoglobins, but tends to vanish in those proteins in which, as a result of extensive digestion, the conformational change accompanying ligand binding is abolished. The dissociation of normal and digested hemoglobins is affected by solvent composition, is at a minimum at pH near 8, and is decreased by the addition of inositol hexaphosphate. Parallel flash photolysis experiments, performed under conditions identical with those used in the gel filtration studies, indicate that the appearance of quickly reacting material parallels dissociation into dimers in normal hemoglobin. Both in normal and digested hemoglobins conditions which decrease dissociation decrease the fraction of rapidly reacting material. In the digested hemoglobins the fraction of rapidly reacting material may be much higher than can be accounted for by the amount of dimers, indicating in these cases that the tetramers may be rapidly reacting. The data point once again to the critical role of the COOH-terminal residues in maintaining the subunit structure of hemoglobin and the interaction effects associated with it

Conteúdo citocrômico mitocondrial e atividade da oxidase citocrômica em alguns peixes amazônicos

Resumo O conteúdo em citocromo de partículas Keilin-Hartree isoladas de peixes respiradores de água e respiradores de ar facultativos é anotado o comparado com dados para mitocondrias de mamíferos. Medições cinéticas de "steady state" foram realizadas sobre o sistema de oxidase. Os resultados podem ser interpretados em termos de dois locais de ligação do citocromo C cineticamente significativos associados com o citocromo-oxidase com valores Km de ~1x10-6 e ~5x10-8 M. Estes valores são semelhantes aos encontrados em mitocondrias de boi (Ferguson-Miller et al, 1976), Os valores de "turnover" para os citocromo-oxidases de vários peixes são comparados, encontrando-se que são similares e próximos aos encontrados para a enzima de mamíferos. A constância do conteúdo de citocromo e os parâmetros cinéticos entre espécies sugerem que os processos respiratórios constituem um sistema conservador desde o ponto de vista evolutivo

O efeito Root na hemoglobina do jaraqui (Prochilodus sp.) um peixe teleósteo

Resumo O sistema de hemoglobina do jaraquí, Prochilodus sp. consiste em componentes múltiplos de hemoglobina. O derivado oxidado elui como uma espécie de único peso molecular em experimentos de filtração. O derivado carboxi tem um peso molecular aparente de 60.000 determinado por filtração de gel. Uma mudança de 44 vezes no p1/2 ocorre entre pH 6,4 e 8,6 na solução da hemoglobina. Esta mudança aumenta 388 vezes sob a mesma faixa de pH na presença de 1mM de ATP. A cooperatividade da união do oxigênio, medida por n na equação de Hill, é maior que um em pH acima de 6,7, porém menor que um quando abaixo deste valor. O hemolisado apresentou efeito "Root", sendo apenas 44% saturado a pH 6,4 em presença de 1mM de ATP e equilibrado com ar a uma atmosfera. Em 30°C e pH 7,6 o sangue total processa-se abaixo de p1/2, 4,7 mm de Hg, menor que o de maioria dos outros teleósteos amazônicos. Ambas as velocidades de combinação do monóxido de carbono e velocidade de dissociação do oxigênio são dependentes do pH e ATP. Entre pH 6,2 e 8,8 a velocidade do COon aumenta 10 vezes. ATP reduz a velocidade em valores de pH Intermediários. A velocidade de Ooff aumenta 2,4 vezes entre pH 8,8 e 6,7. A adição de 1 mM de ATP produz um aumento de 4,5 vezes sobre a mesma faixa de pH. Os baixos valores de n abaixo de pH 6,7 e a heterogeneidade da combinação de CO e o processo de dissociação da combinação de O2 sugerem que os componentes da hemoglobina podem ser funcionalmente diferenciados e/ou existem diferenças intramolecular nas propriedades cinéticas das cadelas oc e β

Molecular basis of a novel adaptation to hypoxic-hypercapnia in a strictly fossorial mole

<p>Abstract</p> <p>Background</p> <p>Elevated blood O₂affinity enhances survival at low O₂pressures, and is perhaps the best known and most broadly accepted evolutionary adjustment of terrestrial vertebrates to environmental hypoxia. This phenotype arises by increasing the intrinsic O₂affinity of the hemoglobin (Hb) molecule, by decreasing the intracellular concentration of allosteric effectors (e.g., 2,3-diphosphoglycerate; DPG), or by suppressing the sensitivity of Hb to these physiological cofactors.</p> <p>Results</p> <p>Here we report that strictly fossorial eastern moles (<it>Scalopus aquaticus</it>) have evolved a low O₂affinity, DPG-insensitive Hb - contrary to expectations for a mammalian species that is adapted to the chronic hypoxia and hypercapnia of subterranean burrow systems. Molecular modelling indicates that this functional shift is principally attributable to a single charge altering amino acid substitution in the β-type δ-globin chain (δ136Gly→Glu) of this species that perturbs electrostatic interactions between the dimer subunits via formation of an intra-chain salt-bridge with δ82Lys. However, this replacement also abolishes key binding sites for the red blood cell effectors Cl^-, lactate and DPG (the latter of which is virtually absent from the red cells of this species) at δ82Lys, thereby markedly reducing competition for carbamate formation (CO₂binding) at the δ-chain N-termini.</p> <p>Conclusions</p> <p>We propose this Hb phenotype illustrates a novel mechanism for adaptively elevating the CO₂carrying capacity of eastern mole blood during burst tunnelling activities associated with subterranean habitation.</p

Molecular basis of a novel adaptation to hypoxic-hypercapnia in a strictly fossorial mole

Background: Elevated blood O2 affinity enhances survival at low O2 pressures, and is perhaps the best known and most broadly accepted evolutionary adjustment of terrestrial vertebrates to environmental hypoxia. This phenotype arises by increasing the intrinsic O2 affinity of the hemoglobin (Hb) molecule, by decreasing the intracellular concentration of allosteric effectors (e.g., 2,3-diphosphoglycerate; DPG), or by suppressing the sensitivity of Hb to these physiological cofactors. Results: Here we report that strictly fossorial eastern moles (Scalopus aquaticus) have evolved a low O2 affinity, DPG-insensitive Hb - contrary to expectations for a mammalian species that is adapted to the chronic hypoxia and hypercapnia of subterranean burrow systems. Molecular modelling indicates that this functional shift is principally attributable to a single charge altering amino acid substitution in the β-type δ-globin chain (δ136Gly→Glu) of this species that perturbs electrostatic interactions between the dimer subunits via formation of an intra-chain salt-bridge with δ82Lys. However, this replacement also abolishes key binding sites for the red blood cell effectors Cl-, lactate and DPG (the latter of which is virtually absent from the red cells of this species) at δ82Lys, thereby markedly reducing competition for carbamate formation (CO2 binding) at the δ-chain N-termini. Conclusions: We propose this Hb phenotype illustrates a novel mechanism for adaptively elevating the CO2 carrying capacity of eastern mole blood during burst tunnelling activities associated with subterranean habitation

Association between first-year virological response to raltegravir and long-term outcomes in treatment-experienced patients with HIV-1 infection

Background: We explored the relationship between virological response in the first year of treatment and long-term outcomes in the BENCHMRK studies. Methods: Patients failing antiretroviral treatment with 3-class resistant HIV-1 received double-blinded raltegravir (or placebo) with optimized background therapy (OBT) until week 156, followed by open-label raltegravir with OBT up to week 240. In this exploratory analysis of patients randomized to raltegravir, virological response over weeks 16-48 was categorized as continuous suppression (CS; viral RNA [vRNA] always 50 copies/ml at least once), or not suppressed (NS; vRNA >400 copies/ml at least once). The association between these first-year vRNA response categories and baseline factors was analysed with univariate and multivariate models. Virological and immunological outcomes for years 2-5 were assessed by first-year vRNA response category (observed failure approach). Results: Baseline vRNA, baseline CD4(+) T-cell count and rapid viral decay (vRNA <50 copies/ml between weeks 2-12) correlated with first-year vRNA response (P<0.001); only rapid viral decay remained significant by multiple regression. Virological response rates were similar in the LLV and CS groups and lowest in the NS group. CD4(+) T-cell count increased through week 240 in the CS and LLV groups. Time to loss of virological response (confirmed vRNA ≥400 copies/ml) through week 240 did not support as strong a difference between the LLV and CS groups (log-rank P=0.11) as previously reported through weeks 156 and 192 (P<0.05). Conclusions: Treatment-experienced patients on a raltegravir-based regimen with early LLV may have long-term virological and immunological benefit when their therapy is maintained

Separação e caracterização dos componentes de hemoglobina de Pterygoplichthys pardalis, o acaribodó

The four main hemoglobin components of the hemolysate of Pterygoplichthys pardalis have been isolated and characterized. The functional properties investigated for the isolated components comprise the effect of pH and ATP on (i) the O2 equilibrium, (ii) the O2 dissociation kinetics, (iii) the CO combination kinetics. Component I, corresponding to approximately 50% of the total hemoglobin, is characterized by functional properties which are distinctly different from those of Components II, III and IV, which are alike. Thus it is shown, once more, that multiple components in an hemolysate fall into categories of hemoglobins characterized by distinct and complementary functional properties.Os quatro componentes de hemoglobina principais do hemolisado de Pterygoplichthys pardalis foram isolados e caracterizados. As propriedades funcionais investigadas para os componentes isolados compreendem o efeito de pH e ATP sobre(i) o equilíbrio de O2, (ii) a cinética da dissociação de O2 (iii) a cinética da combinação de CO. O componente I, corresponde a aproximadamente 50% da hemoglobina total, é caracterizado por propriedades funcionais que são diferentes das dos componentes II, III e IV, os quais são semelhantes. Assim, é provado, uma vez mais, que componentes múltiplos num hemolisado caem na categoria de hemoglobinas caracterizadas por propriedades funcionais distintas e complementares

Isolamento e caracterização dos componentes da hemoglobina de Mylossoma sp., um teleósteo da Amazônia

Resumo Foram isoladas duas hemoglobinas de um peixe caracídeo, Mylossoma sp. O componente eletroforético de maior migração anódica constitui 89% do hemolisado total. As duas hemoglobinas nativas têm peso molecular aparente de 57.000 por cromatografia em gel. Os pesos moleculares aparentes das subunidades desnaturadas são 14.000 por eletroforese em gel de dodecil sulfato de sódio. Não ocorre polimerização depois da oxidação com ferrocianeto de potássio. O estudo da união de oxigênio indica que o componente mais "anódico" da hemoglobina possui um efeito Root. Em pH 5,9 na presença de 1 mM ATP a hemoglobina fica saturada apenas 45% quando equilibrada com ar a 1 atmosfera. O componente mais anódico possui um efeito Bohr normal que é aumentado em presença de 1 mM ATP. A cooperatividade, determinada por n na equação de Hill, varia com o pH. No pH 6,7 e abaixo deste na presença de 1 mM ATP, n < 1. A presença de 1 mM ATP causa uma redução em n em pH abaixo de 8,2. O comportamento menos anódico evidencia um comportamento muito diferente tendo um efeito Bohr reverso Δ log P1/2/Δ pH=0,14, entre pH 7,0 e 8,0 o qual muda para um efeito Bohr normal, Δ log P1/2/Δ pH= —0,13 com adição de 1 mM ATF. Esta hemoglobina mostra cooperatividade em todos os valores de pH estudados. Não mostra efeito Root. Estudos de cinética rápida da ligação CO e da dissociação do O2dos componentes isola dos da hemoglobina mostraram que ambos processos são dependentes do pH para cada componente. Estes resultados são consistentes com as análises dos dados de equilíbrio do oxigênio. As hemoglobinas de Mylossoma sp. se assemelham às de Hoplosternum, truta, salmão, remora e cadozete, no grau de sua diferenciação funcional e podem representar especializações evolutivas designadas para servir funções fisiológicas diversas

The Chandra Source Catalog

The Chandra Source Catalog (CSC) is a general purpose virtual X-ray astrophysics facility that provides access to a carefully selected set of generally useful quantities for individual X-ray sources, and is designed to satisfy the needs of a broad-based group of scientists, including those who may be less familiar with astronomical data analysis in the X-ray regime. The first release of the CSC includes information about 94,676 distinct X-ray sources detected in a subset of public ACIS imaging observations from roughly the first eight years of the Chandra mission. This release of the catalog includes point and compact sources with observed spatial extents <~ 30''. The catalog (1) provides access to the best estimates of the X-ray source properties for detected sources, with good scientific fidelity, and directly supports scientific analysis using the individual source data; (2) facilitates analysis of a wide range of statistical properties for classes of X-ray sources; and (3) provides efficient access to calibrated observational data and ancillary data products for individual X-ray sources, so that users can perform detailed further analysis using existing tools. The catalog includes real X-ray sources detected with flux estimates that are at least 3 times their estimated 1 sigma uncertainties in at least one energy band, while maintaining the number of spurious sources at a level of <~ 1 false source per field for a 100 ks observation. For each detected source, the CSC provides commonly tabulated quantities, including source position, extent, multi-band fluxes, hardness ratios, and variability statistics, derived from the observations in which the source is detected. In addition to these traditional catalog elements, for each X-ray source the CSC includes an extensive set of file-based data products that can be manipulated interactively.Comment: To appear in The Astrophysical Journal Supplement Series, 53 pages, 27 figure