Fernanda R. Alves

pH effect upon HbGp oligomeric stability: characterization of the dissociated species by AUC and DLS studies

Glossoscolex paulistus (HbGp) extracellular hemoglobin is a giant oligomeric protein. It is constituted by 144 heme containing subunits and non-heme structures (linkers), with a total molecular mass of 3.6MDa. AUC and DLS studies were developed for three HbGp forms, oxy-, met- and cyanomet-, at several pH values, in order to characterize the species in solution upon oligomeric dissociation. Isolated SEC fractions, trimer and dodecamer, are less stable as compared to the whole oxy-HbGp. The monomer d displays a large thermal stability up to 59°C. Hydrodynamic properties of the isolated subunits are very similar to those described for them in the whole protein, in the presence of urea or at pH 10.0. The degree of HbGp oligomeric dissociation, in alkaline pH, depends significantly on the iron oxidation state. Also on the ligand coordinated to the heme iron. Thus, at pH 8.0, the oxy-HbGp is partially dissociated, while the met-form is fully dissociated. The cyanomet-HbGp remains undissociated. Our present results show that the effect of pH on the HbGp oligomeric stability is similar to that associated to the urea-induced unfolding. Simultaneous use of AUC and DLS allowed the characterization of the species in the SEC fractions of isolated HbGp subunits.

Sodium dodecyl sulfate (SDS) effect on the thermal stability of oxy-HbGp: Dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) studies

Glossoscolex paulistus (HbGp) hemoglobin is an oligomeric protein, presenting a quaternary structure constituted by 144 globin and 36 non-globin chains (named linkers) with a total molecular mass of 3.6 MDa. SDS effects on the oxy-HbGp thermal stability were studied, by DLS and SAXS, at pH 5.0, 7.0 and 9.0. DLS and SAXS data show that the SDS-oxy-HbGp interactions induce a significant decrease of the protein thermal stability, with the formation of larger aggregates, at pH 5.0. At pH 7.0, oxy-HbGp undergoes complete oligomeric dissociation, with increase of temperature, in the presence of SDS. Besides, oxy-HbGp 3.0mg/mL, pH 7.0, in the presence of SDS, has the oligomeric dissociation process reduced as compared to 0.5mg/mL of protein. At pH 9.0, oxy-HbGp starts to dissociate at 20 °C, and the protein is totally dissociated at 50 °C. The thermal dissociation kinetic data show that oxy-HbGp oligomeric dissociation at pH 7.0, in the presence of SDS, is strongly dependent on the protein concentration. At 0.5mg/mL of protein, the oligomeric dissociation is complete and fast at 40 and 42 °C, with kinetic constants of (2.1 ± 0.2) × 10(-4) and (5.5 ± 0.4) × 10(-4) s(-1), respectively, at 0.6 mmol/L SDS. However, at 3.0mg/mL, the oligomeric dissociation process starts at 46 °C, and only partial dissociation, accompanied by aggregates formation is observed. Moreover, our data show, for the first time, that, for 3.0mg/mL of protein, the oligomeric dissociation, denaturation and aggregation phenomena occur simultaneously, in the presence of SDS. Our present results on the surfactant-HbGp interactions and the protein thermal unfolding process correspond to a step forward in the understanding of SDS effects.

Denaturant effects on HbGp hemoglobin as monitored by 8-anilino-1-naphtalene-sulfonic acid (ANS) probe.

Glossoscolex paulistus extracellular hemoglobin (HbGp) stability has been monitored in the presence of denaturant agents. 8-Anilino-1-naphtalene-sulfonic acid (ANS) was used, and spectroscopic and hydrodynamic studies were developed. Dodecyltrimethylammonium bromide (DTAB) induces an increase in ANS fluorescence emission intensity, with maximum emission wavelength blue-shifted from 517 to 493 nm. Two transitions are noticed, at 2.50 and 9.50 mmol/L of DTAB, assigned to ANS interaction with pre-micellar aggregates and micelles, respectively. In oxy-HbGp, ANS binds to protein sites less exposed to solvent, as compared to DTAB micelles. In DTAB-HbGp-ANS ternary system, at pH 7.0, protein aggregation, oligomeric dissociation and unfolding were observed, while, at pH 5.0, aggregation is absent. DTAB induced unfolding process displays two transitions, one due to oligomeric dissociation and the second one, probably, to the denaturation of dissociated subunits. Moreover, guanidine hydrochloride and urea concentrations above 1.5 and 4.0 mol/L, respectively, induce the full HbGp denaturation, with reduction of ANS-bound oxy-HbGp hydrophobic patches, as noticed by fluorescence quenching up to 1.0 and 5.0 mol/L of denaturants. Our results show clearly the differences in probe sensitivity to the surfactant, in the presence and absence of protein, and new insights into the denaturant effects on HbGp unfolding.

Guanidine hydrochloride and urea effects upon thermal stability of Glossoscolex paulistus hemoglobin (HbGp).

Glossoscolex paulistus hemoglobin (HbGp) has a molecular mass of 3600kDa. It belongs to the hexagonal bilayer hemoglobin class, which consists of highly cooperative respiratory macromolecules found in mollusks and annelids. The present work focusses on oxy-HbGp thermal stability, in the presence of urea and guanidine hydrochloride (GuHCl), monitored by several techniques. Initially, dynamic light scattering data show that the presence of GuHCl induces the protein oligomeric dissociation, followed by a significant 11-fold increase in the hydrodynamic diameter (DH) values, due to the formation of protein aggregates in solution. In contrast, urea promotes the HbGp oligomeric dissociation, followed by unfolding process at high temperatures, without aggregation. Circular dichroism data show that unfolding critical temperature (Tc) of oxy-HbGp decreases from 57°C, at 0.0 mol/L of the denaturant, to 45°C, in the presence of 3.5 mol/L of urea, suggesting the reduction of HbGp oligomeric stability. Moreover, differential scanning calorimetry results show that at lower GuHCl concentrations, some thermal stabilization of the hemoglobin is observed, whereas at higher concentrations, the reduction of stability takes place. Besides, HbGp is more stable in the presence of urea when compared with the guanidine effect, as deduced from the differences in the concentration range of denaturants.

Glossoscolex paulistus Extracellular Hemoglobin (HbGp) Oligomeric Dissociation upon Interaction with Sodium Dodecyl Sulfate: Isothermal Titration Calorimetry (ITC)

Annelid erythrocruorins are respiratory proteins with high cooperativity and low autoxidation rates. The giant extracellular hemoglobin of the earthworm, Glossoscolex paulistus (HbGp), has a molecular mass of 3.6 MDa. In this work, isothermal titration calorimetry (ITC), together with DLS and fluorescence emission have been used to investigate the interaction of SDS with the HbGp in the oxy‐form, at pH 7.0. Our ITC and DLS results show that addition of SDS induces oxy‐HbGp oligomeric dissociation, while a small amount of protein aggregation is observed only by DLS. Moreover, the oligomeric dissociation process is favored at lower protein concentrations. The temperature effect does not influence significantly the interaction of SDS with the hemoglobin, due to the similarities presented by the critical aggregation concentration (cac) and critical micelle concentration (cmc′) for the mixtures. The increase of oxy‐HbGp concentration leads to a slight variation of the cac values for the SDS‐oxy‐HbGp mixture, attributed mainly to the noncooperative electrostatic binding of surfactant to protein. However, the cmc′ values increase considerably, associated to a more cooperative hydrophobic binding. Complementary pyrene fluorescence emission studies show formation of pre‐micellar structures of the mixture already at lower SDS concentrations. This study opens the possibility of the evaluation of the surfactant effect on the hemoglobin stability by ITC, which is made for the first time with this extracellular hemoglobin.

Population size assessment of the Endangered red-billed curassow Crax blumenbachii : accounting for variation in detectability and sex-biased estimates

This study was funded by Conservation des Especes et des Population Animales, the Mohamed bin Zayed Species Conservation Fund, Idea Wild and Vale Nature Reserve. FA and LFS were supported by Sao Paulo Research Foundation (FAPESP) and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq).

Habitat selection by the endangered Red-billed Curassow (Crax blumenbachii) in an Atlantic forest remnant

ABSTRACT Understanding habitat selection is important for informing conservation management actions. However, many endangered species are data deficient, especially in tropical forests. Wild populations of the endangered Red-billed Curassow are one such data-deficient species. We investigated habitat selection by Red-billed Curassows in an important Atlantic forest remnant in Espírito Santo state, Brazil. We sampled vegetation plots to test fine-scale habitat associations and used GIS tools to identify landscape-scale variables that may influence curassow habitat use. We modelled the occurrence of Red-billed Curassows to test the contribution of these variables using hierarchical partitioning analysis in R. Abundance of standing dead trees, decaying log and leaf litter depth had a negative effect on the occurrence of Red-billed Curassows. The species preferred tall forests and abundant trees with diameter at breast height of 11–30 cm. Our results indicated that the Red-billed Curassow can utilise some secondary forest habitats, and suggest a preference for more open forest habitats that may facilitate terrestrial foraging. This is the first scientific examination of habitat requirements of Red-billed Curassows and our results will aid conservation activities by improving site selection for reintroduction efforts.

Interaction of cationic dodecyl‐trimethyl‐ammonium bromide with oxy‐HbGp by isothermal titration and differential scanning calorimetric studies: Effect of proximity of isoelectric point

In this work, isothermal titration and differential scanning calorimetric methods, in combination with pyrene fluorescence emission and dynamic light scattering have been used to investigate the interaction of dodecyltrimethylammonium bromide (DTAB) with the giant extracellular Glossoscolex paulistus hemoglobin (HbGp) in the oxy‐form, at pH values around the isoelectric point (pI ≈ 5.5). Our ITC results have shown that the interaction of DTAB with the hemoglobin is more intense at pH 7.0, with a smaller cac (critical aggregation concentration) value. The increase of protein concentration does not influence the cac value of the interaction, at both pH values. Therefore, the beginning of the DTAB‐oxy‐HbGp premicellar aggregates formation, in the cac region, is not affected by the increase of protein concentration. HSDSC studies show higher Tm values at pH 5.0, in the absence and presence of DTAB, when compared with pH 7.0. Furthermore, at pH 7.0, an aggregation process is observed with DTAB in the range from 0.75 to 1.5 mmol/L, noticed by the exothermic peak, and similar to that observed for pure oxy‐HbGp, at pH 5.0, and in the presence of DTAB. DLS melting curves show a decrease on the hemoglobin thermal stability for the oxy‐HbGp‐DTAB mixtures and formation of larger aggregates, at pH 7.0. Our present data, together with previous results, support the observation that the protein structural changes, at pH 7.0, occur at smaller DTAB concentrations, as compared with pH 5.0, due to the acidic pI of protein that favors the oxy‐HbGp‐cationic surfactant interaction at neutral pH.

Evaluation of methodological protocols using point counts and mist nets: a case study in southeastern Brazil

Embora muito utilizado com a finalidade de estimar a abundância de especies de aves, pontos de escuta e redes de neblina seguem protocolos desenvolvidos em regioes temperadas, com pouca atencao para modificacoes para sistemas tropicais. Para averiguar por quanto tempo e necessaria amostragem por pontos de escuta para o registro da maior parte da avifauna (ao menos 90% de todas as especies e individuos), assim como para determinar se as capturas com redes de neblina em intervalos de 1 h detectam igualmente numeros de especies e individuos, ambas as metodologia foram utilizadas a cada tres meses entre dezembro de 2009 e janeiro de 2011 em dois fragmentos florestais do sudeste do Brasil. Quatro pontos de escuta de 20 min conduzidos durante cinco dias consecutivos acumularam 90% da riqueza estimada apos 20 h (60 pontos de 20 minutos) em uma mata ombrofila densa (MC), enquanto 17 h (51 pontos de 20 minutos) foram insuficientes para o registro da mesma porcentagem de especies em uma mata semidecidual (IT). Os primeiros 5 min dos pontos de escuta detectaram significativamente mais especies em MC (63% do total de especies) e em IT (65%) em comparacao com os minutos restantes, mas foram necessarios 15 min para o registro de 86% do total de contatos em ambas as florestas. Cinco dias consecutivos (~ 9 h/dia) com redes de neblina abertas resultaram em 70,5 horas-rede/m2 (MC) e 74,8 horas-rede/m2 (IT) de esforco amostral, de modo que 80 a 85% do numero estimado de especies foram capturados. Embora curvas de acumulacao nao tenham apresentado tendencia a estabilizacao do numero de especies observado, o numero de especies estimado demonstrou assintota a partir das primeiras 20 h em ambas as florestas. Nao houve diferenca significativa na captura de especies ou individuos entre horarios de revisoes a cada hora, mas notou-se uma tendencia na qual tais parâmetros mostraram-se mais elevados entre as segundas e quartas revisoes do dia. Redes de neblina abertas durante tres dias (43,8 e 63,3 horas-rede/m2 em MC e IT, respectivamente) foram suficientes para o registro de 90% das especies capturadas. Essa diminuicao do esforco amostral nao prejudicou a estimativa do numero de especies, ao passo que o numero de individuos capturados diminuiu em 34% em MC e 38% em IT. As revisoes ate as 1100 h capturaram 90% de todas as especies registradas com redes de neblina em ambos os fragmentos, porem 67% de todos os individuos foram capturados ate este horario. Nossos resultados demonstram que o inventario e a estimativa de abundância da avifauna nessas localidades requerem delineamentos unicos, os quais devem ser testados antes do inicio da coleta de dados com pontos de escuta ou redes de neblina.

Interaction of Glossoscolex paulistus extracellular hemoglobin with hydrogen peroxide: Formation and decay of ferryl-HbGp

The giant extracellular hemoglobin from earthworm Glossoscolex paulistus (HbGp) reacts with hydrogen peroxide, displaying peroxidase activity in the presence of guaiacol. The formation of ferryl-HbGp (compound II) from the peroxidase cycle was studied in the present work. The hypervalent ferryl-HbGp species was formed directly by the reaction of oxy-HbGp and hydrogen peroxide. The oxy-HbGp heme groups (144) under different excess of H2O2, relative to heme, showed an influence in the total amount of ferryl-HbGp at the end of the reaction. The ferryl-HbGp was formed with second order rate constant of 27.1±0.5M-1s-1, at pH7.0 and 25°C. The increase of the pH value to 8.0 induces both faster formation and decay of ferryl-HbGp, together with oligomeric dissociation induced by the presence of H2O2, as observed by DLS. This effect of dissociation increases the heme exposure and decreases the ferryl-HbGp stability, affecting the rate constant as a parallel reaction. At pH7.0, high excess of H2O2, above 1:5 oxy-HbGp heme: H2O2, produces the aggregation of the protein. Our results show for the first time, for an extracellular giant hemoglobin, the possible effects of oxidative stress induced by hydrogen peroxide.