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Made available in DSpace on 2022-04-29T08:36:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-12-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) In this work we synthesize and characterize AuNPs using two chemical routes: via sodium citrate and via sodium citrate/borohydride. The cytotoxicity of AuNPs was tested by traditional in vitro assays and the in vivo toxicity was evaluated using alternative animals (Galleria mellonella and Caenorhabditis elegans). Avoidance tests with Folsomia candida were carried out to evaluate possible toxic effects in the soil ecosystem. Through Scanning Electron Microscopy and Transmission Electron Microscopy we verified the spherical shape of the AuNPs, with diameters of 34.8 ± 5.5 nm (via sodium citrate) and 7.9 ± 2.2 nm (via sodium citrate/borohydride). The hydrodynamic diameters values were compatible with those obtained microscopically, 30.1 ± 10.1 nm (method A) and 8.3 ± 2.8 nm (method B). Stability of up to 6 months was verified for AuNPs synthesized via sodium citrate, while synthesis via citrate/sodium borohydride was stable for only 1 month. The AuNPs from method A showed better efficacy in the NPs formation with higher absorbance in the plasmonic band at 523 nm, higher concentration and size, spherical shape, homogeneity and stability. The cytotoxicity of the two AuNPs exhibited a slight dose- and size-dependent behavior, with cell viability greater than 80%. The in vivo toxicity in the G. mellonella model showed a survival rate of 100% for 7 days, however, with C. elegans, the percent of survival increased as the concentration of AuNPs decreased. All concentrations of AuNPs, except for the AuNPs synthesized by the citrate pathway (1 mg/kg), showed non-avoidance responses when exposed to contaminated soil. School of Pharmaceutical Sciences Department of Clinical Analysis São Paulo State University (UNESP) Institute of Chemistry Department of Biochemistry & Chemical Technology São Paulo State University (UNESP) School of Pharmaceutical Sciences Department of Bioprocesses and Biotechnology Engineering São Paulo State University (UNESP) School of Chemistry and Molecular Biosciences The University of Queensland Postgraduate Program in Agricultural and Natural Ecosystems (PPGEAN) Federal University of Santa Catarina (UFSC) Area of Exact Sciences and Engineering University of Caxias do Sul (UCS) Institute of Exact Sciences Federal University of Southern and Southeastern Pará (UNIFESSPA) School of Pharmaceutical Sciences Department of Clinical Analysis São Paulo State University (UNESP) Institute of Chemistry Department of Biochemistry & Chemical Technology São Paulo State University (UNESP) School of Pharmaceutical Sciences Department of Bioprocesses and Biotechnology Engineering São Paulo State University (UNESP) FAPESP: 2016/11836-0 FAPESP: 2017/19603-8 CNPq: 470261/2012-9

Made available in DSpace on 2020-12-12T02:19:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-09-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The aim of the present work was the biophysical characterization of the Amynthas gracilis hemoglobin (HbAg). The oxy-HbAg optical absorption data, with Soret and Q bands centered at 415, 540 and 575 nm, were stable and unchanged at pH 7.0. An increase in pH promotes decrease in the intensity in the optical absorption bands, suggesting an oligomeric dissociation and partial oxidation. Identical stability at pH 7.0 was observed in DLS results that presented a hydrodynamic diameter of 28 nm, characteristic of the whole oligomer. DLS shows that HbAg undergoes oligomeric dissociation and an aggregation/denaturation process that corroborates spectroscopic data. Our results showed that the monomer d presents four isoforms with molecular mass (MM) ranging from 16,244 to 16,855 Da; the trimer subunit presents two isoforms, (abc)1 and (abc)2, with MM of 51,415 ± 20 Da and 51,610 ± 14 Da, respectively, and a less intense species, at 67,793 Da, assigned to the tetramer abcd. Monomeric chains a, obtained from reduction of the disulfide-bonded trimer abc, present four isoforms with MM 17,015 Da, 17,061 Da, 17,138 Da and 17,259 Da. DLS and LSI revealed an isoeletric point (pI) of oxy-HbAg of 6.0 ± 0.3 and 5.5, respectively. Data analysis by IEF–SDS–PAGE revealed that the pI of oxy-HbAg is 6.11, correlating with DLS and LSI data. These studies indicate that oxy-HbAg is very stable, at pH 7.0, and has differing properties from orthologous giant hemoglobins. São Paulo State University (Unesp) Campus of Registro Institute of Chemistry São Paulo State University (Unesp) Federal University of South and Southeast Pará (Unifesspa) State University of Mato Grosso São Paulo State University (Unesp) Campus of Registro Institute of Chemistry São Paulo State University (Unesp) FAPESP: 2015/11447-1

Glossoscolex paulistus hemoglobin structure is composed of 144 globin chains and 36 polypeptide chains lacking the heme group, with a total molecular mass of 3600 kDa. The current study focuses on the oxy-HbGp oligomeric stability, as a function of the storage time, at pH 7.0, using dynamic light scattering, analytical ultracentrifugation (AUC), optical absorption and size exclusion chromatography (SEC). HbGp stored in Tris-HCl buffer, pH 7.0, at 4 °C, for two years remains in the native form, while 4–6 years HbGp stocks present typical hemichrome species absorption spectra. AUC and SEC analyses show that the contribution of HbGp-subunits, such as, dodecamer (abcd)3, tetramer abcd, trimer abc and monomer d, increases with the protein aging due to the lower stability of the HbGp with the time. The dissociation and the oxidation of the iron noted for the older protein solutions indicate that HbGp storage for periods of time longer than two years changes its ability to carry oxygen. Despite the reduction of HbGp stability and oxygen carrying capacity with aging, the protein stability is still larger as compared to mammalian hemoglobins. Thus, the extracellular hemoglobins are quite stable and resistant to the auto-oxidation process, making them of interest for biotechnological applications.

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.

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.

Glossoscolex paulistus extracellular hemoglobin (HbGp) stability has been followed, in the presence of urea, using fluorescein isothiocyanate (FITC). Binding of FITC to HbGp results in a significant quenching of probe fluorescence. Tryptophan emission decays present four characteristic lifetimes: two in the sub-nanosecond/picosecond, and two in the nanosecond time ranges. Tryptophan decays for pure HbGp and HbGp-FITC systems are similar. In the absence of denaturant, and up to 2.5mol/L of urea, the shorter lifetimes predominate. At 3.5 and 6.0mol/L of urea, the longer lifetimes increase significantly their contribution. Urea-induced unfolding process is characterized by protein oligomeric dissociation and denaturation of dissociated subunits. FITC emission decays for FITC-HbGp system are also multi-exponential with three lifetimes: two in the sub-nanosecond and one in the nanosecond range with a value similar to free probe in buffer. Increase of urea concentration leads to increase of the longer lifetime contribution, implying the removal of the quenching observed for the native HbGp-FITC system. Anisotropy decays are characterized by two rotational correlation times associated to re-orientational motions of the probe relative to protein. Our results suggest that FITC bound to HbGp is useful to monitor denaturant effects on the protein.

Made available in DSpace on 2018-11-26T17:06:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-09-01 Oxy-HbRa thermal stability was evaluated by dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS) at pH 5.0, 7.0, 8.0, and 9.0. DLS results show that oxy-HbRa, at pH 7.0 and 5.0, remains stable up to 56 degrees C, undergoing denaturation/aggregation in acidic media above 60 degrees C, followed by partial sedimentation of aggregates. At alkaline pH values 8.0 and 9.0, oxy-HbRa oligomeric dissociation is observed above 30 degrees C, before denaturation. SAXS data show that oxy-HbRa, at 20 degrees C, is in its native form, displaying radius of gyration (R-g) and particle maximum dimension (D-max) of 108 +/- 1 and 300 +/- 10 angstrom, respectively. Oxy-HbRa, at pH 7.0, undergoes denaturation/aggregation at 60 degrees C. At pH 5.0-6.0, HbRa thermal denaturation/aggregation start earlier, at 50 degrees C, accompanied by an increase of R-g and D-max values. However, an overlap of oligomeric dissociation and denaturation in the system is observed upon temperature increase, with an increase in R-g and D-max. Analysis of experimental p(r) curves as a linear combination of theoretical curves obtained for HbGp fragments from the crystal structure shows an increasing contribution of dodecamer (abcd)(3) and tetramer (abcd) in solution, as a function of pH values (8.0 and 9.0) and temperature. Finally, our data show, for the first time, that oxy-HbRa, in neutral and acidic media, does not undergo oligomeric dissociation before denaturation, while in alkaline media the oligomeric dissociation process is an important step in the thermal denaturation. Inst Quim Sao Carlos, Sao Carlos, SP, Brazil Univ Estado Mato Grosso, Barra Do Bugres, MT, Brazil Univ Estadual Paulista, Registro, SP, Brazil Univ Estadual Paulista, Registro, SP, Brazil

Glossoscolex paulistus hemoglobin (HbGp) is an oligomeric multisubunit protein with molecular mass of 3600kDa. In the current study, the interaction of sodium dodecyl sulfate (SDS) and cetyl trimethylammonium chloride (CTAC) surfactants with the monomer d and the whole oxy-HbGp, at pH 7.0, was investigated. For pure monomer d solution, SDS promotes the dimerization of subunit d, and the monomeric and dimeric forms have sedimentation coefficient values, s20,w, around 2.1-2.4 S and 2.9-3.2 S, respectively. Analytical ultracentrifugation (AUC) and isothermal titration calorimetry (ITC) data suggest that up to 26 DS- anions are bound to the monomer. In the presence of CTAC, only the monomeric form is observed in solution for subunit d. For the oxy-HbGp, SDS induces the dissociation into smaller subunits, such as, monomer d, trimer abc, and tetramer abcd, and unfolding without promoting the protein aggregation. On the other hand, lower CTAC concentration promotes protein aggregation, mainly of trimer, while higher concentration induces the unfolding of dissociated species. Our study provides strong evidence that surfactant effects upon the HbGp-subunits are different, and depend on the surfactant: protein concentration ratio and the charges of surfactant headgroups.

In this work we investigate the presence of divalent cations bound to the Glossoscolex paulistus (HbGp) hemoglobin and their effect over the protein stability and the peroxidase (POD) activity. Atomic absorption studies show that the HbGp iron content is consistent with the presence of 144 ions per protein. Moreover, using iron as a reference, the content of calcium was estimated as 30±4 ions per protein, independently of the EDTA pre-treatment or not prior to the acidic treatment performed in the protein digestion. The zinc content was 14±2 ions in the absence of EDTA pre-treatment, and 3±1 ions per protein in the presence of EDTA pre-treatment, implying the presence of one zinc ion per protomer (1/12 of the whole molecule). Finally, the copper concentration is negligible. Different from the vertebrate hemoglobins, where the effectors are usually organic anions, the hexagonal bilayer hemoglobins have as effectors inorganic cations that increase the oxygen affinity and stabilize the structure. Previous studies have suggested that the presence of divalent cations, such as copper and zinc, is related to the different types of antioxidant enzymatic activities as the superoxide dismutase (SOD) activity shown by giant hemoglobin from Lumbricus terrestris (HbLt). Recently, studies on HbGp crystal structure have confirmed the presence of Zn(2+) and Ca(2+) binding sites. The Ca(2+) sites are similar as observed in the HbLt crystal structure. Otherwise, the Zn(2+) sites have no relation with those observed in Cu/Zn SODs. Our peroxidase assays with guaiacol confirm the POD activity and the effect of the zinc ions for HbGp. Our present results on HbGp metal content and their stability effects is the first step to understand the role of these cations in HbGp function in the future.

A hemoglobina de Glossoscolex paulistus (HbGp) é caracterizada por uma massa molecular de 3,6 MDa, alta estabilidade oligomérica, resistência a auto-oxidação, e alta afinidade em ligar oxigênio. A estrutura quaternária desta macromolécula apresenta 144 cadeias com grupo heme (globinas) e 36 cadeias sem grupo heme (linkers), dispostos em duas camadas hexagonais. No presente trabalho estudos de caracterização das subunidades da HbGp, bem como da estabilidade da HbGp em diferentes formas, em função do pH, e em diferentes concentrações de ureia, por diferentes técnicas biofísicas, foram realizados. Os estudos de caracterização por eletroforese SDS-PAGE, MALDI-TOF-MS e ultracentrifugação analítica (AUC) das subunidades isoladas mostraram que apenas o monômero d obtido da cromatografia de exclusão por tamanho (SEC) tem alto grau de pureza. Para as demais frações mais de uma contribuição foi observada em solução. Assim, para a fração trimérica, duas espécies estão presentes em solução, a espécie predominante (87 %) é atribuída ao trímero abc e a outra espécie (13 %) pode ser associada ao complexo (abc + L). Os dados espectroscópicos e de AUC mostraram que a estabilidade da HbGp depende fortemente do estado de oxidação do heme, do ligante coordenado no centro metálico e da concentração de proteína. Assim, a forma oxidada, a meta-HbGp, mostrou-se menos estável em meio alcalino e na presença de ureia, seguida pelas formas oxi- e cianometa-HbGp. Desta forma, no pH 8,0, a meta-HbGp está totalmente dissociada em trímero abc e monômero d, enquanto a oxi-HbGp está apenas parcialmente dissociada com uma contribuição de 88 % de proteína íntegra em solução e a cianometa-HbGp não sofre dissociação oligomérica. Os valores de coeficiente de sedimentação s20,w e massa molecular (MM) determinados para as espécies em solução são similares aos observados para as correspondentes espécies isoladas por SEC. Na presença de ureia a mesma tendência foi observada para as três formas da HbGp. Porém, para uma caracterização melhor de processo de desnaturação, os dados espectroscópicos foram analisados usando modelos de dois e três estados para obter informações sobre os parâmetros termodinâmicos do sistema. Assim, bons ajustes foram obtidos usando ambos os modelos, no entanto, o modelo de três estados foi mais adequado para descrever o processo. Por este modelo o processo de desnaturação da HbGp pode ser descrito por duas etapas. A primeira etapa, na faixa de 1,0 - 3,0 mol/L de ureia, está associada a transição do estado nativo para o estado intermediário (N → I), e é caracterizada pela dissociação do oligômero nas diferentes subunidades da HbGp. O estado intermediário apresenta propriedades físico-químicas similares ao estado nativo, sugerindo que o processo de dissociação oligomérica não induz mudanças significativas na estrutura secundária e na região do grupo heme da proteína. Os parâmetros termodinâmicos associados à primeira transição apresentaram erros consideráveis, que podem ser atribuídos à complexidade do estado intermediário com diferentes espécies em solução bem como à semelhança ao estado nativo. A segunda etapa (I → U) com transição bem definida entre 4,5 - 5,0 mol/L de desnaturante é caracterizada pela desnaturação das subunidades dissociadas. Os dados de AUC e SAXS são consistentes com os dados obtidos por espectroscopia, onde a primeira etapa do processo foi caracterizada pela dissociação oligomérica do oligômero em dodecâmero (abcd)3, tetrâmero abcd, trímero abc e monômero d. Para concentrações acima de 4,0 e 5,0 mol/L de ureia, para oxi-HbGp e cianometa-HbGp, respectivamente, aumentos significativos nos valores de I(0), Dmax e Rg sugerem que as subunidades da HbGp estão desnaturadas em solução. As massas moleculares (MM) obtidas por espectrometria de massas e AUC, e os coeficientes de sedimentação s20,w são consistentes com outros resultados reportados para hemoglobinas ortólogas. Além disso, os resultados aqui apresentados representam um avanço importante na caracterização do processo de desnaturação de proteínas oligoméricas complexas. Glossoscolex paulistus hemoglobin (HbGp) is characterized by a molecular mass of 3.6 MDa, a high oligomeric stability, a high resistance to oxidation and a high affinity to oxygen. The quaternary structure of this macromolecule consists of 144 globin chains, and 36 additional chains lacking the heme group, named linkers, organized in a double-layered hexagonal structure. In this current work the characterization of the HbGp subunits and the effect of pH and urea upon the oligomeric stability were studied by several biophysical techniques. Our results obtained by electrophoresis SDS-PAGE, MALDI-TOF-MS and analytical ultracentrifugation (AUC) showed that only the monomer d isolated by size exclusion chromatography (SEC) presented high purity. For the other fractions various species were observed in the solution. Thus, for the trimeric fraction, two species are present in the equilibrium, the main species with percentage contribution of 87 % is assigned to the trimer abc and the species with 13 % in the solution is associated to the complex (abc + L). Additionally, the data obtained by several spectroscopic techniques and AUC show clearly that the oligomeric stability of HbGp depends on the iron oxidation state, the specific ligand coordinated to the iron and the protein concentration. Therefore, our results show that the met-HbGp form is the less stable one in the alkaline medium and in the presence of urea, followed by the oxy- and cyanomet- forms. In this way, at pH 8.0, the met- form is fully dissociated into smaller subunits, such as, trimer abc and monomer d, while the oxy-HbGp is partially dissociated with a significant percentage contribution (88 %) of undissociated protein, and the cyanomet-HbGp does not undergo oligomeric dissociation. The sedimentation coefficients (s20,w) and molecular masses (MM) values for species present in the solution, at different pH, are very close to the values obtained for isolated species. In the presence of urea the same behavior was observed for the three HbGp forms as compared to the alkaline medium. However, for a full characterization of the unfolding process the thermodynamic parameters were obtained by spectroscopic data analysis using models of two and three states. Adequate fits were obtained for both models, but the three states model was very appropriate to describe the HbGp denaturation process. Thus, the denaturation process of HbGp is defined by two phases. The first phase between 1.0 and 3.0 mol/L, of urea is assigned to the transition of native state to an intermediate state (N → I), and is characterized by dissociation of the oligomer in several subunits. The strong similarity of the intermediate state to the native one suggests that oligomeric dissociation induces little changes in the secondary structure and the region of heme group of the protein. As a consequence, the thermodynamic parameters associated to the first transition have large errors due to the complexity of the intermediate state with different species in the solution, as well as its great similarity to the native state. The second phase (I → U), associated with a cooperative transition at 4,5 - 5,0 mol/L of denaturant agent, is attributed to the unfolding of the dissociated subunits. Our AUC and SAXS data are very consistent with spectroscopic data. Thus, in the first phase the oligomeric dissociation of whole protein in dodecamer (abcd)3, tetramer abcd, trimer abc and monomer d was observed. For urea concentrations above 4.0 - 5,0 mol/L, for oxy-HbGp and cyanomet-HbGp, respectively, the significant increase in I(0), Dmax and Rg values suggests that the HbGp subunits are denatured in the solution. The molecular masses values (MM) obtained by mass spectrometry and AUC, and the sedimentation coefficients (s20,w) are consistent with others results reported in the literature for orthologous hemoglobins. In addition, the results of this work correspond to an important advance in the characterization of the denaturation process of this complex oligomeric protein.

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 (HbGp) hemoglobin is an oligomeric protein, displaying a quaternary structure constituted by 144 globin and 36 non-globin chains (named linkers) with a total molecular mass of 3.6MDa. CTAC effects on the oxy-HbGp thermal stability were investigated, by DLS and SAXS, at pH 5.0, 7.0 and 9.0. DLS data show that the oxy-HbGp-CTAC interactions induce a significant decrease of the protein thermal stability, with the formation of larger aggregates, at pH 5.0 and 7.0. In the acidic pH, oxy-HbGp 0.5mg/mL, undergoes a partial oligomeric dissociation, on going from 0.2 to 0.6mmol/L of CTAC, accompanied by a decrease in the Dh values from 27±1 to 22±1nm. It is observed, for the first time, that in the absence and in the presence of CTAC, oxy-HbGp undergoes a partial oligomeric dissociation, with increase of temperature, before denaturation and aggregation at pH values 7.0 and 5.0. SAXS data show that oxy-HbGp undergoes denaturation at 60°C, in the presence of CTAC, pH 5.0. At neutral pH 7.0, the aggregation process starts at 20°C, with increase of Rg and Dmax parameters. At both pH values, 5.0 and 7.0, the denaturation and aggregation are accompanied by the sedimentation of the aggregates. At pH 9.0, oxy-HbGp is totally dissociated at 40°C, in the presence of 0.2mmol/L of CTAC, while in the presence of 0.4mmol/L of surfactant the aggregation process starts at 20°C, with the full denaturation of protein at higher temperature. Finally, our data show, for the first time, that the oligomeric dissociation is an important step in the thermal denaturation of oxy-HbGp, in the presence of CTAC, independently of both the pH and the protein concentration.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:27:28Z No. of bitstreams: 0 Made available in DSpace on 2014-05-27T11:27:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-01-01 The urea effect on the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) stability was studied by analytical ultracentrifugation (AUC) and small angle X-ray scattering (SAXS). AUC data show that the sedimentation coefficient distributions curves c (S), at 1.0mol/L of urea, display a single peak at 57 S, associated to the undissociated protein. The increase in urea concentration, up to 4.0mol/L, induces the appearance of smaller species, due to oligomeric dissociation. The sedimentation coefficients and molecular masses are 9.2S and 204kDa for the dodecamer (abcd)3, 5.5S and 69kDa for the tetramer (abcd), 4.1S and 52kDa for the trimer (abc) and 2.0 S and 17kDa for the monomer d, respectively. SAXS data show initially a decrease in the I(0) values due to the oligomeric dissociation, and then, above 4.0mol/L of denaturant, for oxy-HbGp, and above 6.0mol/L for cyanomet-HbGp, an increase in the maximum dimension and gyration radius is observed, due to the unfolding process. According to AUC and SAXS data the HbGp unfolding is described by two phases: the first one, at low urea concentration, below 4.0mol/L, characterizes the oligomeric dissociation, while the second one, at higher urea concentration, is associated to the unfolding of dissociated species. Our results are complementary to a recent report based on spectroscopic observations. © 2012 Elsevier B.V. Instituto de Química de São Carlos Universidade de São Paulo, SP Universidade Estadual Paulista Julio de Mesquita Filho Registro, São Paulo, SP Universidade Estadual Paulista Julio de Mesquita Filho Registro, São Paulo, SP

Giant extracellular hemoglobins, also known as erythrocruorins, have been investigated as a model of extreme complexity in oxygen-binding heme proteins [1,2]. They are characterized by a very high molecular mass (MM) of several megadalton (MDa), and their oligomeric structure together with the crowded and protected heme environment are two of the main factors responsible for the high redox stability [3,4]. Superoxide dimustase (SOD)-like intrinsic activity, observed for hemoglobins of Lumbricus terrestris (HbLt) and of Arenicola marina (HbAm), is another important factor [5,6]. These hemoglobins present a highly cooperative oxygen binding and a peculiar behavior associated to their oligomeric dissociation into smaller subunits and possible rearrangement back into the native oligomeric structure [7,8].

Made available in DSpace on 2013-09-27T14:53:26Z (GMT). No. of bitstreams: 1 WOS000301028300007.pdf: 449071 bytes, checksum: ee6e3e8698bad8f0d45e90aa250142ff (MD5) Previous issue date: 2012-03-01 Made available in DSpace on 2013-09-30T19:24:40Z (GMT). No. of bitstreams: 1 WOS000301028300007.pdf: 449071 bytes, checksum: ee6e3e8698bad8f0d45e90aa250142ff (MD5) Previous issue date: 2012-03-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T15:34:43Z No. of bitstreams: 1 WOS000301028300007.pdf: 449071 bytes, checksum: ee6e3e8698bad8f0d45e90aa250142ff (MD5) Made available in DSpace on 2014-05-20T15:34:43Z (GMT). No. of bitstreams: 1 WOS000301028300007.pdf: 449071 bytes, checksum: ee6e3e8698bad8f0d45e90aa250142ff (MD5) Previous issue date: 2012-03-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) The stability of the Glossoscolex paulistus hemoglobin (HbGp), in two iron oxidation states (and three forms), as monitored by optical absorption, fluorescence emission and circular dichroism (CD) spectroscopies, in the presence of the chaotropic agent urea, is studied. HbGp oligomeric dissociation, denaturation and iron oxidation are observed. CD data show that the cyanomet-HbGp is more stable than the oxy-form. Oxy- and cyanomet-HbGp show good fits on the basis of a two state model with critical urea concentrations at 220-222 nm of 5.1 +/- 0.2 and 6.1 +/- 0.1 mol/L, respectively. The three-state model was able to reveal a subtle second transition at lower urea concentration (1.0-2.0 mol/L) associated to partial oligomeric dissociation. The intermediate state for oxy- and cyanomet-HbGp is very similar to the native state. For met-HbGp, a different equilibrium, in the presence of urea, is observed. A sharp transition at 1.95 +/- 0.05 mol/L of denaturant is observed, associated to oligomeric dissociation and hemichrome formation. In this case, analysis by a three-state model reveals the great similarity between the intermediate and the unfolded states. Analysis of spectroscopic data, by two-state and three-state models, reveals consistency of obtained thermodynamic parameters for HbGp urea denaturation. (C) 2012 Elsevier B.V. All rights reserved. Univ São Paulo, Inst Quim São Carlos, São Paulo, Brazil Univ Estadual Paulista Julio de Mesquita Filho Re, São Paulo, Brazil Univ Estadual Paulista Julio de Mesquita Filho Re, São Paulo, Brazil FAPESP: 09/17261-6

The giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) has a molecular mass (M) of 3600 ± 100 kDa and a standard sedimentation coefficient ( s 20 , w 0 ) of 58 S, estimated by analytical ultracentrifugation (AUC). In the present work, further AUC studies were developed for HbGp, at pH 10.0, which favors oligomeric dissociation into lower M species. The HbGp oligomer is formed by globin chains a, b, c and d plus the linker chains. The pure monomeric fraction, subunit d, and HbGp at pH 10.0, in the presence of β-mercaptoethanol, were also studied. Our results indicate that for samples of pure subunit d, besides the monomeric species with s 20 , w 0 of 2.0 S, formation of dimer of subunit d is observed with s 20 , w 0 of around 2.9 S. For the whole HbGp at pH 10.0 contributions from monomers, trimers and linkers are observed. No contribution from 58 S species was observed for the sample of oxy-HbGp at pH 10.0, showing its complete dissociation. For cyanomet-HbGp form a contribution of 17% is observed for the un-dissociated oligomer, consistent with data from other techniques that show the cyanomet-form is more stable as compared to oxy-HbGp. Masses of HbGp subunits, especially trimer abc and monomeric chains a, b, c and d, were also estimated from sedimentation equilibrium data, and are in agreement with the results from MALDI-TOF-MS.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:26:06Z No. of bitstreams: 0Bitstream added on 2014-05-27T14:41:55Z : No. of bitstreams: 1 2-s2.0-80053956145.pdf: 943856 bytes, checksum: ddf821677a97459ba29bef1736ec5e18 (MD5) Made available in DSpace on 2014-05-27T11:26:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-11-01 Further characterization of hemoglobin of Glossoscolex paulistus (HbGp) subunits was performed based on SDS-PAGE, size exclusion chromatography (SEC) and MALDI-TOF-MS analysis. SDS-PAGE has shown a total of four linker chains, two quite intense and two of lower intensity. HbGp fractions (I-VI), obtained by size exclusion chromatography (SEC), from oligomeric dissociation at alkaline pH 9.6, were monitored. Fraction I is identical to the whole protein. The monomeric chains c, obtained from the trimer abc reduction, present four isoforms with MM 17,336 Da, 17,414 Da, 17,546 Da and 17,620 Da. Furthermore, the trimer subunit presents two isoforms, T 1 and T 2, with MM 51,200 ± 60 and 51,985 ± 50 Da, respectively. Based on SDS-PAGE, the linker chains seem to be distributed along the different fractions of the SEC chromatogram, appearing along the peaks corresponding to fractions I-V. The fraction IV contains, predominantly, trimers with some linkers contamination. The strong interaction of linker chains L with the trimers abc, makes it difficult to obtain these subunits in pure form. The monomer d in fraction VI appears to be quite pure, in agreement with previous studies. © 2011 Elsevier Ltd. All rights reserved. Instituto de Química de São Carlos Universidade de São Paulo, São Carlos, SP Universidade Estadual Paulista Júlio de Mesquita Filho Campus Experimental de Registro, SP Universidade Estadual Paulista Júlio de Mesquita Filho Campus Experimental de Registro, SP

© 2010 American Chemical Society, The extracellular hemoglobin from Glossoscolex paulistus (HbGp) has a molecular mass of 3.6 MDa. It has a high oligomeric stability at pH 7.0 and low autoxidation rates, as compared to vertebrate hemoglobins. In this work, fluorescence and light scattering experiments were performed with the three oxidation forms of HbGp exposed to acidic pH. Our focus is on the HbGp stability at acidic pH and also on the determination of the isoelectric point (pI) of the protein. Our results show that the protein in the cyanomet form is more stable than in the other two forms, in the whole pH range. Our zeta-potential data are consistent with light scattering results. Average values of pI obtained by different techniques were 5.6±0.5, 5.4±0.2 and 5.2±0.5 for the oxy, met, and cyanomet forms. Dynamic light scattering (DLS) experiments have shown that, at pH 6.0, the aggregation (oligomeric) state of oxy-, met- and cyanomet-HbGp remains the same as that at pH 7.0. The interaction between the oxy-HbGp and ionic surfactants at pH 5.0 and 6.0 was also monitored in the present study. At pH 5.0, below the protein pI, the effects of sodium dodecyl sulfate (SDS) and cetyltrimethylammonium chloride (CTAC) are inverted when compared to pH 7.0. For CTAC, in acid pH 5.0, no precipitation is observed, while for SDS an intense light scattering appears due to a precipitation process. HbGp interacts strongly with the cationic surfactant at pH 7.0 and with the anionic one at pH 5.0. This effect is due to the predominance, in the protein surface, of residues presenting opposite charges to the surfactant headgroups. This information can be relevant for the development of extracellular hemoglobin-based artificial blood substitutes., The authors are grateful to Brazilian agencies FAPESP, CNPq, and CAPES and to the Portuguese FCTMCTES for partial financial support.M.M.D. also acknowledges the Ph.D. fellowship SFRH/BD/41750/2007 from FCT-MCTES.

Diorchidiella verlandii sp. nov. on Mimosa schomburqkii is described as the second species of Diorchidiella. It has spermogonia of type 5 and at least one beaked cell on its bicellulur teliospores. This is the first report of a spermogonial state in Diorchidiella.

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.

Background Color assessment in aesthetic dentistry is one of the most challenging steps for direct restorative treatment. Shade selection tools should be able to mimic closely the materials and layering technique used in the final restoration, hence the development of prefabricated anatomic dual-laminate shade guides. Objective This study aims to compare different shade selection techniques and determine the suitability of a prefabricated anatomic dual-laminate shade guide and its best mode of use compared to a conventional guide and a layered custom guide. Materials and methods CIELab coordinates of different shade guides were assessed: Vitapan Classical (tab A2; Vita); Miris2 prefabricated anatomic dual-laminate shade guide, enamel WR tab on top of dentine S3 tab and nothing in-between (M2air) or glycerin gel (M2gly) or water (M2w); custom shade guide using prefabricated silicon moulds, Miris2 enamel WR composite resin moulded directly on dentine S3 pre-polymerised base (M2cus). The average values were obtained to calculate Δ E and compare the different shade selection techniques. Additional samples and measurements were made to compare Vitapan Classical shade tabs A1, A2 and A3 and all possible combinations of Miris2 and establish the closest matching shade (Δ E ≤ 3.3). Results High Δ E values were found (6.51–9.11) when comparing M2air to Vita, M2gly M2w M2cus. Differences appeared acceptable (Δ E 2.09–2.99) between Vita, M2gly and M2w and M2cus. Seven combinations of M2 were found to match Vita tab A1 and A2 and three Miris2 combinations for Vita A3 (Δ E ≤ 3.3). Conclusions The use of Miris2 prefabricated anatomic dual-laminate shade guide with interposition of water or glycerin between the enamel–dentine tabs demonstrated acceptable Δ E values when compared to Vitapan Classical and custom guides. A chart for matching Vita shades with various combinations of Miris2 enamel/dentine shades was produced to assist the clinician in obtaining acceptable restorations. Clinical significance The prefabricated anatomic dual-laminate shade guide is as efficient as a custom shade guide, facilitating clinical steps and saving material when doing composite resin restorations.

The giant extracellular hemoglobin (HbAg) of the annelid Amynthas gracilis has a molecular mass (MM) of 3400kDa. In the current work, the characterization of MM values of HbAg and its subunits is presented. Electrophoresis, MALDI-TOF-MS and AUC show that the MM values of HbAg subunits are very close, but not identical to those of Glossoscolex paulistus (HbGp) and Rhinodrilus alatus (HbRa) hemoglobins. Analytical ultracentrifugation (AUC) sedimentation velocity experiments were performed to obtain M for HbAg in oxy- form. value of 59.3 ± 0.2 S was obtained for native HbAg. From the ratio between sedimentation and diffusion coefficients values for M of approximately 3400 ± 100 kDa for oxy-HbAg was obtained. MALDI-TOF-MS data gave MM for HbAg subunits. Monomer d is found to exist in, at least, four isoforms with MM 16,244±3Da, 16,459±5Da, 16,667±5Da and 16,855±3Da, as noticed for HbGp, and not observed for HbRa. Furthermore, the trimer subunit presents two isoforms ((abc)1 and (abc)2) with MM 51,415±20Da and 51,610±14Da, respectively. This might indicate that the monomers a, b and c do have isoforms, as found for HbGp and not for HbRa. The monomeric chains a, obtained from the trimer abc reduction, present three isoforms with MM 17,015Da, 17,061Da and 17,138Da, differing from HbGp that presents four isoforms. A less intense species is observed at 67,717, and is due to the tetramer abcd contribution. Finally, AUC and MALDI-TOF-MS data are very close as compared to that obtained for HbGp and HbRa. Our results show total consistency between M obtained by AUC and recent partial characterization by mass spectrometry. Finantial support: FAPESP and CNPq Brazilian agencies.