Further characterization of the subunits of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) by SDS-PAGE electrophoresis and MALDI-TOF-MS

Carvalho, Fábio Silva de; Carvalho, José Wilson Pires; Santiago, Patrícia S.; Tabak, Marcel

doi:10.1016/j.procbio.2011.08.013

Cited by 28 publications

(31 citation statements)

References 23 publications

(38 reference statements)

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“…The predictions of the s 0 20,w for the linkers, based on the aminoacid sequences of the known HbLt chains, give values very close to the sedimentation coefficients obtained for the monomeric subunit d [32]. Thus, it is quite possible that the contributions from these polypeptide chains overlap with the contribution of the monomer d and trimer (abc) and, for this reason, the contribution of isolated linker chains is not observed.…”

Section: Auc Datasupporting

confidence: 70%

“…1B, Table 1). Furthermore, taking into account the limitations of the technique and the complexity of the samples, the values of MM for the tetramer (abcd) obtained from our SV data, by global analysis, are quite reasonable, as compared to the MM value of 68 kDa determined by MALDI-TOF-MS [13,32]. In Table 1 the relative percentages for the three species in equilibrium are displayed: they are, respectively, 20 ± 4, 15 ± 2 and 65 ± 7%, for the tetramer, dodecamer and whole protein.…”

Section: Auc Datasupporting

confidence: 58%

“…The fact that, in the distributions curves, the contributions of the linkers chains, L 1 , L 2 and L 3 are not observed, is, probably, due to the overlap of the contributions of these polypeptide chains with other species, being, for this reason, unresolved. In a recent paper, describing the characterization of HbGp species by SDS-PAGE electrophoresis and MALDI-TOF-MS, it is suggested that a linker chain is strongly bound to the trimer (abc) [32]. Besides, AUC measurements of isolated trimeric fractions show that 80% of the solution composition corresponds to the trimer (abc), and the remaining 20% are due to the trimer, associated to a linker chain (abc + L, data not shown, [32]).…”

Section: Auc Datamentioning

confidence: 99%

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Urea-induced unfolding of Glossoscolex paulistus hemoglobin, in oxy- and cyanomet-forms: A dissociation model

Carvalho

Santiago

et al. 2013

International Journal of Biological Macromolecules

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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 1.0 mol/L of urea, display a single peak at 57 S, associated to the undissociated protein. The increase in urea concentration, up to 4.0 mol/L, induces the appearance of smaller species, due to oligomeric dissociation. The sedimentation coefficients and molecular masses are 9.2S and 204 kDa for the dodecamer (abcd)(3), 5.5S and 69 kDa for the tetramer (abcd), 4.1S and 52 kDa for the trimer (abc) and 2.0 S and 17 kDa for the monomer d, respectively. SAXS data show initially a decrease in the I(0) values due to the oligomeric dissociation, and then, above 4.0 mol/L of denaturant, for oxy-HbGp, and above 6.0 mol/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 4.0 mol/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.

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Section: Auc Datasupporting

confidence: 70%

Section: Auc Datasupporting

confidence: 58%

Section: Auc Datamentioning

confidence: 99%

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Urea-induced unfolding of Glossoscolex paulistus hemoglobin, in oxy- and cyanomet-forms: A dissociation model

Carvalho

Santiago

et al. 2013

International Journal of Biological Macromolecules

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“…A dissociação dessa estrutura pode dar origem a doze unidades de massa molecular 1/12 da molécula íntegra, de cerca de 300 kDa (Figs. 2B e 2C, [3,6] 2B). O papel das subunidades linkers está provavelmente relacionado com a manutenção da estrutura íntegra na forma de bicamada hexagonal [7][8][9].…”

Section: Hemoglobina Extracelular De Glossoscolex Paulistus (Hbgp)unclassified

“…Além disso, estudos por ultracentrifugação analítica [20], e cromatografia de exclusão por tamanho [3,6] …”

Section: Fonte-unclassified

Estudo da estabilidade térmica da hemoglobina extracelular gigante de Glossoscolex paulistus (HbGp): efeitos do estado de oxidação do ferro do grupo heme, pH e presença de surfactante

Carvalho¹

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AgradecimentosAgradeço! Aos meus pais Maria Gorete Pires Carvalho e José de Ribamar Gomes Carvalho e a minha esposa Sumária Sousa e Silva pela dedicação, carinho, motivação nas horas mais difíceis, sem eles não teria forças para seguir essa jornada; Ao Professor Marcel Tabak por sua orientação e pelos laços de amizade e respeito que se formaram nesta convivência e também pela oportunidade de poder desenvolver esse trabalho;A Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) pela bolsa de doutorado;Aos meus irmãos Genilson, Genilda e Gesilda pelo apoio, carinho e afeto que temos e pelas palavras amigas;Às minhas tias Eva (segunda mãe), Maria da Guia e Raimunda Teresa e os primos (as) Rita de Kássia, Karla Karolina, Raiza, Rairone, Railson, Amanda e Constância pelo apoio e carinho que sempre me motiva; Aos meus amigos de laboratório Adriano, Fernanda, Ana Eliza, Diógenes e Ezer Biazin, pelo apoio moral e pela contribuição direta e indireta na minha formação e neste trabalho. Obrigado em especial a vocês Adriano e Ezer Biazin; Aos meus amigos Orlando, Ana Célia, Adriane, Washington, Alexandre, Tairo, Adriana, Elenice, Jairo e Edvan pelas discussões bem humoradas e pela amizade; Aos professores Dr.(a) (s) Leandro R. S. Barbosa (IF-USP), Rosângela Itri (IF-USP), Patríca S. Santiago (UNESP-Registro), Carlos Ernesto Garrido Salmon (USP-Riberão Preto) e Tatiana Batista (UEL-PR) pelas discussões e colaborações durante meu curso de doutorado, que foi muito importante para minha formação; Ao Ezér Biazin, técnico do laboratório, por toda ajuda na parte instrumental e que sem nenhuma dúvida foi muito importante para o bom desenvolvimento deste trabalho e pela amizade que se construiu nestes cinco anos; A Andressa Patrícia Alves Pinto, técnica do laboratório de biofísica do IFSC por toda ajuda nos experimentos de CD que foi muito importante para o bom desenvolvimento deste trabalho; Ao Laboratório Nacional de Luz Síncrotron (LNLS), em especial ao Dr. Mateus Cardoso, coordenador da linha SAXS2, pelo auxílio na realização das medidas de SAXS;Ao pessoal da pós-graduação, Sílvia, Andréia e Gustavo, por toda a atenção, carinho e pelos bons serviços prestados;A dona Suely e seu José de Ribamar pelos conselhos e apoio nos momentos difíceis.

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Thermal stability of extracellular hemoglobin of Rhinodrilus alatus (HbRa): DLS and SAXS studies

Carvalho

Santiago

et al. 2016

Eur Biophys J

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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 °C, undergoing denaturation/aggregation in acidic media above 60 °C, followed by partial sedimentation of aggregates. At alkaline pH values 8.0 and 9.0, oxy-HbRa oligomeric dissociation is observed above 30 °C, before denaturation. SAXS data show that oxy-HbRa, at 20 °C, is in its native form, displaying radius of gyration (R g) and particle maximum dimension (D max) of 108 ± 1 and 300 ± 10 Å, respectively. Oxy-HbRa, at pH 7.0, undergoes denaturation/aggregation at 60 °C. At pH 5.0-6.0, HbRa thermal denaturation/aggregation start earlier, at 50 °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.

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Further characterization of the subunits of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) by SDS-PAGE electrophoresis and MALDI-TOF-MS

Cited by 28 publications

References 23 publications

Urea-induced unfolding of Glossoscolex paulistus hemoglobin, in oxy- and cyanomet-forms: A dissociation model

Urea-induced unfolding of Glossoscolex paulistus hemoglobin, in oxy- and cyanomet-forms: A dissociation model

Estudo da estabilidade térmica da hemoglobina extracelular gigante de Glossoscolex paulistus (HbGp): efeitos do estado de oxidação do ferro do grupo heme, pH e presença de surfactante

Thermal stability of extracellular hemoglobin of Rhinodrilus alatus (HbRa): DLS and SAXS studies

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