No description available.

Durum wheat (<i>Triticum turgidum</i> L. var. durum, 2n = 4x = 28, AABB genomes) is used predominantly for semolina and pasta products, but there is increasing interest in using durum for bread-making to provide alternative markets during periods of overproduction. The goal of this study was to characterize the bread-making quality of durum wheat cultivars and emmer (<i>Triticum turgidum</i> L. var. dicoccum, 2n = 4x = 28) derived breeding lines derived from crosses of durum wheat with an Emmer land race 97Emmer19 from Iran. Emmer-derived breeding lines were evaluated along with three high quality bread wheat (<i>Triticum aestivum</i> L., 2n = 6x = 42, AABBDD genomes) cultivars and seven durum wheat cultivars across three environments in replicated yield trials in the 2005 and 2006 growing seasons. Four 1AS.1AL-1DL translocation lines which carry the Glu-D1d allele [high molecular weight glutenin subunit (HMW-GS) pair 1Dx5+1Dy10] from chromosome 1D of bread wheat were also evaluated. In general, durum wheat cultivars with elevated gluten strength and/or increased dough extensibility were noted to have higher loaf volume (LV) than those with weaker gluten. The 1AS.1AL-1DL translocation line L252 carrying the LMW-1 banding pattern had better dough mixing stability and LV than the translocation lines with the LMW-2 banding pattern. The 1AS.1AL-1DL translocation lines had higher grain protein concentrations (GPC), but the lowest loaf volumes of all the lines tested. These translocation lines also exhibited unappealing external loaf quality (loaf shape and appearance) and poor internal loaf quality (crumb structure). Variation in bread-making quality attributes were observed among durum genotypes. 97Emmer19 exhibited higher LV than all the durum wheats evaluated and approached the loaf volume achieved with the bread wheat cultivar AC Superb. Breeding lines derived from crosses of 97Emmer19 to strong gluten durum cultivars (WB881 or AC Navigator) had higher LV than those of the durum checks. 97Emmer19 carried Glu-A1a* (HMW-GS 1Ax1) and the progeny carrying that allele generally exhibited higher loaf volumes. Durum wheat genotypes expressing the Glu-B1d (HMW-GS pair Bx6+By8) allele exhibited better overall bread-making quality compared with those expressing the Glu-B1b (HMW-GS pair Bx7+By8) allele. The durum cultivar Arcola and the emmer-derived breeding line 2000EB4, showed higher alveograph extensibility (L) values than did the bread wheat check AC Barrie. The durum wheat genotypes (with the exception of Stewart-63) and emmer-derived breeding lines exhibited better dough extensibility than the USDA-ARS 1AS.1AL-1DL translocation lines. These results indicate that there is potential to select for genotypes with improved baking quality in durum breeding programs.

Durum wheat (<i>Triticum turgidum</i> L. var. durum, 2n = 4x = 28, AABB genomes) is used predominantly for semolina and pasta products, but there is increasing interest in using durum for bread-making to provide alternative markets during periods of overproduction. The goal of this study was to characterize the bread-making quality of durum wheat cultivars and emmer (<i>Triticum turgidum</i> L. var. dicoccum, 2n = 4x = 28) derived breeding lines derived from crosses of durum wheat with an Emmer land race 97Emmer19 from Iran. Emmer-derived breeding lines were evaluated along with three high quality bread wheat (<i>Triticum aestivum</i> L., 2n = 6x = 42, AABBDD genomes) cultivars and seven durum wheat cultivars across three environments in replicated yield trials in the 2005 and 2006 growing seasons. Four 1AS.1AL-1DL translocation lines which carry the Glu-D1d allele [high molecular weight glutenin subunit (HMW-GS) pair 1Dx5+1Dy10] from chromosome 1D of bread wheat were also evaluated. In general, durum wheat cultivars with elevated gluten strength and/or increased dough extensibility were noted to have higher loaf volume (LV) than those with weaker gluten. The 1AS.1AL-1DL translocation line L252 carrying the LMW-1 banding pattern had better dough mixing stability and LV than the translocation lines with the LMW-2 banding pattern. The 1AS.1AL-1DL translocation lines had higher grain protein concentrations (GPC), but the lowest loaf volumes of all the lines tested. These translocation lines also exhibited unappealing external loaf quality (loaf shape and appearance) and poor internal loaf quality (crumb structure). Variation in bread-making quality attributes were observed among durum genotypes. 97Emmer19 exhibited higher LV than all the durum wheats evaluated and approached the loaf volume achieved with the bread wheat cultivar AC Superb. Breeding lines derived from crosses of 97Emmer19 to strong gluten durum cultivars (WB881 or AC Navigator) had higher LV than those of the durum checks. 97Emmer19 carried Glu-A1a* (HMW-GS 1Ax1) and the progeny carrying that allele generally exhibited higher loaf volumes. Durum wheat genotypes expressing the Glu-B1d (HMW-GS pair Bx6+By8) allele exhibited better overall bread-making quality compared with those expressing the Glu-B1b (HMW-GS pair Bx7+By8) allele. The durum cultivar Arcola and the emmer-derived breeding line 2000EB4, showed higher alveograph extensibility (L) values than did the bread wheat check AC Barrie. The durum wheat genotypes (with the exception of Stewart-63) and emmer-derived breeding lines exhibited better dough extensibility than the USDA-ARS 1AS.1AL-1DL translocation lines. These results indicate that there is potential to select for genotypes with improved baking quality in durum breeding programs.

In wheat (Triticum aestivum L), high molecular weight glutenin subunits (HMW -GS) are synthesized by the loci Glu-A1, Glu-B1, and Glu-D1 on the long arm of group 1 chromosome, and their variants play a significant role in the functional properties of flour; hence dough properties and tortilla quality. This study was conducted to understand the effects of HMW-GS on dough properties and tortilla quality using 40 different wheat lines from two different locations; Texas Agrilife Experiment Station at McGregor, and at Castroville, Texas, in 2010. Wheat lines in which one or more of these loci were absent (deletion lines) and non-deletion lines were used. Flours were evaluated for insoluble polymeric protein (IPP) content and mixograph properties. Dough properties; compression force, stress relaxation test, and dough extensibility, were determined using a texture analyzer. Tortillas were produced by hot-pressed method and evaluated for physical properties and textural change during 16 days of storage. Flour from deletion lines had lower average IPP content (38.4%) than non-deletion lines (41.9%). Dough from deletion lines were more extensible (44.8 mm) and required lower equilibrium force from stress relaxation test (4.91 N) compared to non-deletion lines (34.2 mm, and 6.56 N, respectively). Deletion lines produced larger diameter tortillas (177 mm) than non-deletion lines (165 mm) and had lighter color (L* = 82.3) than tortillas from non-deletion lines (L* = 81.0). Most of the deletion lines interestingly produced tortillas with acceptable flexibility scores on day 16 of storage (>= 3.0). Flour IPP content (r = -0.57) and equilibrium force (r = -0.80) were negatively correlated with tortilla diameter, but positively correlated with 16 day flexibility scores (r = 0.72, and r = 0.68, respectively). In general, deletion at Glu-A1 or Glu-D1 or presence of 2+12 instead of 5+10 allelic pair at Glu-D1 locus produced large diameter tortillas, but with poor day 16 flexibility. However, combination of 7+9 at Glu-B1 locus with deletions at Glu-A1 or Glu-D1 or 2+12 at Glu-D1 consistently produced tortillas that had large diameter and retained good flexibility scores during 16 days of storage. The results indicate the presence of 7+9 at Glu-B1 may play a crucial role in selection of wheat varieties for tortilla making.

A Na,K-ATPase é uma proteína encontrada na membrana plasmática de praticamente todas as células animais, que utiliza a energia derivada da hidrólise do ATP para transportar 3 íons Na+ e 2 íons K+. É composta por duas subunidades denominadas e . Um aspecto que ainda gera controvérsias se refere à sua forma de associação nativa e funcional como um protômero ou ainda na forma de oligômeros ()2 ou ()4. Uma forma de estudar essa enzima é pela sua solubilização da membrana, e posteriormente reconstituição em lipossomos de DPPC:DPPE. A caracterização cinética e estrutural desse sistema mostra que a enzima se apresenta na forma oligomérica ()2. O objetivo desse trabalho foi avaliar os mecanismos de dissociação e de desnaturação da Na,K-ATPase solubilizada bem como da reconstituída em lipossomos de DPPC:DPPE, por agentes físicos (temperatura) e químicos (relação proteína:detergente, uso de agentes caotrópicos como a guanidina e mudanças de pH), para interpretar as suas formas de associação e regulação. Para isso, foram realizados experimentos de dicroísmo circular (CD), calorimetria (DSC), infravermelho (FTIR), fluorescência de emissão do triptofano, tensão superficial, elasticidade, atividade catalítica (ATPase e pNPPase). Os estudos de CD em função da variação de temperatura mostraram que ocorre uma transição na curva de elipticidade (222 nm) a 43,7°C para a enzima solubilizada e a 42,0°C para a enzima reconstituída em lipossomos. Estas transições foram também encontradas pela técnica de FTIR. Os experimentos por DSC para a enzima solubilizada revelaram a presença de três picos em 54,7; 64,7 e 67,8°C. Já para a enzima reconstituída observam-se transições em menores temperaturas entre 30 a 40ºC (referentes aos lipídios) e ainda a preservação do pico de transição para proteína em 68,0°C. A análise de fluorescência de triptofano para ambas formas de enzima revelou deslocamentos de pico máximo de emissão a partir de 60°C. Já a presença de guanidina mostrou dois pontos de transição em 3 e 5 mol.L-1 para a Na,K-ATPase solubilizada. O efeito de diferentes meios tamponantes revelou que a enzima apresenta maiores conteúdos em -hélice em pH 7,5, concomitante com um aumento na intensidade de emissão de fluorescência do triptofano na faixa de pH de 5,0 a 8,5. Analisando conjuntamente todas as técnicas podemos propor um mecanismo de dissociação/desnaturação da enzima em função da temperatura. Primeiramente a enzima passa do seu estado oligomérico ()2 e forma protômeros . A atividade ATPase é perdida completamente (acima de 60ºC) quando as subunidades são completamente separadas, ocorrendo então uma agregação das subunidades , através dos domínios citoplasmáticos. Finalmente, a análise da enzima em diferentes proporções de proteína:detergente revela que a Na,K-ATPase, na presença de concentrações abaixo da CMC, se encontra na forma ()2 ou ainda ()4 (dependendo da concentração de proteína). Já para concentrações acima da CMC ocorre a separação das subunidades e consequente perda de atividade catalítica. Devido à dependência da atividade ATPase com seu estado conformacional e seu estado de oligomerização, este estudo realizado por técnicas bioquímicas e biofísicas, resulta em novas informações acerca da compreensão dos mecanismos que controlam o processo de associação, o qual é importante para a função da enzima na membrana natural. / Na,K-ATPase is a protein found in the plasmatic membrane of almost all animal cells and it uses the energy from ATP hydrolysis to transport 3 Na+ ions and 2 K+ ions. It is formed by subunits called and. One controversial aspect refers to its native and functional association form as a protomer or still in ()2 or ()4 oligomers form. One way to study this protein in our laboratory is by its solubilization from membrane, and later reconstitution in liposome from DPPC:DPPE. The kinetic and structural characterization fo this system shows that the enzyme presents itself in the oligomeric form ()2. The aim of this work was to evaluate the dissociation and denaturation mechanisms of the solubilized NA,K-ATPase as well as the one reconstituted in DPPC:DPPE liposome, by physic (temperature) and chemical agents (relation protein:detergent, use of chaotropic agents as Guanidine chloride, or still by the pH changes, to interpret its association and regulation forms. To that end, experiments of circular dichroism (CD), calorimetry (DSC), superficial tension, elasticity , catalytic activity (ATPase an pNPPase) were done. The CD studies in function of temperature variation have shown that a transition occurs in the ellipticity curve (222 nm) at 43.7ºC for the solubilized enzyme and at 42.0ºC for the enzyme reconstituted in liposome. These transitions were also found by the FTIR technique. The experiments by DSC for the solubilized enzyme have shown the presence of three peaks at 54.7ºC, 64.7ºC and 67.8ºC. As for the reconstituted enzyme, transitions in lower temperatures between 30ºC and 40ºC (concerning the lipids) and also the preservation of the transition peak for the protein at 68.0ºC were observed. The Tryptophane fluorescence analysis for both enzyme forms has revealed emission maximum peak shifts starting from 60ºC. The Guaniddine presence has shown two transition points at 3 and 5 mol.L-1 for the solubilized Na,K-ATPase. The effect of different buffer media has shown that the enzyme presents higher contents in -helix at pH 7.5, concomitant with an increase of the intensity of tryptophane fluorescence emission in the pH range of 5.0 to 8.5. Analyzing all the techniques together we can propose a dissociation/denaturation mechanism in function of the temperature. First, the enzyme goes from its oligomeric ()2 state and forms protomers. The ATPase activity é totally lost ( over 60ºC) when the subunits are completely separated, when an subunits aggregation then occurs, through the cytoplasmatic domains. Finally, the analysis of the enzyme in different proportions of protein:detergent reveals that the NA,K-ATPase, in the presence of concentrations bellow CMS, is in the ()2 or yet in the ()4 form (Depending on protein concentration). Now for concentrations above CMS, the separation of the subunits occurs and consequent catalytic activity loss. Due to the ATPase activity dependence on its conformational form and oligomerization state, this study done with biophysical and biochemistry techniques, results in new information on the comprehension of the mechanisms that control the association processes, which is important to the enzyme function in the natural membrane.

A Na,K-ATPase é uma proteína encontrada na membrana plasmática de praticamente todas as células animais, que utiliza a energia derivada da hidrólise do ATP para transportar 3 íons Na+ e 2 íons K+. É composta por duas subunidades denominadas e . Um aspecto que ainda gera controvérsias se refere à sua forma de associação nativa e funcional como um protômero ou ainda na forma de oligômeros ()2 ou ()4. Uma forma de estudar essa enzima é pela sua solubilização da membrana, e posteriormente reconstituição em lipossomos de DPPC:DPPE. A caracterização cinética e estrutural desse sistema mostra que a enzima se apresenta na forma oligomérica ()2. O objetivo desse trabalho foi avaliar os mecanismos de dissociação e de desnaturação da Na,K-ATPase solubilizada bem como da reconstituída em lipossomos de DPPC:DPPE, por agentes físicos (temperatura) e químicos (relação proteína:detergente, uso de agentes caotrópicos como a guanidina e mudanças de pH), para interpretar as suas formas de associação e regulação. Para isso, foram realizados experimentos de dicroísmo circular (CD), calorimetria (DSC), infravermelho (FTIR), fluorescência de emissão do triptofano, tensão superficial, elasticidade, atividade catalítica (ATPase e pNPPase). Os estudos de CD em função da variação de temperatura mostraram que ocorre uma transição na curva de elipticidade (222 nm) a 43,7°C para a enzima solubilizada e a 42,0°C para a enzima reconstituída em lipossomos. Estas transições foram também encontradas pela técnica de FTIR. Os experimentos por DSC para a enzima solubilizada revelaram a presença de três picos em 54,7; 64,7 e 67,8°C. Já para a enzima reconstituída observam-se transições em menores temperaturas entre 30 a 40ºC (referentes aos lipídios) e ainda a preservação do pico de transição para proteína em 68,0°C. A análise de fluorescência de triptofano para ambas formas de enzima revelou deslocamentos de pico máximo de emissão a partir de 60°C. Já a presença de guanidina mostrou dois pontos de transição em 3 e 5 mol.L-1 para a Na,K-ATPase solubilizada. O efeito de diferentes meios tamponantes revelou que a enzima apresenta maiores conteúdos em -hélice em pH 7,5, concomitante com um aumento na intensidade de emissão de fluorescência do triptofano na faixa de pH de 5,0 a 8,5. Analisando conjuntamente todas as técnicas podemos propor um mecanismo de dissociação/desnaturação da enzima em função da temperatura. Primeiramente a enzima passa do seu estado oligomérico ()2 e forma protômeros . A atividade ATPase é perdida completamente (acima de 60ºC) quando as subunidades são completamente separadas, ocorrendo então uma agregação das subunidades , através dos domínios citoplasmáticos. Finalmente, a análise da enzima em diferentes proporções de proteína:detergente revela que a Na,K-ATPase, na presença de concentrações abaixo da CMC, se encontra na forma ()2 ou ainda ()4 (dependendo da concentração de proteína). Já para concentrações acima da CMC ocorre a separação das subunidades e consequente perda de atividade catalítica. Devido à dependência da atividade ATPase com seu estado conformacional e seu estado de oligomerização, este estudo realizado por técnicas bioquímicas e biofísicas, resulta em novas informações acerca da compreensão dos mecanismos que controlam o processo de associação, o qual é importante para a função da enzima na membrana natural. / Na,K-ATPase is a protein found in the plasmatic membrane of almost all animal cells and it uses the energy from ATP hydrolysis to transport 3 Na+ ions and 2 K+ ions. It is formed by subunits called and. One controversial aspect refers to its native and functional association form as a protomer or still in ()2 or ()4 oligomers form. One way to study this protein in our laboratory is by its solubilization from membrane, and later reconstitution in liposome from DPPC:DPPE. The kinetic and structural characterization fo this system shows that the enzyme presents itself in the oligomeric form ()2. The aim of this work was to evaluate the dissociation and denaturation mechanisms of the solubilized NA,K-ATPase as well as the one reconstituted in DPPC:DPPE liposome, by physic (temperature) and chemical agents (relation protein:detergent, use of chaotropic agents as Guanidine chloride, or still by the pH changes, to interpret its association and regulation forms. To that end, experiments of circular dichroism (CD), calorimetry (DSC), superficial tension, elasticity , catalytic activity (ATPase an pNPPase) were done. The CD studies in function of temperature variation have shown that a transition occurs in the ellipticity curve (222 nm) at 43.7ºC for the solubilized enzyme and at 42.0ºC for the enzyme reconstituted in liposome. These transitions were also found by the FTIR technique. The experiments by DSC for the solubilized enzyme have shown the presence of three peaks at 54.7ºC, 64.7ºC and 67.8ºC. As for the reconstituted enzyme, transitions in lower temperatures between 30ºC and 40ºC (concerning the lipids) and also the preservation of the transition peak for the protein at 68.0ºC were observed. The Tryptophane fluorescence analysis for both enzyme forms has revealed emission maximum peak shifts starting from 60ºC. The Guaniddine presence has shown two transition points at 3 and 5 mol.L-1 for the solubilized Na,K-ATPase. The effect of different buffer media has shown that the enzyme presents higher contents in -helix at pH 7.5, concomitant with an increase of the intensity of tryptophane fluorescence emission in the pH range of 5.0 to 8.5. Analyzing all the techniques together we can propose a dissociation/denaturation mechanism in function of the temperature. First, the enzyme goes from its oligomeric ()2 state and forms protomers. The ATPase activity é totally lost ( over 60ºC) when the subunits are completely separated, when an subunits aggregation then occurs, through the cytoplasmatic domains. Finally, the analysis of the enzyme in different proportions of protein:detergent reveals that the NA,K-ATPase, in the presence of concentrations bellow CMS, is in the ()2 or yet in the ()4 form (Depending on protein concentration). Now for concentrations above CMS, the separation of the subunits occurs and consequent catalytic activity loss. Due to the ATPase activity dependence on its conformational form and oligomerization state, this study done with biophysical and biochemistry techniques, results in new information on the comprehension of the mechanisms that control the association processes, which is important to the enzyme function in the natural membrane.

The somatosensory corticothalamic (CT) circuit processes ascending sensory signals, and disruption to the balance of excitation and inhibition (E/I) within CT circuitry leads to absence seizures, sleep disorders, and attention deficits. E/I balance may be restored by independently modulating excitatory CT input to the ventral posteromedial (VPM) thalamus and inhibitory input to the VPM through the CT-thalamic reticular nucleus (nRT)-VPM pathway. This work revealed novel N-methyl-D-aspartate receptor (NMDAR) nucleus-specific and frequency-dependent functional diversity in the somatosensory CT circuit. Specifically, these findings illustrate the different effects of NMDAR negative modulation in the nRT and the VPM, which offers a method to preferentially decrease high frequency excitatory CT input to the VPM while having no significant effect on nRT activity. These results demonstrate the potential of utilizing NMDAR selective modulators to decrease overall excitation within the somatosensory CT circuit. Further investigation is required to elucidate the precise mechanisms underlying this phenomenon, including where NMDARs are localized at CT synapses and the effect of positive NMDAR modulators on nRT and VPM activity. / Master of Science / The sensory gating mechanism helps our brain to select essential sensory information to process. Impairment of this sensory gating has been reported in epilepsy, schizophrenia, and autism. The somatosensory corticothalamic (CT) circuit oversea the sensory gating process by adjusting how much excitation and inhibition signals are integrated into the thalamus. Disruption of the balance of excitation and inhibition (E/I) within CT circuitry leads to the absence seizures, sleep disorders, and attention deficits. Our work revealed one of the glutamate receptors N-methyl-D-aspartate receptor (NMDAR), has nucleus-specific and frequency-dependent functional diversity in the somatosensory CT circuit. By targeting the different NMDAR subunits in the circuit, we were able to preferentially decrease high-frequency excitatory input to the thalamus while having no significant effect on inhibitory input. These results offer the potential to utilize NMDAR selective modulators to decrease overall excitation within the somatosensory CT circuit, which is useful to restore the disrupted E/I balance in the thalamus from a variety of neurological diseases. Further investigation is required to elucidate the precise mechanisms underlying this phenomenon.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 4 uppsatser.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2004. / Vita. Bibliography: References located at the end of each chapter.

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 &rarr; 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 &rarr; 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 &rarr; 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 &rarr; 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.