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Caracterização estrutural de sistemas biológicos de diferentes classes: um estudo pela técnica de SAXS / Structural characterization of biological systems from different classes: A study by the SAXS techniqueOseliero Filho, Pedro Leonidas 28 November 2018 (has links)
Esta tese apresenta resultados da caracterização estrutural de três sistemas de classes diferentes por meio, principalmente, da modelagem de dados de espalhamento de raios X a baixos ângulos (SAXS). No sistema surfactante-surfactante as micelas mistas são formadas por dodecil sulfato de sódio (SDS) e um dos surfactantes da série Tween (Tween 20, 40, 60 e 80). A modelagem adotada impôs vínculos moleculares uma vez que os dados de SAXS estavam em escala absoluta. Isso reduziu a ambiguidade nos valores dos parâmetros ajustáveis e permitiu verificar que os dados de SAXS são satisfatoriamente descritos considerando-se que as micelas são elipsoides de revolução core-shell, podendo ser prolatas ou oblatas dependendo do tipo de Tween empregado. Para o sistema proteína-surfactante, a metodologia experimental utilizada permitiu um estudo estrutural e termodinâmico dos complexos formados por meio do acompanhamento do processo de ligação de SDS às proteínas lisozima e alfa-lactalbumina. A técnica de calorimetria de titulação isotérmica (ITC) forneceu um panorama geral sobre a desnaturação proteica e norteou os experimentos seguintes de SAXS e dicroísmo circular (CD). Por meio da análise dos dados de CD concluiu-se que as proteínas perdem quase totalmente sua estrutura terciária, mas não a secundária (esse estado é conhecido como molten globule). Já a modelagem de SAXS em escala absoluta com imposição de vínculos permitiu concluir que os complexos proteína-surfactante podem ser entendidos micelas decoradas, isto é, a proteína está distribuída sobre a superfície de uma micela de SDS. Esse modelo, aliado à abordagem experimental empregada, permitiu a caracterização sistemática dos complexos durante a desnaturação proteica. Em relação ao sistema lipossomas-(bio)ativos, a análise dos dados de SAXS por meio do Método da Deconvolução Gaussiana usando bicamadas simétricas, para os sistemas Phospholipon 90H curcumina/vitamina D3 e dipalmitoilfosfatidilcolina de soja (DPPC) ácido láurico (LA), e assimétricas, para o caso fosfatidilcolina de ovo (EPC) sumatriptano (SMT), permitiu acompanhar mudanças na estrutura das mesmas ocasionadas pela presença dos (bio)ativos. Verificou-se em todos os casos que a espessura da bicamada se mantém praticamente constante. A flexibilidade membranar aumenta, seja em função da temperatura, para o sistema Phospholipon 90H curcumina/ vitamina D3, seja em função da concentração de (bio)ativos, como nos outros dois casos. Para estes, concluiu-se pela análise dos perfis de contraste de densidade eletrônica que os (bio)ativos interagem preferencialmente com as cabeças polares dos fosfolipídeos que constituem os lipossomas, possivelmente causando defeitos topológicos nessa região e ocasionando o aumento da flexibilidade membranar mencionada antes. LA, diferentemente de SMT, induz uma transição de lipossomas multilamelares para unilamelares, e esse fenômeno é grandemente influenciado pelo pH do meio. / This thesis presents a structural characterization of three systems of different classes through, mainly, the small angle X-ray scattering (SAXS) technique. In the surfactant-surfactant system the mixed micelles are composed by sodium dodecyl sulfate (SDS) and one of the Tween surfactants (Tween 20, 40, 60 and 80). The adopted modeling imposed molecular constraints since the SAXS data was in absolute scale. This procedure reduced the ambiguity in the values of the adjustable parameters and allowed to verify that SAXS data is satisfactorily described considering that the micelles are core-shell revolution ellipsoids which can be prolate or oblate depending on the type of Tween used in the micelle. For the protein-surfactant system, the applied experimental methodology allowed a structural and thermodynamic study of the complexes formed through monitoring the binding of SDS to the proteins lysozyme and alpha-lactalbumin. Isothermal titration calorimetry (ITC) technique provided an overview of proteic denaturation and guided the following experiments of SAXS and circular dichroism (CD). From CD data analysis it was concluded that the proteins lose almost totally their tertiary structure, but not the secondary one (this state is known as \"molten globule\"). On the other hand, SAXS data modeling in absolute scale with molecular constraints leaded to the conclusion that protein-surfactant complexes can be considered decorated micelles in which the protein is distributed over a SDS micelle surface. This model, combined to the adopted experimental procedure, allowed the systematic characterization of the complexes along the protein denaturation. In the liposome-(bio)actives system, the SAXS data analysis using the Gaussian Deconvolution Method assuming symmetric bilayers, for the systems Phospholipon 90H curcumin /vitamin D3 and soybean dipalmitoyl phosphatidylcholine (DPPC) lauric acid (LA), and asymmetric bilayers, in the case of egg phosphatidylcholine (EPC) sumatriptan (SMT) system, allowed to follow changes in the lipid bilayer structure induced by the presence of the (bio)actives. It has been found, in all cases, that the bilayer thickness remains approximately. Membrane flexibility increases, depending on the temperature, for the Phospholipon 90H curcumin /vitamin D3 system, or as a function of the (bio)actives concentrations, as in the other two cases. For those, it was concluded, by the analysis of electron density contrast profiles, that the (bio)actives preferentially interact with the polar heads of the phospholipids forming the liposomes, possibly causing topological defects in that region and leading the membrane flexibility increase. LA, unlike SMT, induces a transition from multilamellar to unilamellar liposomes, and this phenomenon is greatly influenced by the pH of the medium.
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Síntese e propriedades de soluções de tensoativos catiônicos derivados de (3-dimetilaminopropil)amidas de ácidos carboxílicos / Synthesis and solution properties of cationic surfactants derivated from fat acids of (3-dimethylaminopropyl)amides.Pires, Paulo Augusto Rodrigues 06 December 2002 (has links)
Duas séries homólogas de tensoativos foram sintetizadas, os cloretos de benzil-(3-acilaminopropil)dimetilamônio, RCONH(CH'ind.2')3N'pot.+'(CH'ind.3')]pot.2'CH'pot.2'C'pot.6'H'pot.5'Cl'pot.-'e (3-acilaminopropil)trimetilamônio, RCONH[(CH'pot.2')]'ind.3'N'pot.+'(CH'ind.3')'pot.3'Cl'ind. -' , onde RCO = C'ind.10' a C'ind.16'. A síntese foi realizada a partir de precursor comum, RCONH(CH'ind.2')'pot.3'N'(CH'ind.3')ind.2', sendo a quaternização feita pela reação com cloreto de benzila e iodeto de metila (seguido de troca iônica), respectivamente. Investigou-se a adsorção na interface solução/ar, a micelização e a estrutura dos agregados formados. Os resultados foram comparados com os de tensoativos catiônicos. Algumas das propriedades micelares das séries estudadas (ex: concentrações micelares críticas, graus de dissociação, números de agregação) tem valores na mesma faixa de grandeza dos outros tensoativos catiônicos com o mesmo tamanho de cadeia hidrofóbica. Outras, por exemplo, a energias livres de Gibbs de adsorção e micelização são diferentes (mais favoráveis). Os resultados apontam para o fato de que o grupo 3-amidopropila, [-CO-NH-CH'ind.2'-CH'ind.2'-CH'ind.2'-], quando inserido na molécula do tensoativo, acaba constituindo parte do seu grupo hidrofílico e facilita a micelização do mesmo. A polaridade microscópica da água interfacial foi determinada usando sondas solvatocrômicas e mostra que a água interfacial é menos polar que água 'bulk'. / The following surfactants have been synthesized: benzyl-(3-acyl-aminopropyl)dimethylammonium chloride, RCONH(CH'ind.2')'ind.3'N'pot.+'(CH'ind.3')'ind.2'CH'ind.2'C'ind.6'H'ind.5' Cl'pot.-' and (3-acylaminopropyl) trimethylammonium chloride, RCONH(CH'ind.2')'ind.3'N'pot.+'(CH'ind.3')'ind.3' Cl'ind.-', where RCO = C'ind.10' to C'ind.16'. The two series have been synthesized from the precursor RCONH(CH2)3N(CH3)2, by quaternization with benzyl chloride, and/or methyl iodide (followed by ion exchange), respectively. Adsorption at solution/air interface and micellization in aqueous solution has been studied, and the results compared with those of other cationic surfactants. Some properties, e.g., critical micelle concentrations, degrees of micelle dissociation, and aggregation numbers are similar to those of other cationic surfactants of equivalent hydrophobic chain length. The Gibbs free energies of adsorption at solution/air interface, and of micellization are, however, more favorable. The group [-CO-NH-CH'ind.2'-CH'ind.2'-CH'ind.2'-] is a part of the interface, and lead to favorable micellization. The microscopic polarity of interfacial water has been measured by appropriate solvatochromic probes, and is less than that of bulk water.
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Trifluorometilsulfonato como contra-íon de micelas catiônicas / Trifluoromethylsulfonate as counterion of cationic micellesFilipe da Silva Lima 20 June 2013 (has links)
Micelas são agregados coloidais formados por moléculas anfifílicas i.e., moléculas com uma região hidrofóbica e uma porção hidrofílica (surfactantes). Efeitos específicos de íons (EEIs) são observados em micelas iônicas, uma vez que as propriedades físico-químicas de agregado micelares, como tamanho e geometria, dependem da natureza do contra-íon. Diferentes ânions inorgânicos produzem alterações modestas nas propriedades de agregados micelares catiônicos, mas ânions orgânicos podem induzir efeitos mais pronunciados, como transições de forma do agregado ou separações de fase. Em sistemas micelares, os EEIs podem estar relacionados a: (a) diferenças na localização dos ânions nas micelas; (b) diferenças na hidratação micellar e de íons; e (c) possível formação de pares iônicos entre surfactantes e contra-íons na interface micelar. Diversos modelos foram desenvolvidos para descrever a formação e estabilidade de agregados micelares, considerando diferentes termos energéticos que possivelmente contribuem para a formação/estabilidade de micelas. Contudo, os termos descritos acima (a - c) geralmente não são incluídos nos modelos micelares. Assim, não deve ser possível predizer as propriedades de micelas catiônicas, usando os modelos atuais, caso o contra-íon seja pequeno, desidratado e capaz de formar pares iônicos, como o ânion trifluorometilsulfonato (triflato, Tf). Tendo isso em vista, determinamos as propriedades micelares de triflato de dodeciltrimetilamônio (DTATf) e comparamos com micelas análogas formadas por brometo, cloreto e metanosulfonato, visando identificar diferenças estruturais das micelas e suas origens. Para determinação de propriedades micelares, utilizamos uma série de técnicas experimentais: fluorescência resolvida no tempo, espalhamento de raio-X de baixo ângulo, condutometria, cinética química, ressonâncias paramagnética eletrônica e magnética nuclear e espectroscopia de relaxação dielétrica, entre outras. Observamos que o agregado de DTATf possui uma estrutura discoidal altamente empacotada, ordenada e desidratada e estas propriedades foram reproduzidos em simulações de dinâmica molecular. A análise do conjunto de resultados obtidos para DTATf demonstrou que a formação de pares iônicos na interface micelar induz severas alterações nas propriedades micelares, como a desidratação dos agregados. Os dados obtidos com DTATf demonstram claramente que, para um modelo teórico de sistemas micelares ser capaz de predizer propriedades micelares de diferentes agregados, a possibilidade de formação de pares iônicos na interface micelar e as interações específicas entre contra-íons e surfactantes devem ser modeladas. Adicionalmente, devido aos resultados aqui reportados e analisando outros sistemas interfaciais, propomos um papel mais fundamental para a água (interfacial ou de hidratação) nas propriedades micelares / Micelles are colloidal aggregates formed by amphiphilic monomers i.e., molecules with a hydrophobic and a hydrophilic moiety (surfactants). Specific ion effects (SIEs) are observed in cationic micelles, because the physicochemical properties of the micellar aggregates, such as size and shape, depend on the nature of the counterion. Different inorganic counterions lead to small changes in micellar properties of cationic aggregates, but organic counterions can induce more pronounced effects, such as shape transitions of the aggregates or phase separation. In micellar systems, the SIEs can be related with: (a) differences in the location of anions in micelles; (b) differences in the hydration of micelles and ions; and (c) possible ion-pair formation between surfactants and counterions at the micellar interface. Several models have been developed to describe the formation and stability of micellar aggregates, considering different energy terms that possibly contribute to the formation/stability of micelles. However, the terms described above (a - c) are generally not included in micellar models. Thus, it should not be possible to predict the properties of cationic micelles, using the current models, if the counterion is small, dehydrated and capable of forming ion-pairs, such as the trifluoromethylsulfonate anion (triflate, Tf). In this context, we have determined the micellar properties of dodecyltrimethylammonium triflate (DTATf) micelles and we have compared the results with similar micelles formed by bromide, chloride and methanesulfonate, aiming to identify their structural differences and its origins. To determine the micellar properties, we have used several techniques: time resolved fluorescence, small angle X-ray scattering, conductometry, kinetic assays, electron paramagnetic and nuclear magnetic resonances and dielectric relaxation spectroscopy, among others. We have observed that the DTATf aggregate presents a highly packed, ordered and dehydrated disk-like geometry and these properties were reproduced in molecular dynamics simulations. The analysis of the DTATf properties showed that the formation of ion-pairs at the micellar interface induces severe changes in micellar properties, such as micellar dehydration. The DTATf properties clearly demonstrate that for a theoretical model of micellar system to be accurate and general, the possibility of ion-pair formation at the micellar interface and the counterions-surfactant specific interactions must be modeled. Additionally, due to the results reported herein and by analyzing other systems, we suggest a more fundamental role of water (interfacial or hydrating water) in the micellar properties.
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Trifluorometilsulfonato como contra-íon de micelas catiônicas / Trifluoromethylsulfonate as counterion of cationic micellesLima, Filipe da Silva 20 June 2013 (has links)
Micelas são agregados coloidais formados por moléculas anfifílicas i.e., moléculas com uma região hidrofóbica e uma porção hidrofílica (surfactantes). Efeitos específicos de íons (EEIs) são observados em micelas iônicas, uma vez que as propriedades físico-químicas de agregado micelares, como tamanho e geometria, dependem da natureza do contra-íon. Diferentes ânions inorgânicos produzem alterações modestas nas propriedades de agregados micelares catiônicos, mas ânions orgânicos podem induzir efeitos mais pronunciados, como transições de forma do agregado ou separações de fase. Em sistemas micelares, os EEIs podem estar relacionados a: (a) diferenças na localização dos ânions nas micelas; (b) diferenças na hidratação micellar e de íons; e (c) possível formação de pares iônicos entre surfactantes e contra-íons na interface micelar. Diversos modelos foram desenvolvidos para descrever a formação e estabilidade de agregados micelares, considerando diferentes termos energéticos que possivelmente contribuem para a formação/estabilidade de micelas. Contudo, os termos descritos acima (a - c) geralmente não são incluídos nos modelos micelares. Assim, não deve ser possível predizer as propriedades de micelas catiônicas, usando os modelos atuais, caso o contra-íon seja pequeno, desidratado e capaz de formar pares iônicos, como o ânion trifluorometilsulfonato (triflato, Tf). Tendo isso em vista, determinamos as propriedades micelares de triflato de dodeciltrimetilamônio (DTATf) e comparamos com micelas análogas formadas por brometo, cloreto e metanosulfonato, visando identificar diferenças estruturais das micelas e suas origens. Para determinação de propriedades micelares, utilizamos uma série de técnicas experimentais: fluorescência resolvida no tempo, espalhamento de raio-X de baixo ângulo, condutometria, cinética química, ressonâncias paramagnética eletrônica e magnética nuclear e espectroscopia de relaxação dielétrica, entre outras. Observamos que o agregado de DTATf possui uma estrutura discoidal altamente empacotada, ordenada e desidratada e estas propriedades foram reproduzidos em simulações de dinâmica molecular. A análise do conjunto de resultados obtidos para DTATf demonstrou que a formação de pares iônicos na interface micelar induz severas alterações nas propriedades micelares, como a desidratação dos agregados. Os dados obtidos com DTATf demonstram claramente que, para um modelo teórico de sistemas micelares ser capaz de predizer propriedades micelares de diferentes agregados, a possibilidade de formação de pares iônicos na interface micelar e as interações específicas entre contra-íons e surfactantes devem ser modeladas. Adicionalmente, devido aos resultados aqui reportados e analisando outros sistemas interfaciais, propomos um papel mais fundamental para a água (interfacial ou de hidratação) nas propriedades micelares / Micelles are colloidal aggregates formed by amphiphilic monomers i.e., molecules with a hydrophobic and a hydrophilic moiety (surfactants). Specific ion effects (SIEs) are observed in cationic micelles, because the physicochemical properties of the micellar aggregates, such as size and shape, depend on the nature of the counterion. Different inorganic counterions lead to small changes in micellar properties of cationic aggregates, but organic counterions can induce more pronounced effects, such as shape transitions of the aggregates or phase separation. In micellar systems, the SIEs can be related with: (a) differences in the location of anions in micelles; (b) differences in the hydration of micelles and ions; and (c) possible ion-pair formation between surfactants and counterions at the micellar interface. Several models have been developed to describe the formation and stability of micellar aggregates, considering different energy terms that possibly contribute to the formation/stability of micelles. However, the terms described above (a - c) are generally not included in micellar models. Thus, it should not be possible to predict the properties of cationic micelles, using the current models, if the counterion is small, dehydrated and capable of forming ion-pairs, such as the trifluoromethylsulfonate anion (triflate, Tf). In this context, we have determined the micellar properties of dodecyltrimethylammonium triflate (DTATf) micelles and we have compared the results with similar micelles formed by bromide, chloride and methanesulfonate, aiming to identify their structural differences and its origins. To determine the micellar properties, we have used several techniques: time resolved fluorescence, small angle X-ray scattering, conductometry, kinetic assays, electron paramagnetic and nuclear magnetic resonances and dielectric relaxation spectroscopy, among others. We have observed that the DTATf aggregate presents a highly packed, ordered and dehydrated disk-like geometry and these properties were reproduced in molecular dynamics simulations. The analysis of the DTATf properties showed that the formation of ion-pairs at the micellar interface induces severe changes in micellar properties, such as micellar dehydration. The DTATf properties clearly demonstrate that for a theoretical model of micellar system to be accurate and general, the possibility of ion-pair formation at the micellar interface and the counterions-surfactant specific interactions must be modeled. Additionally, due to the results reported herein and by analyzing other systems, we suggest a more fundamental role of water (interfacial or hydrating water) in the micellar properties.
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Emprego de sondas solvatocrômicas no estudo de solvatação em solventes puros, misturas de solventes e soluções micelares / Employment solvatochromic probes in the study of solvation in pure solvents, solvent mixtures and micellar solutionsTada, Erika Batista 08 October 2004 (has links)
Neste trabalho, sondas solvatocrômicas foram empregadas no estudo da solvatação em solventes puros e misturas aquosas de solventes polares práticos e apráticos. A partir da polaridade de misturas aquosas de solventes orgânicos, determinou-se a concentração de água interfacial de micelas catiônicas. Finalmente, avaliou-se o efeito da polaridade e força iônica interfaciais de micelas catiônicas sobre a velocidade da reação entre p-nitrofenildifenilfosfato e o íon fluoreto. No estudo de misturas aquosas de solventes orgânicos, um novo modelo de solvatação preferencial foi elaborado, segundo o qual três espécies presentes em solução competem pela camada de solvatação da sonda: água, solvente orgânico e o \"complexo\" formado por uma molécula de água e outra de solvente orgânico (Solv-Água). Através deste modelo, analisou-se o efeito da temperatura e das propriedades das sondas e dos solventes orgânicos sobre o fenômeno da solvatação. Em soluções aquosas de micelas catiônicas, observou-se uma desidratação da interface em função do aumento do grupo hidrofílico do tensotativo e da mudança de geometria micelar de esférica para cilíndrica. Verificou-se que a velocidade da reação entre pnitrofenildifenilfosfato e fluoreto é pouco afetada pela interface micelar e pode ser reproduzida, na ausência de micelas, em soluções com mesma concentração de água e força iônica que as micelas. / Solvatochromic probes have been employed to study the polarity of pure solvents and binary mixtures of water with protic and aprotic polar solvents. From polarity data of aqueous organic mixtures, the concentration of interfacial water of cationic micelles has been determined. In aqueous solutions of cationic micelles, the dehydration of interfacial region has been observed as a result of increasing the volume of the surfactant head group and changing micellar geometry from spherical to cylindrical. Finally, the effect of interfacial polarity and ionic force on the rate of the reaction between 4-nitrophenyldiphenylphosphate and fluoride ion has been evaluated. In studying aqueous organic mixtures, a new preferential solvation model has been developed, that considers the competition between three species in solution for the probe micro-solvation shell: water, organic solvent and a 1:1 \"complex\" formed by water and organic solvent (Solv-Água). Based on this new model, the effect of temperature, as well as probe and organic solvent properties on solvation has been analyzed. It has been observed that the rate of the reaction between p-nitrophenyldiphenylphosphate and fluoride ion shows little dependence on the properties of interfacial region of cationic micelles and can be reproduced, in the absence of micelles, in solutions containing the same water concentration and ionic force as the micellar pseudo-phase.
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Aplica??o da espectroscopia de absor??o de raios X no estudo do comportamento do ?on Cu2+ em micelas inversasGomes, Washington Charles de Macedo 29 April 2009 (has links)
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Previous issue date: 2009-04-29 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / This work deals with the application of X-Ray Absorption Spectroscopy on the study of the behavior of Cu2+ ions in inverse micelles. The formation of copper nanoparticles in water-in-oil microemulsions in pseudo-ternary systems of cetyl
trimethylammonium Bromide (CTAB) surfactant, butanol co-surfactant, heptane as oil phase and aqueous solutions of CuSO4.5H2O, and NaBH4. The microemulsions were prepared with a fixed percentage (60 %) of oil phase and a variable water to tensoative proportion. It was observed an increase on Cu2+ reduction by the sodium borohydride in microemulsions with 13 % of aqueous phase, independent of the reaction time. For the microemulsions in which the aqueous phase is composed only by the CuSO4 solution, it was observed that the color of the solution depends on the
water to surfactant ratio. These changes in color were attributed to a competition for the hidratation water between the polar head of the tensoative and Cu2+ ions with the
eventual substitution of oxygen by bromine atoms in the first coordination shell of Cu2+ ions / Neste trabalho estudou-se aplica??o da espectroscopia de absor??o de raios x no estudo do comportamento dos ?ons Cu2+ em micelas inversas. Estudou-se a forma??o de nanopart?culas de cobre em microemuls?es ?gua-em-?leo de sistemas pseudo-tern?rios tensoativo (brometo de hexadeciltrimetilam?nio, CTAB)-cotensoativo (butanol) / fase oleosa (heptano) / fase aquosa (solu??es de CuSO4.5H2O, e NaBH4). As microemuls?es foram preparadas fixando em 60% a
fase ?leo (Heptano) e variando 40% de fase aquosa com tensoativo/cotensoativo na mesma propor??o, sendo que a fase aquosa do agente redutor NaBH4 foi usada em
excesso de 0,4 mol.L-1 e 0,8 mol.L-1 em rela??o 0,2 mol.L-1 de fase aquosa de CuSO4.5H2O. Observou-se uma maior redu??o dos ?ons Cu2+ pelo boridreto de s?dio em microemuls?es com 13% de ?gua, independente do tempo de rea??o. Para as microemuls?es em que a fase aquosa corresponde a solu??o de CuSO4, observou-se uma depend?ncia na colora??o em fun??o da rela??o entre as
concentra??es de ?gua/tensoativo. Essa mudan?a de colora??o foi atribu?da a uma competi??o pelas mol?culas de ?guas entre a cabe?a polar do tensoativo e os ?ons
Cu2+ com uma eventual substitui??o do oxig?nio da primeira esfera de coordena??o por bromo
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Caracterização estrutural de sistemas biológicos de diferentes classes: um estudo pela técnica de SAXS / Structural characterization of biological systems from different classes: A study by the SAXS techniquePedro Leonidas Oseliero Filho 28 November 2018 (has links)
Esta tese apresenta resultados da caracterização estrutural de três sistemas de classes diferentes por meio, principalmente, da modelagem de dados de espalhamento de raios X a baixos ângulos (SAXS). No sistema surfactante-surfactante as micelas mistas são formadas por dodecil sulfato de sódio (SDS) e um dos surfactantes da série Tween (Tween 20, 40, 60 e 80). A modelagem adotada impôs vínculos moleculares uma vez que os dados de SAXS estavam em escala absoluta. Isso reduziu a ambiguidade nos valores dos parâmetros ajustáveis e permitiu verificar que os dados de SAXS são satisfatoriamente descritos considerando-se que as micelas são elipsoides de revolução core-shell, podendo ser prolatas ou oblatas dependendo do tipo de Tween empregado. Para o sistema proteína-surfactante, a metodologia experimental utilizada permitiu um estudo estrutural e termodinâmico dos complexos formados por meio do acompanhamento do processo de ligação de SDS às proteínas lisozima e alfa-lactalbumina. A técnica de calorimetria de titulação isotérmica (ITC) forneceu um panorama geral sobre a desnaturação proteica e norteou os experimentos seguintes de SAXS e dicroísmo circular (CD). Por meio da análise dos dados de CD concluiu-se que as proteínas perdem quase totalmente sua estrutura terciária, mas não a secundária (esse estado é conhecido como molten globule). Já a modelagem de SAXS em escala absoluta com imposição de vínculos permitiu concluir que os complexos proteína-surfactante podem ser entendidos micelas decoradas, isto é, a proteína está distribuída sobre a superfície de uma micela de SDS. Esse modelo, aliado à abordagem experimental empregada, permitiu a caracterização sistemática dos complexos durante a desnaturação proteica. Em relação ao sistema lipossomas-(bio)ativos, a análise dos dados de SAXS por meio do Método da Deconvolução Gaussiana usando bicamadas simétricas, para os sistemas Phospholipon 90H curcumina/vitamina D3 e dipalmitoilfosfatidilcolina de soja (DPPC) ácido láurico (LA), e assimétricas, para o caso fosfatidilcolina de ovo (EPC) sumatriptano (SMT), permitiu acompanhar mudanças na estrutura das mesmas ocasionadas pela presença dos (bio)ativos. Verificou-se em todos os casos que a espessura da bicamada se mantém praticamente constante. A flexibilidade membranar aumenta, seja em função da temperatura, para o sistema Phospholipon 90H curcumina/ vitamina D3, seja em função da concentração de (bio)ativos, como nos outros dois casos. Para estes, concluiu-se pela análise dos perfis de contraste de densidade eletrônica que os (bio)ativos interagem preferencialmente com as cabeças polares dos fosfolipídeos que constituem os lipossomas, possivelmente causando defeitos topológicos nessa região e ocasionando o aumento da flexibilidade membranar mencionada antes. LA, diferentemente de SMT, induz uma transição de lipossomas multilamelares para unilamelares, e esse fenômeno é grandemente influenciado pelo pH do meio. / This thesis presents a structural characterization of three systems of different classes through, mainly, the small angle X-ray scattering (SAXS) technique. In the surfactant-surfactant system the mixed micelles are composed by sodium dodecyl sulfate (SDS) and one of the Tween surfactants (Tween 20, 40, 60 and 80). The adopted modeling imposed molecular constraints since the SAXS data was in absolute scale. This procedure reduced the ambiguity in the values of the adjustable parameters and allowed to verify that SAXS data is satisfactorily described considering that the micelles are core-shell revolution ellipsoids which can be prolate or oblate depending on the type of Tween used in the micelle. For the protein-surfactant system, the applied experimental methodology allowed a structural and thermodynamic study of the complexes formed through monitoring the binding of SDS to the proteins lysozyme and alpha-lactalbumin. Isothermal titration calorimetry (ITC) technique provided an overview of proteic denaturation and guided the following experiments of SAXS and circular dichroism (CD). From CD data analysis it was concluded that the proteins lose almost totally their tertiary structure, but not the secondary one (this state is known as \"molten globule\"). On the other hand, SAXS data modeling in absolute scale with molecular constraints leaded to the conclusion that protein-surfactant complexes can be considered decorated micelles in which the protein is distributed over a SDS micelle surface. This model, combined to the adopted experimental procedure, allowed the systematic characterization of the complexes along the protein denaturation. In the liposome-(bio)actives system, the SAXS data analysis using the Gaussian Deconvolution Method assuming symmetric bilayers, for the systems Phospholipon 90H curcumin /vitamin D3 and soybean dipalmitoyl phosphatidylcholine (DPPC) lauric acid (LA), and asymmetric bilayers, in the case of egg phosphatidylcholine (EPC) sumatriptan (SMT) system, allowed to follow changes in the lipid bilayer structure induced by the presence of the (bio)actives. It has been found, in all cases, that the bilayer thickness remains approximately. Membrane flexibility increases, depending on the temperature, for the Phospholipon 90H curcumin /vitamin D3 system, or as a function of the (bio)actives concentrations, as in the other two cases. For those, it was concluded, by the analysis of electron density contrast profiles, that the (bio)actives preferentially interact with the polar heads of the phospholipids forming the liposomes, possibly causing topological defects in that region and leading the membrane flexibility increase. LA, unlike SMT, induces a transition from multilamellar to unilamellar liposomes, and this phenomenon is greatly influenced by the pH of the medium.
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[en] MECHANISMS OF NITROSYLATION OF IRON PORPHYRINS BY SNPA: KINETICS OF THE REACTION AND STABILITY UNDER AEROBIC ENVIRONMENT / [pt] MECANISMOS DE NITROSILAÇÃO DE FERRO PORFIRINAS POR SNAP: CINÉTICA DE REAÇÃO E ESTABILIDADE EM AMBIENTE AERÓBICOFERNANDO DE SOUZA DIAS DOS SANTOS VILHENA 07 November 2006 (has links)
[pt] Vários complexos metálicos têm sido identificados como
alvos para a ligação de óxido nítrico. Para que esses
complexos possam ser usados com eficiência e segurança em
aplicações medicinais, tem-se como meta compreender suas
propriedades cinéticas e estruturais em modelos cada vez
mais realísticos. Neste trabalho foram utilizadas as
técnicas de absorção ótica e de ressonância paramagnética
eletrônica para estudar a nitrosilação de duas porfirinas
férricas solúveis em água, FeTMPyP, porfirina catiônica, e
FeTPPS4, porfirina aniônica, pelo doador de óxido nítrico
S-nitroso-acetilpenicilamina (SNAP). Foram obtidas as
taxas de formação dos complexos nitrosilados em função da
concentração de SNAP. Foram encontradas as constantes
cinéticas de segunda ordem para ambas porfirinas. A
semelhança entre essas constantes, KTMPyP = 0.84 x 10(3) M(-1)s(-1) e KTPPS4 =
0.97 x 10(3) M(-1)s(-1), sugeriram que o
mecanismo da reação é independente da natureza dos meso
substituintes das porfirinas. Demonstrou-se que a
nitrosilação por SNAP é diferente daquela obtida por
reação com NO e propô-se um mecanismo para a reação. A
reação foi investigada na presença de oxigênio. Ao
contrário do que acontece com gás NO, SNAP foi capaz de
produzir ferro-porfirinas nitrosiladas em ambiente
aeróbico. A influência da interação com micelas iônicas na
estabilidade dos complexos foi avaliada em atomosfera
aeróbica. A associação das forro-porfirinas com albumina
sérica bovina e sua influência de fatores eletrostáticos
na ligação da ferro-porfirina com a BSA. A interação com
BSA exerceu forte influência sobre as taxas de / [en] Serveal metalic complexes have been identified as tergets
for the binding of nitric oxide. In order that these
complexes can be efficiently and safely used in medical
applications, it is important to understand their kinetic
and structural properties in realistic models. In this
work, the optical absorption and electron paramagnetic
resonance techniques were used to study the nitrosylation
of two water soluble ferric porphyrins, FeTMPyP, cationic,
and FeTPPS4, anionic, by the NO donor S-nitrous-
Nacetylpenecillamine (SNAP). For both porphyrins,
nitrosylation rates as a function of SNAP concentration
were obtained, and the second order rate constants were
found. The similarity between these constants, KTMPYP =
0.84 x 10(3) M(-1)s(-1) and KTPPS4 = 0.97 x 10(3) M(-1)s(-1),
suggests a mechanism independent of the nature of the
porphyrin meso-substituents. It was demonstrated tha the
nitrosylation by SNAP is different from that by NO gas,
and a reaction mechanism was proposed. The reaction was
also studied in aerobic environment. In contrast to NO
gas, SNAP was able to produce nitrosylated iron porphyrins
even in the presence of oxygen. The influence of ionic
micelles on the stability of nitrosylated complexes was
evaluated. The association of iron porphyrins with BSA and
its influence on nitrosylation by SNAP were studied. The
results demonstrated strong electrostatic influence on the
binding of iron porphyrins to BSA. The interaction with
BSA influenced the nitrosylation rates. The nitrosylated
porphyrins were characterized by EPR spectroscopy.
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Studies of UHT-plant fouling by fresh, recombined and reconstituted whole milk : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food EngineeringSrichantra, Arunee January 2008 (has links)
The objective of this study was to investigate the effects of preheat treatments on fouling by fresh whole milk (FWM), recombined whole milk (RCB) and reconstituted whole milk (Recon) in the high-temperature heater of indirect UHT plants. Various preheat treatments prior to evaporation during milk powder manufacture were applied to skim milk powder (SMP, 75 °C 2 s, 85 °C, 155 s and 95 °C, 155 s) and whole milk powder (WMP, 95 °C, 33 s). These preheat treatments were so-called “evaporator preheat treatments”. Skim milk powder (SMP) and whole milk powder (WMP) were derived from the same original batch of pasteurised FWM to remove the effects of the variation in milk composition between different milk batches. These SMPs were recombined with anhydrous milk fat and water to prepare RCB, and WMPs were reconstituted with water to prepare Recon. Then, (homogenized) FWM, RCB and Recon were subjected to various preheat treatments (75 °C, 11 s, 85 °C, 147 s and 95 °C, 147 s) prior to UHT processing. These preheat treatments were so-called “UHT preheat treatments”. Temperature difference (hot water inlet temperature – milk outlet temperature) was taken as a measure of the extent of fouling in the high-temperature heater. The slope of the linear regression of temperature difference versus time (for two hours of UHT processing) was taken as fouling rate (°C/h). Increasing both evaporator and UHT preheat treatments resulted in increasing fouling rate and total deposit weight for all three whole milk types for several milk batches. In the case of FWM, there was no reduction in fouling rate with increasing UHT preheat treatment whether FWM was homogenized then preheated, preheated then homogenized or not homogenized at all. These findings, which are wholly consistent and well replicated, are in apparent conflict with the results of most previous comparable studies. Possible reasons for this are explained. Further investigations of the effects of homogenization relating to the role of whey protein on the surface of the fat globules showed that whey protein associated with the membrane covering the surface of fat globules for homogenized then preheated FWM, RCB and Recon and that association increased with increasing heating process stage. The increasing association of whey protein with the milk fat globules membrane with increasing severity of heating process stage became faster when preheat treatment was more severe: the association of whey protein plateaued on intermediate temperature heating when the milks were preheated at 75°C, 11 s and on preheating when the milks were preheated at 95°C, 147 s. In the case of FWM, the thickness of the membrane covering the surface of fat globules for homogenized then preheated FWM, which increased with the severity of heating process stage, was greater than the thickness of the membrane in preheated then homogenized FWM. Preheating then homogenization resulted in the greater interfacial spreading of small molecules on the surface of fat globules, i.e. whey protein or small molecules from the disintegration of casein micelles during preheating. Possible basic mechanisms for UHT fouling in the high-temperature heater include: the reduction in the solubility of calcium phosphate and the deposition of protein as fat-bound protein and non-fat-bound protein. When non-fat-bound protein in milk plasma deposited, it could be a carrier for the deposition of mineral, such as, the precipitate of calcium phosphate in the casein micelles or the deposition of complexes between whey protein and casein micelles.
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Studies of UHT-plant fouling by fresh, recombined and reconstituted whole milk : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food EngineeringSrichantra, Arunee January 2008 (has links)
The objective of this study was to investigate the effects of preheat treatments on fouling by fresh whole milk (FWM), recombined whole milk (RCB) and reconstituted whole milk (Recon) in the high-temperature heater of indirect UHT plants. Various preheat treatments prior to evaporation during milk powder manufacture were applied to skim milk powder (SMP, 75 °C 2 s, 85 °C, 155 s and 95 °C, 155 s) and whole milk powder (WMP, 95 °C, 33 s). These preheat treatments were so-called “evaporator preheat treatments”. Skim milk powder (SMP) and whole milk powder (WMP) were derived from the same original batch of pasteurised FWM to remove the effects of the variation in milk composition between different milk batches. These SMPs were recombined with anhydrous milk fat and water to prepare RCB, and WMPs were reconstituted with water to prepare Recon. Then, (homogenized) FWM, RCB and Recon were subjected to various preheat treatments (75 °C, 11 s, 85 °C, 147 s and 95 °C, 147 s) prior to UHT processing. These preheat treatments were so-called “UHT preheat treatments”. Temperature difference (hot water inlet temperature – milk outlet temperature) was taken as a measure of the extent of fouling in the high-temperature heater. The slope of the linear regression of temperature difference versus time (for two hours of UHT processing) was taken as fouling rate (°C/h). Increasing both evaporator and UHT preheat treatments resulted in increasing fouling rate and total deposit weight for all three whole milk types for several milk batches. In the case of FWM, there was no reduction in fouling rate with increasing UHT preheat treatment whether FWM was homogenized then preheated, preheated then homogenized or not homogenized at all. These findings, which are wholly consistent and well replicated, are in apparent conflict with the results of most previous comparable studies. Possible reasons for this are explained. Further investigations of the effects of homogenization relating to the role of whey protein on the surface of the fat globules showed that whey protein associated with the membrane covering the surface of fat globules for homogenized then preheated FWM, RCB and Recon and that association increased with increasing heating process stage. The increasing association of whey protein with the milk fat globules membrane with increasing severity of heating process stage became faster when preheat treatment was more severe: the association of whey protein plateaued on intermediate temperature heating when the milks were preheated at 75°C, 11 s and on preheating when the milks were preheated at 95°C, 147 s. In the case of FWM, the thickness of the membrane covering the surface of fat globules for homogenized then preheated FWM, which increased with the severity of heating process stage, was greater than the thickness of the membrane in preheated then homogenized FWM. Preheating then homogenization resulted in the greater interfacial spreading of small molecules on the surface of fat globules, i.e. whey protein or small molecules from the disintegration of casein micelles during preheating. Possible basic mechanisms for UHT fouling in the high-temperature heater include: the reduction in the solubility of calcium phosphate and the deposition of protein as fat-bound protein and non-fat-bound protein. When non-fat-bound protein in milk plasma deposited, it could be a carrier for the deposition of mineral, such as, the precipitate of calcium phosphate in the casein micelles or the deposition of complexes between whey protein and casein micelles.
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