Solubilização de misturas lipídicas ternárias do tipo raft por detergentes não-iônicos
Arquivos
Data
2015-11-27
Autores
Carita, Amanda Costa 
Orientadores
Riske, Karin do Amaral
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
O processo de solubilização de membranas biológicas é relevante para inúmeros protocolos bioquímicos. Em determinadas condições, algumas membranas apresentam resistência à solubilização, mesmo sob alta concentração de detergente. Esses fragmentos insolúveis, chamados de DRMs (membranas resistentes a detergentes), são ricos em esfingolipídios, colesterol e proteínas com preferência por ambientes mais organizados. A composição dos DRMs é similar à dos rafts, que são domínios funcionais de membrana envolvidos em diversos mecanismos celulares importantes, e à da fase líquido-ordenada de bicamadas lipídicas. Devido à complexidade do estudo dos rafts in vivo, as membranas modelo representam uma interessante opção para elucidar seu funcionamento. Aqui, realizamos o estudo de aspectos físico-químicos que regem o processo de solubilização de misturas lipídicas ternárias compostas pelos lipídios POPC (palmitoil oleoil fosfatidilcolina, um lipídio insaturado), SM (esfingomielina, um lipídio saturado) e colesterol em uma grande gama de composições. Vesículas unilamelares gigantes (GUVs) e grandes (LUVs) foram escolhidas como modelos de membrana para os ensaios de solubilização. O principal detergente utilizado neste trabalho foi o Triton X-100 (TX-100), amplamente empregado como agente solubilizador de membranas biológicas. Adicionalmente, os detergentes Brij 98, maltosídeo e octil-glicopiranosídeo (OG) também foram investigados. A principal técnica utilizada foi a microscopia óptica de GUVs. Em paralelo, alguns estudos de fluorescência e espalhamento de luz foram feitos em LUVs. Este trabalho teve como objetivo principal verificar se o TX-100 induzia a separação de fases líquido-desordenada/líquido-ordenada e quantificar a extensão de solubilização, à medida que a proporção dos lipídios era alterada na composição ternária. Foi possível observar que a adição de TX-100 aumenta consideravelmente a região de coexistência de fases macroscópica e a presença de SM e col tornam a mistura cada vez mais insolúvel. A técnica de FRET foi empregada para verificar a existência de domínios sub-microscópicos e compreender se a adição de detergente ampliava os domínios pré-existentes ou induzia sua formação. O espalhamento de luz estático também foi utilizado para monitorar o perfil de solubilização na mesma amostra. 5 Ademais, GUVs compostas por POPC puro e POPC:SM:col (4:2:4) foram escolhidas para estudos por microscopia óptica com os detergentes OG, maltosídeo e Brij 98. O foco aqui foi comparar os efeitos observados com o TX-100 com os demais detergentes não-iônicos. No processo de solubilização de GUVs de POPC ficou evidente como a natureza química do detergente influencia no processo de solubilização: o TX-100 possui a capacidade de ser incorporado em grande quantidade na bicamada e de solubilizá-la completamente. A partição do OG na membrana parece ser menor e seu caráter solubilizador foi menor, pois fragmentos insolúveis foram verificados. O maltosídeo, cuja cabeça polar é composta por um dissacarídeo, levou à ruptura da vesícula e o Brij 98 apresentou uma pequena partição na membrana mas não foi capaz de solubilizá-la. Os resultados com as GUVs de composição ternária mostraram que além do TX-100, os detergentes maltosídeo e OG induziram a formação de domínios, mas em proporções distintas dependendo do detergente. Dessa maneira, apesar de o uso de detergentes ser um fator importante para o estudo dos lipid rafts não é possível realizar uma associação direta entre as frações insolúveis resultantes desse processo e os rafts de membrana in vivo.
The solubilization process of biological membranes is important for many biochemical protocols. Under certain conditions, some membranes exhibit resistance to solubilization, even at high detergent concentration. These insoluble fragments, named DRMs (detergent resistant membranes), are rich in sphingolipids, cholesterol and proteins with a preference for more organized environments. The composition of DRMs is similar to that of rafts, which are functional membrane domains involved in several important cellular mechanisms and of the liquid-ordered phase of lipid bilayers. Due to the complexity of the study of rafts in vivo, model membranes represent an interesting option to elucidate its functioning. Here, we conducted a study of the physical-chemical aspects that govern the solubilization process of lipid ternary mixtures composed of POPC (palmitoyl oleoyl phosphatidylcholine, an unsaturated lipid), SM (sphingomyelin, a saturated lipid) and cholesterol in a wide range of compositions. Giant (GUVs) and large (LUVs) unilamellar vesicles were chosen as model membranes in the solubility assays. The main detergent used in this work was Triton X-100 (TX-100), widely used as a solubilizing agent of biological membranes. Additionally, the detergents Brij 98, maltoside and octyl-glucopyranoside (OG) were also investigated. The main technique used was optical microscopy of GUVs. In parallel, some fluorescence and light scattering studies were performed in LUVs. The main goal of this work was to verify if TX-100 induced separation of liquid-disordered/liquid-ordered phases and to quantify the extent of solubilization, as the proportion of lipids in the ternary composition was varied. It was shown that the addition of TX-100 significantly increases the region of macroscopic phase coexistence and the presence of SM and cholesterol render the mixture increasingly insoluble. The FRET technique was employed to verify the existence of sub-microscopic domains and understand if the addition of detergent amplified the pre-existing domains or induced its formation. Static light scattering was also used to monitor the solubilization profile in the same sample. Moreover, GUVs composed of pure POPC and POPC:SM:chol (4: 2 4) were chosen for optical microscopy studies with the detergents OG, maltoside and Brij 98. The focus here was to compare the effects observed with TX-100 with other non- 7 ionic detergents. In the solubilization process of GUVs composed of POPC it was evident that the chemical nature of the detergent influences the solubilization process: TX-100 has the ability to be incorporated in the bilayer in large to complete solubilize it completely. The membrane partition of OG and seems to be smaller, and its solubilizing character was smaller, because insoluble fragments were found. Maltoside, whose polar head comprises a disacharide, causes vesicle rupture and Brij 98 exhibited a small membrane partition and was not able to solubilize it. The results with GUVs of the ternary composition showed that in addition to TX-100, maltoside and OG induced the formation of domains, but in different proportions depending on the detergent. Thus, despite the fact that the use of detergents is an important factor in the study of lipid rafts, it is not possible to make a direct association between the insoluble fractions resulting from this process and membrane rafts in vivo.
The solubilization process of biological membranes is important for many biochemical protocols. Under certain conditions, some membranes exhibit resistance to solubilization, even at high detergent concentration. These insoluble fragments, named DRMs (detergent resistant membranes), are rich in sphingolipids, cholesterol and proteins with a preference for more organized environments. The composition of DRMs is similar to that of rafts, which are functional membrane domains involved in several important cellular mechanisms and of the liquid-ordered phase of lipid bilayers. Due to the complexity of the study of rafts in vivo, model membranes represent an interesting option to elucidate its functioning. Here, we conducted a study of the physical-chemical aspects that govern the solubilization process of lipid ternary mixtures composed of POPC (palmitoyl oleoyl phosphatidylcholine, an unsaturated lipid), SM (sphingomyelin, a saturated lipid) and cholesterol in a wide range of compositions. Giant (GUVs) and large (LUVs) unilamellar vesicles were chosen as model membranes in the solubility assays. The main detergent used in this work was Triton X-100 (TX-100), widely used as a solubilizing agent of biological membranes. Additionally, the detergents Brij 98, maltoside and octyl-glucopyranoside (OG) were also investigated. The main technique used was optical microscopy of GUVs. In parallel, some fluorescence and light scattering studies were performed in LUVs. The main goal of this work was to verify if TX-100 induced separation of liquid-disordered/liquid-ordered phases and to quantify the extent of solubilization, as the proportion of lipids in the ternary composition was varied. It was shown that the addition of TX-100 significantly increases the region of macroscopic phase coexistence and the presence of SM and cholesterol render the mixture increasingly insoluble. The FRET technique was employed to verify the existence of sub-microscopic domains and understand if the addition of detergent amplified the pre-existing domains or induced its formation. Static light scattering was also used to monitor the solubilization profile in the same sample. Moreover, GUVs composed of pure POPC and POPC:SM:chol (4: 2 4) were chosen for optical microscopy studies with the detergents OG, maltoside and Brij 98. The focus here was to compare the effects observed with TX-100 with other non- 7 ionic detergents. In the solubilization process of GUVs composed of POPC it was evident that the chemical nature of the detergent influences the solubilization process: TX-100 has the ability to be incorporated in the bilayer in large to complete solubilize it completely. The membrane partition of OG and seems to be smaller, and its solubilizing character was smaller, because insoluble fragments were found. Maltoside, whose polar head comprises a disacharide, causes vesicle rupture and Brij 98 exhibited a small membrane partition and was not able to solubilize it. The results with GUVs of the ternary composition showed that in addition to TX-100, maltoside and OG induced the formation of domains, but in different proportions depending on the detergent. Thus, despite the fact that the use of detergents is an important factor in the study of lipid rafts, it is not possible to make a direct association between the insoluble fractions resulting from this process and membrane rafts in vivo.
Descrição
Citação
CARITA, Amanda Costa. Solubilização de misturas lipídicas ternárias do tipo raft por detergentes não-iônicos. 2015. 83 f. Dissertação (Mestrado em Biologia Molecular) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2015.