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Advisor(s)
Abstract(s)
Diferentes áreas da biologia dependem da cromatografia para obtenção de biomoléculas, sendo a cromatografia de afinidade o método de purificação mais seletivo para proteínas e outras biomoléculas.
O desenvolvimento de novos suportes cromatográficos tem sido um tema investigado intensivamente no intuito de se encontrar um bom suporte, que reúna um equilíbrio ideal de características, tais como custos baixos, eficiência, elevada capacidade e estabilidade físico-química.
Utilizando a esterificação de Steglich, que em anteriores trabalhos se tinha mostrado eficaz para a imobilização de ligandos a uma matriz de agarose, foram sintetizados vários suportes cromatográficos com dois ligandos e diferentes densidades de ligando.
Como ligandos imobilizados foram utilizadas duas carbocianinas simétricas que diferem no número de anéis aromáticos da sua constituição. As carbocianinas simétricas são constituídas por dois anéis heterocíclicos iguais ligados por uma cadeia de três átomos de carbono com ligações duplas conjugadas.
Posteriormente, foram estudadas as interações de afinidade entre os suportes cromatográficos obtidos e as várias proteínas modelo, dando preferência às interações iónicas e hidrofóbicas. Dentro de todos os resultados obtidos, o suporte com concentração inicial de 0,078mmol de ligando, apresentou picos mais bem definidos, tanto na separação da mistura proteica como nos testes com as proteínas isoladas, indicando ser o melhor suporte numa separação cromatográfica.
Estes estudos permitiram demonstrar o potencial da aplicabilidade de ligandos catiónicos, tais como as carbocianinas, para a separação e purificação de proteínas por cromatografia de afinidade.
Different areas of biology depend on the chromatography in order to obtain bio-molecules, being the affinity chromatography the most selective method of purification for proteins and other biomolecules. The development of new chromatographic supports has been a topic investigated intensively in order to find a good support, bringing together an ideal balance of features, such as low cost, efficiency, high capacity and physical and chemical stability. Using the Steglich esterification, which in previous work had shown itself effective for immobilization of ligands to an agarose matrix, were synthesized various chromatographic supports with two ligands and different densities of ligand. As ligands immobilized were used two symmetric carbocianinas which differ in the number of aromatic rings. The symmetrical carbocianines consist of two equal heterocyclic rings connected by a chain of three carbon atoms with conjugated double bonds. Later, the affinity chromatographic interactions between the chromatographic supports and the various model proteins were studied, giving preference to ionic and hydrophobic interactions. Within all results obtained, the support with initial concentration of 0, 078mmol of ligand, presented more clearly defined peaks, both in the separation of the protein mixture as in the tests with isolated proteins, indicating the best support a chromatographic separation. These studies have made it possible to demonstrate the potential of the applicability of cationic ligands, such as the carbocianines, for the separation and purification of proteins by affinity chromatography.
Different areas of biology depend on the chromatography in order to obtain bio-molecules, being the affinity chromatography the most selective method of purification for proteins and other biomolecules. The development of new chromatographic supports has been a topic investigated intensively in order to find a good support, bringing together an ideal balance of features, such as low cost, efficiency, high capacity and physical and chemical stability. Using the Steglich esterification, which in previous work had shown itself effective for immobilization of ligands to an agarose matrix, were synthesized various chromatographic supports with two ligands and different densities of ligand. As ligands immobilized were used two symmetric carbocianinas which differ in the number of aromatic rings. The symmetrical carbocianines consist of two equal heterocyclic rings connected by a chain of three carbon atoms with conjugated double bonds. Later, the affinity chromatographic interactions between the chromatographic supports and the various model proteins were studied, giving preference to ionic and hydrophobic interactions. Within all results obtained, the support with initial concentration of 0, 078mmol of ligand, presented more clearly defined peaks, both in the separation of the protein mixture as in the tests with isolated proteins, indicating the best support a chromatographic separation. These studies have made it possible to demonstrate the potential of the applicability of cationic ligands, such as the carbocianines, for the separation and purification of proteins by affinity chromatography.
Description
Keywords
Carbocianinas Cromatografia de Afinidade Esterificação de Steglish Purificação de Proteínas Sais de Amónio Quaternário