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Master's Dissertation
DOI
https://doi.org/10.11606/D.9.2009.tde-02102009-130108
Document
Author
Full name
Letícia Célia de Lencastre Novaes
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2009
Supervisor
Committee
Vessoni Penna, Thereza Christina (President)
Gurpilhares, Daniela de Borba
Stephano, Marco Antônio
Title in Portuguese
Efeito de polímeros e sais na estabilidade térmica da proteína verde fluorescente (GFP)
Keywords in Portuguese
Citrato
Estabilidade térmica
Fosfato
GFP
Polímeros
Proteínas de fluorescência verde
Sais
Tecnologia farmacêutica
Abstract in Portuguese
O emprego de aditivos hidrossolúveis como açúcares, tensoativos, sais e polímeros é prática comum na tentativa de se estabilizar proteínas durante aquecimento. Diversos polímeros têm sido utilizados para estabilizar proteínas, sendo seu efeito dependente das características da proteína. Sais podem estabilizar, desestabilizar ou não ter efeito na estabilidade de proteínas; dependendo do tipo, concentração, natureza das interações iônicas e resíduos carregados da proteína. A termoestabilidade da proteína verde fluorescente (GFP) tem sido demonstrada ao calor úmido, à temperaturas elevadas (T ≥ 95°C), à valores de pH alcalinos e a alguns agentes químicos. Sua denaturação térmica é altamente reprodutível e a variação da intensidade de fluorescência pode ser facilmente determinada por espectrofluorimetria. O objetivo deste trabalho foi estudar o comportamento da GFP na presença de diferentes soluções aquosas de polímeros (polietileno glicol, DEAE-Dextrana e ácido poliacrílico) e sais (citrato e fosfato). A partir dos dados obtidos, pode-se concluir que o citrato favoreceu a preservação da estrutura nativa da GFP nas temperaturas estudadas (70 a 95ºC), em concentrações acima de 10% m/m. O ácido poliacrílico também auxiliou na manutenção da estrutura nativa da GFP, porém em menor intensidade, e com concentrações acima de 20% m/m.
Title in English
Effect of polymers and salts in thermal stability of green fluorescent protein (GFP)
Keywords in English
Citrate
GFP
Green fluorescent proteins
Phosphate
Polymers
Salts
Technology pharmaceutical
Thermal stability
Abstract in English
The addition of hydrosoluble excipients, such as, sugars, surfactants, salts and polymers is a common practice in the intent of stabilization of proteins during heating. Several polymers have been used to proteins stabilization, being their effect dependent of protein characteristics, however in some cases, it could cause a reduction of stability. Salts can stabilize proteins, or have no influence in their stability, and these behaviors depend on the type, concentration, ionic interaction and charged protein residues. Thermal stability of green protein fluorescent (GFP) have been demonstrated to humid heat, elevated temperatures (T ≥ 95°C), alkaline pH and to some chemical agents. Its thermal denaturation is highly reproducible and the variation of fluorescence intensity can be easily determinate by spectrofluorometry. The objective of this work was study the behavior of GFP in the presence of different aqueous solutions of polymers (polyethylene glycol, DEAE-Dextran and acid polyacrylic) and salts (citrate and phosphate). From the results, it may be concluded that the citrate favored the preservation of native structure of GFP in the temperatures studied (70ºC to 95ºC), in concentrations above 10% m/m. The PAA polymer also favored the GFP thermal stability, but in a minor intensity and in concentrations above 20% m/m.
 
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Novaes_LCL.pdf (1.09 Mbytes)
Publishing Date
2010-03-30
 
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