Effect of surfactants and methodologic alterations in the rat inner mitochondrial membrane solubilization analysed by native and two dimensional electrophoresis / Efeito de surfatantes e modificações metodologicas na solubilização de membrana mitocondrial interna de ratos analisada por eletroforese nativa e bidimensional

AUTOR(ES)
DATA DE PUBLICAÇÃO

2008

RESUMO

The majority of the chemical energy produced inside aerobic cells is a result of the oxidative phosphorylation of ATP, inside the mitochondria. A great part of mitochondria proteins are organized into complexes located in the inner mitochondria membrane. Isolation and identification of those proteins have been conducted by electrophoresis, both in native as in denaturant condition. In this work we analysed solubilization of the inner mitochondrial membrane proteins of rat?s gastrocnemius muscles, through the use of zwitterionic (ASB-14, ASB-16, CHAPS), non-ionic (Digitonin, C12E8, Triton X-100) and anionic (sodium cholate), by native and two-dimensional electrophoresis. We have developed a new protocol for the native gel electrophoresis, using the buffer sample described in BN-PAGE but changing other steps of the technique according to the protocol of Laemmli et al (1970). With this novel protocol, the gels prepared at ambient temperature had a better resolution than the classic BNPAGE technique; with low costs and short-time runs (1-2 hours). As for the effectiveness of surfactants studied to solubilize inner mitochondrial membrane proteins, when used at the same concentration (43.2 mM), the zwitterionic ASB-16 was the best, both accordingly to the total amount of solubilized protein, as for the number of "spots" detected in the two-dimensional electrophoresis, followed by non-ionic Triton X-100. The non-ionic surfactant dodecyl maltoside was used in the 2D electophoresys preparation at lower concentrations (20%) than that recommended in the literature (10% or 195.8 mM) without prejudice in the efficiency of protein solubilization. The results with native gels proved that all the studied surfactants can be used to separate the proteins of MMI, but with different selectivity for each enzymatic complex. This specificity should be better studied in the future, looking for the determination of specific protocols for better isolation of each types of MMI complex

ASSUNTO(S)

proteinas de membrana solubilização solubilization agentes ativos de superficies eletroforese electrophoresis surface active agents mitocondria membrane proteins mitochondria

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