Abstract
The present paper reviews the reversed micellar extraction of protein on the basis of the author's research work on the occasion to be accorded the 1997 young investigator's award by the Membrane Society of Japan. The extraction of protein using AOT (di-2-ethylhexyl sodium sulfosuccinate) /isooctane reversed micellar system was investigated from the viewpoint of the solubilization of protein and water by amphiphilic molecules. The minimal AOT concentration required for 100% forward extraction of protein was found. At the minimal AOT concentration, the hydrophilic surroundings, i.e., the molar ratio of water to extracted protein in the organic phase, were independent of the protein concentration. The hydrophilic surroundings were related with Fisher's polarity ratio as an index of the hydrophobicity of protein. Using this relation, a procedure to estimate the sufficient AOT concentration for the protein extraction was proposed. In the cases of cytochrome c and lysozyme extraction, the water concentration was larger than that in the protein-free system in spite of the same AOT concentration. On the contrary, in the case of ribonuclease A, this large water uptake was not observed. These differences of water uptake were discussed in relation to the location of the protein in the AOT reversed micelles. The forward extraction was accompanied by a conformational change of protein, whereas the backward extracted protein consequently refolded in the native conformation in the aqueous phase. Therefore, the relative reactivity of recovered cytochrome c and ribonuclease A after forward and backward extractions showed almost 100%. The recovered activity yield showed a maximum with respect to AOT concentration because of the combined effect of both decreases in forward and backward extraction yields at low and high AOT concentrations, respectively.
