Precipitation Proteins with organic solvents
Proteins are maintained in solution by the interaction of surface hydrophilic groups with the water solvent. Consequently, if the polarity of the solvent is reduced by the addition of an organic solvent less polar than water, the protein will tend to become less soluble. Concurrently, the protein usually becomes less stable because a major contributor to protein stability is the distribution of hydrophilic and hydrophobic groups into the lowest energy conformation, based on the solvent being water.
To minimize denaturation of the protein in the less polar solvent, it is usually necessary to conduct the fractionation at a low temperature. Two solvents that have been commonly used in the fractional precipitation of proteins are ethanol and acetone. Ethanol is widely used in the low temperature fractionation of blood proteins, for example, while acetone was formerly commonly used to make “acetone powders”, a means of preserving proteins.
To effect the precipitation of a protein with an organic solvent, the protein solution should be chilled close to 0∞C and the solvent to at least -20C. The solvent is slowly but smoothly added to the protein solution, with constant stirring to avoid the formation of high local concentrations of solvent. Occasionally, addition of the solvent leads to the formation of a milky colloidal suspension, rather than a precipitate. If this happens, addition of a drop of NaCl solution may be necessary to induce flocculation.
For the preparation of an “acetone powder”, the protein precipitated with acetone is harvested by centrifugation and spread out to air dry. The latent heat of vaporization of the acetone keeps the protein cool as it dries to a powder. Alternatively, the protein precipitate may be stored at low temperature and subsequently reconstituted by dissolving in chilled buffer solution. Some denaturation of the protein during organic solvent precipitation is unavoidable.
References
-Dennison, C. (2002). A guide to protein isolation . School of Molecular mid Cellular Biosciences, University of Natal . Kluwer Academic Publishers new york, Boston, Dordrecht, London, Moscow .
