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 .