I have been working on alpha amylase and tried precipitating it with ammonium sulphate salt.
With ammonium sulphate salt i do get precipitates but they are flaky, float in the supernatant and don't settle at the bottom after centrifugation making the recovery of protein very less. Whereas by using chilled ethanol i get good amount of precipitates instantly.
You are using two precipitation procedures that work differently. You are probably denaturing your protein with ammonium sulfate.
It is not a trivial question, indeed.
The conformational state of a protein in solution is an energetic compromise between protein-protein interactions and protein-solvent interactions to favor the most stable conformation(s). The free energy of transfer, ΔGtr, from water to the various solvents, is a good measure of the most probable conformational state in a specific solution. More negative is the value of the free energy difference between the protein in water and the one in solution (ΔGtr), more the solubility of the protein in solution is favored. For example, ΔGtr for the peptide group from water to urea 2 M is about – 70 (cal/mol), while that for the transfer of the peptide group to ethanol is + 1500 (cal/mol), which means that the peptide group is more soluble in urea solution than in ethanol. This explains why globular proteins are more soluble in urea or GdnHCl solutions (because more unfolded, namely, more prone to interact with the solution molecules) in respect to ethanol solutions. This is the quantitative aspect of the protein's behavior in solution, but the interaction mechanism capable of producing these behaviors requires different considerations.
Ethanol is a polar solvent that influences the conformation of proteins through its direct binding, changing significantly with the ethanol concentration and length of exposure and protein type (due to different composition and residues exposure). In general alcohols act as hydrogen bond donors whose binding to the polypeptide chain is stabilized by hydrophobic interactions where properties of alcohol binding sites have been derived from X-ray crystal structures of alcohol-protein complexes. It seems that at first the protein unfolds in ethanol, but it often refolds into rod-like alpha helix structures probably to bury peptide groups inside the compact helical rods, out of interaction with ethanol. Thus, interacting only with exposed non polar side chains. This suggests that alpha helix should be stable in ethanol. In fact, numerous alcohols are alpha-inductors.
Instead, ammonium sulfate is a very soluble inorganic salt that disassociates into ammonium and sulfate ions in aqueous solutions. It stabilizes protein structure and the protein solubility changes according to the salt concentration (that is, according to the ionic strength). At low salt concentrations (between 0 and about 0.5 M) the protein solubility increases, with increasing salt concentration, but when the salt concentration is further increased, the protein solubility decreases. We have the famous “salting in” and “salting out”. But ammonium and sulphate ions are member of the Hofmeister series where the effect of these ions is on the water, at least in part, by ‘making’ or ‘breaking’ bulk water structure, that is, the water molecules that act as cage around the protein. However, models suggest that this effect depend both upon direct ion–macromolecule interactions and interactions with water molecules in the first hydration shell (the water-cage) of the protein. Models also suggest that ions seem to modulate the ability of the various water layers in order to help out the first water layer solvating the protein surface. Therefore, the mechanisms of action of the two solutions are very different, though the behaviors in solution (the thermodynamic aspect) follow the same law.
I hope that these explanations, though a bit rough for necessity, may be useful.
I agree with the excellent comments of Giovanni and to try to summarize it I would like to say that the precipitation of any protein with Ammonium Sulphate or Ethanol is based on the range of different physico-chemical properties of each particular protein such as hydrophobic properties, isoelectric point, glycosylation, molecular mass, and many other characteristics.
Dear Viktor
I must agree that in a few words you have done an excellent summary of what I have said. However my end was essentially didactic to try to clarify the doubts of our young colleague. Thanks for your appreciation, also because much of modern science forgets fundamental aspects of protein chemistry.
Giovanni
Thank you so much Dr. Giovanni for sparing so much of your time to explain the things in detail.
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