|MadSci Network: Biochemistry|
Thanks for your question. To understand the effects of salt on protein solubility, we have to think about the surface of a protein molecule and how it interacts with ions when in solution.
Take a look at this graphic, which is a surface representation showing a soluble protein fragment. The red areas are negative charge (acidic residues), the blue are positive charge (basic residues). When proteins fold, they tend to bury hydrophobic residues in their core. However, there will always be hydrophobic surface "patches", exposed to the solvent. The surface of a folded protein is therefore a patchwork mosaic of charged, uncharged and hydrophobic regions.
Now, what happens when we add a salt, such as ammonium sulfate, to a protein in solution? At low salt concentration we see an effect called "salting-in", in which the solubility of the protein increases slightly. What's happening here is that ions from the salt associate with the surface of the protein. This shields those areas from the solvent molecules (water). In effect, this means that less water molecules are required to interact with the protein surface and the concentration of "free" water is increased. We say that the "activity" of the water has increased. The net effect is that the protein becomes more soluble.
At higher concentrations of salt we see the reverse effect, "salting-out". What's happening now is that all the binding sites on the protein surface for the salt ions have become occupied and so the ions begin to interact with the solvent. The concentration of "free" solvent molecules decreases as they are used to solvate the salt ions. Protein molecules therefore move closer together and begin to interact with one another via the hydrophobic or charged patches on their surfaces. At some salt concentration (which depends on the salt and the size/charge characteristics of the protein), the protein molecules aggregate and come out of solution.
This behaviour is the basis for some clever techniques in protein purification. You probably know that ammonium sulfate is often used for salting-out; it is highly-soluble and normally non-denaturing (so will have no adverse effects on protein activity). A crude purification can be achieved using an ammonium sulfate "cut", in which the saturation of ammonium sulfate in a crude protein extract is increased incrementally until the protein of interest precipitates. It's then possible to perform "hydrophobic interaction chromatography" (HIC). In HIC, a hydrophobic matrix is equilibrated in buffer containing a high concentration of salt (usually ammonium sulfate). A protein sample from an ammonium sulfate cut in which the protein is just soluble is then applied to the matrix and the protein binds via hydrophobic interactions. By gradually lowering the salt concentration in the buffer, hydrophobic interactions are decreased and proteins elute from the matrix at different salt concentrations and time points depending on the strength of their hydrophobic interactions.
Hope this helps with your question. I've included some links to more detailed information at the end of this answer.Neil
Try the links in the MadSci Library for more information on Biochemistry.