Question

1. In protein salting out experiment, we're told to separate globulin and albumin from protein solution by centrifuge. What causes the difference of extent of centrifuge speed and solubility in water between globulin and albumin?
2. Why for any protein, the extent of precipitation and the fluidity are different    in several organic solvent(including water, acetone, and ethanol.)?
3. Why adding more HCL will increase the extent of precipitation for any protein in different organic solvent?

Answer 1

1. The extent of centrifuge speed and solubility depends on the properties of the protein such as its ionic strength, hydrophilicity, hydrophobicity, etc... In distilled water, globulin is insoluble whereas in dilute salt solutions globulin is slightly soluble and can be precipitated (commonly used salt solution is 50% saturated ammonium sulfate). On the other hand, albumin is readily soluble both in water and in dilute salt solutions. If using saturated ammonium sulfate solution, then albumin precipitates first. This principle of difference in solubility of proteins is used in salting out experiments.  

2. The extent of precipitation and fluidity for different proteins vary because every protien has distinct amino acid compositions. Hence, different proteins precipitate at different salt solution concentrations. Solvents (water, acetone, ethanol, etc...) and salt concentrations are chosen to separate out the proteins at a level at which it becomes insoluble. Solubility of proteins in water varies depending on the level of hydrophilic and hydrophobic properties of the protein surface. Protein surfaces having higher hydrophobic properties will precipitate readily.

3. Adding more HCl will increase the extent of precipitation for any protein in different organic solvents because most of the proteins are insoluble or less soluble in solutions with a higher concentration of salt concentration. This is because as more salt is added the ions present will shield the protein (shielding effect) with the multiple ion charges and nullify the activity.