Solubility of protein in supernatant

[TytoAlba95]

Homework Statement

Details in picture

Relevant Equations

None

Answer: (d)

My understanding:
1. At pH 6.4 i.e. less than 6.5, protonation or deprotonation will just begin (if we imagine that we are adding an acid to the protein soln), so not many protein molecules will be there in the supernatant
2. Glycerol interacts through H-bond with surface polar amino acids, 10% might be significant enough to observe good solubility.
3.10mM CaCl2- I don't know if I'm thinking rightly but CaCl2 in solution will interact with the polar amino acids and increase their solubility in the supernatant.
4.(NH4)2SO4- (NH4)2SO4 is used to both salt in and salt out proteins. At lower concentration it should salt in, I'm unaware of the threshold value at which proteins are solubilised by the salt.

 

[chemisttree]

Each of these treatments lowers the solubility of the protein. There is no salting in or increase in solubility for any of them, just different degrees of reduction of solubility.

I hate questions like these. You need to know which conditions are the most strongly influencing for protein solubility. Is the isoelectric point more strongly influencing than 40% (NH₄)₂SO₄? The fact that the protein binds calcium strongly suggests that calcium treatment will strongly reduce solubility but is that more effective than 10% glycerol?

My guess is that 40% ammonium sulfate is one of the more effective precipitating agents so either b or d on the graph must be that treatment. This leaves only d) as a reasonable answer. If this answer is correct, then CaCl₂ is the least effective precipitant. Why?