Thank you all for the input. I've recently found a paper which
addresses glycerol and protein concentration in the 'selection' for
dimer over monomer, and likewise active over
inactive enzyme. The mechanism of glycerol "activation" may not be
well understood save speculation that glycerol is involved in
"preferential hydration" of the protein; particularly, the partial
specific volume of the protein is increased with increasing glycerol
concentration; this is according to the following paper: The authors
of the paper (Paul L. Darke, J. L. C., Lloyd Waxman, Dawn L. Hall,
Mohinder K. Sardana§, and and L. C. Kuo (1996). "Active Human
Cytomegalovirus Protease Is a Dimer." JBC 271(13): 7445-7449) use
20% vs. 10% glycerol to show an increase in enzyme specific activity
as well as an increase in the dimer (over the monomer) population
(i.e. Kd decreases by ~15x as the glycerol is increased from 10% to
20%). I am currently examining the possibility that if the monomer
population is indeed capable of dimerizing (e.g. it is not unfolded
irreversibly) then I should be able to select for the dimer by
equilibrating the protein sample into buffers of increasing glycerol
concentrations.
It seems like a simple matter--this business of monomer vs. dimer--but
there might be something very interesting in there after all!
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I have not used, during protein prep, more than 10% glycerol.
However, I did follow one protocol, at the end of the prep, to store the
purified enzyme in 50% glycerol at -80 degree frozen. I guess the percentage
of glycerol is not much a concern as long as your procedure/instrument
can endure the high viscosity/back-pressure. I am wondering though
about your rationale for increasing the glycerol. Are you suspecting
hydrophobic, non-specific stickiness of the protein be the culprit for your
problem? Is there other reasons that might cause the issue you observe?
disulfide bond formation? ionic interaction? protein unfolding? etc.
Practically, there are many steps/details that can be altered from one
prep to another, even though one may not realize it. I had many such
experiences, personally and from colleagues. It sure helps to back track
every little details of your operation to trouble-shoot. Hope you can
get over this hurdle soon. Good luck.
Ligong Wang
Yale University
