A friend is studying two subunits of a multi subunit
bacterial enzyme complex. The two subunits that he is
studying form a stable heterodimer in solution and a goal of
his research is to evaluate the nature of the interaction
between these two subunits. Both proteins can be expressed
and purified in large amounts for physical analysis.
He has made mutations in secific sites that directly
decrease the affinity of the two subunits for formation of
the heterodimer. Using gel filtration chromatography, he is
readily able to demonstrate the presence of the dimer but
needs to quantify the magnitude of the association constants
for the various mutants.
He has two questions:
1. How does one measure the association constant for two
proteins? (I have suggested using Biacore but we do not have
access to this methodology.)
2. What is the lower limit for an association constant
below which one would not expect to be able to observe
significant dimerization during gel filtration
chromatography?
Thanks in advance for any input and suggestions that might
point us to the right literature.
Mark Lively