Testing "stepwise" biochemical affinity for multimers?

December 4, 2015 6:43 AM

How would you test whether the affinity of a tetramer for a particular ligand proceeds stepwise, i.e. assuming that the tetramer can bind one ligand per subunit, how would you test whether an initial binding event made subsequent binding events on the same tetramer more likely? I can't think of a good way to isolate a single molecule in solution, so any experiments would have to be on a aggregate population which would make stepwise cascades in affinity impossible to measure. Maybe if the measurement was really really time sensitive? Thoughts?
posted by nicolas léonard sadi carnot to Science & Nature (3 answers total)

You run your assay as normal but fit it to the Hill equation which will determine the level of cooperativity present in the system.

You could also plot it as a logistic regression and by examining the slope of the graph at the inflection point you can determine the Hill coefficient that way as well.
posted by koolkat at 7:40 AM on December 4, 2015

What kind of equipment do you have access to? Can you use LC-MS or tandem MS?
posted by late afternoon dreaming hotel at 8:55 AM on December 4, 2015

Response by poster: I'm planning on doing my kinetics measurements with ITC or microscale thermophoresis. I can use a MALDI type thing downstairs, maybe not a column-based one though.
posted by nicolas léonard sadi carnot at 9:40 AM on December 4, 2015