I need some help to interpret my results…I’m working with anthrax lethal factor, which is a Zn2+ dependent metalloproteinase. I would like to calculate Ki of an inhibitor using a FRET inhibition assay.The FRET assay are run for 30 minutes at 25 °C. At the end I obtain a graph intensity vs time which presents an hump around 10 minutes.What does it mean?Can the protein have two binding site?
You can find attached the ASCII file of the graph…
When I perform the same assay, changing the order of reagents and incubating the enzyme with inhibitor before adding substrate, this humps do not appear.