Need help understanding data for a kinact/Ki study to determine the rate of covalent modification of compounds

[Spezialemic]

I’m doing a study to determine the rate of covalent modification, also known as kinact/Ki. From the literature, I see that it is determined by plotting percent occupancy over time (DOI: 10.1177/1087057116671509). I ran a 3 pt (8 compounds) fluorescent assay over 180 minute and normalized the data to the blank at time t and the final positive control. This activity vs time was converted to percent occupancy over time with a typical (1-value)*100. The graphs this generates are not consistent with what is expected from the literature, and I’m curious as to whether I have done something incorrectly, or if these compounds are not, in fact, covalent; I’m not sure how likely that is.

I have included the spreadsheet. Keep in mind if you backtrack the data that E,G, and H9 and all column 10 values from the plate were excluded due to the availability of the substrate.  Because of this, looking at values besides those in compounds 5, 7, and 9 are most representative of the proper formatting. 
 

Any help would be greatly appreciated! Thank you

Kinetic Study Post.xlsx

[Spezialemic]

For reference, the literature:

[BabcockHall]

The asymptote at low concentrations of I to a graph of the pseudo first order rate constant kobs vs concentration of I  is kinact/K_i.  It is unclear to me how you could find this number without varying .  I am pressed for time right now, but I might be able to say more later.

Edited August 15, 2019 by BabcockHall

[BabcockHall]

My previous message was not edited correction (my apologies for the confusion).  I meant to write: "...without varying the concentration of I."  Are you working at more than one concentration of inhibitor?

Edited August 16, 2019 by BabcockHall