enzymatic inhibition assay - (Aug/23/2010 )

Hello!
I have to test the inhibition potency of same potential inhibitors against a metalloprotease, known as anthrax lethal factor, by means of a spectrophotometric assay.
The enzymatic reactions were performed in 250 mM Na2HPO4, 150 mM NaCl, pH=7.4 at 25°C with a LF concentration of 10 nM.
After the addition of LF to the substrate, the release of p-nitroaniline was monitored recording the absortion at 405 nm.
The inhibitory assays were performed using a substrate final concentration of 5 µM. After addition of the desired quantity of inhibitor (we test them at various concentration), at the time zero, LF was added and the decrease of substrate’s absorbance at 405 nm was followed.
At this point I have a problem because I observe, during the first second, a rapid decrease of assorbance followed by an increase of it. I do not obtain useful curves and I don't know how to analyse them.
Is it a problem of mixing?
help me please!!!

claire84 on Mon Aug 23 12:56:53 2010 said:

Are you doing this by hand? The best way I’ve found for rapidly mixing small amounts of substrate into a spectro-cuvette is to melt a plastic inoculation loop into a spoon shape. Then the substrate can be placed on top and with a single dunk it is both added and mixed in half a second.

DRT on Mon Aug 23 22:56:43 2010 said:

I add the substrate and the inhibitor in a eppendorf and I mix by means of a micro-pipette, then finally I add the enzyme and I move all the reaction mixture in the cuvette.Is this procedure wrong?

when i used to do these reactions i always started the reaction in the final vessel.

Do you add the enzyme in the cuvette?and then do you start the measurement?

claire84 on Wed Aug 25 15:18:09 2010 said:

yes. we add the enzyme with a mixer (plastic stick with foot on the bottom with wells to hold enzyme, pass it up and down a couple of times to mix) and start to read immediately.