enzymatic inhibition assay

[claire84]

Hello, I am new to your forum.
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!!!
thanks

[JackBean]

Hi there,
did you mix it for enough long time? How does the inhibitor work? Is it ir/reversible inhibitor? Could it react with something in the reaction mixture? What if you add only water (blank reaction)?

[claire84]

I mix it in an eppendorf by means of a micropipette before to start the measure.The inhibitor is a reversible one. In the reaction mixture there are only the enzyme, the substrate and the inhibitor dissolved in the buffer described before