Answering the "finding" in first sentence (the finding in the second sentence seems different) your inhibitor gives signal in your measure assay. You may test it by putting everything but not the enzyme to check that the inhibitor increases the signal.

Thank you very much for reply. I have already checked my nanoparticle solution is highly coloured so there is a dignificant increase in the absorbance. But after the reaction, I centrifuge the reaction mixture at high speed and measure absorbance of the supernatant which is almost free of nanoparticle solution's colour. Even then I find same pattern of absorbance. Can it be related in context to the sizes of enzyme molecules and the nanoparticles? As the available number of enzyme molecules is fixed compared to the number of nanoparticles which are variable. High concentration of nanoparticles makes the reaction medium messy and not favorable for interaction between particles and the enzyme molecules. Not sure about it but just thinking a lot.

Hi Muhammad

what are you measuring? Absorbance at wich wavelength?

And do you have data of same nanoparticle concentration at different inhibitor concentration?

And a futher question. Is it possible in your asay to measure real time, i.e. do repeated measures along a time period to see if absorbacnce changes (due to enzyme activity)?

I do not think that it is anything related with more or less favrouable interaccion of enzymes with nanonparticles but a more "unspecific" issue

Firstly, in determining the inhibitory activity of the nanoparticle, did you maintain both Enzyme blank and Substrate blank in the assay?

Secondly, is the nanoparticle the only component of the inhibitor drug (using it in isolation) or a component in a mixed solution? if not, is the concentration the varied with respect to the concentration of the drug?

Thirdly, enzyme inhibition and kinetics are best understood and suited for low concentrations of inhibitor (or substrate). so what are the concentrations used with respect to the enzyme concentration?

if these three questions are critically examined, we can proffer solutions to the question of why the inhibition values are decreasing.

Hi Rafael,

Thanks for reply.

I am doing anti-cholinesterase assay at 412 nm.

I do not have the facility to measure real time but I still record absorbance over a period of 10-30 minutes.