I am trying to standardize de-acetylation assay for my enzyme and I have taken no enzyme reaction as blank. My substrate is labelled with AMC and using NAD as cofactor.
I am not getting saturation curve for both varied substrate and varied enzyme concentrated catalytic reaction.
I am not sure whether my enzyme is working efficiently because only peptide and test reaction with enzyme giving same intensity. One more problem is '0'th time reaction is showing higher florescence intensity instead of the 1hr reaction?
I am not able to understand the problem please help
If you see a linear dependence of the reaction rate on the substrate concentration, rather than saturation, a probable reason is that the Km for the substrate is much higher than the range of substrate concentrations you are using.
You should see that the reaction rate is directly proportional to the enzyme concentration. That is expected.
If you are seeing no reaction, your enzyme may be inactive due to denaturation, misfoldiing or aggregation. Or the buffer conditions you have chosen are far from optimal for the enzyme. Or you have omitted an essential ingredient that the enzyme requires for activity, such as Mg2+.
As for the observation that the fluorescence intensity is higher at t=0 than at t=1 hour, is this a large enough difference to be meaningful, or just random variation? Is it possible that you have allowed the fluorophore to become photobleached during the hour through excessive exposure to light? Has the sample evaporated during that time, reducing the volume of it that the light passes through? Has the peptide adsorbed to the surface of its container, reducing the concentration left in solution?
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