If the time scale were in seconds, instead of minutes, I would suggest that inadequate mixing of the sample might be a factor. However, since the time scale is in minutes, this is unlikely to be the cause.

For the spikes, the most likely cause is air bubbles arising in the light path.

Instability of the product is probably responsible for the time-dependent absorbance decrease on the gray line. The solution to that problem is to run the reaction over a shorter time scale during which product instability is negligible.

To deal with anomalies at the start, you can take the approach of running a blank in parallel that has no enzyme, then subtracting the -enzyme curve from the +enzyme curve to remove the initial artifact. This will probably not be perfect, but it could be sufficient to allow you to better measure the initial rate, which is usually what is desired for enzyme kinetics.

A small absorbance difference of less than 0.10 absorbance units change is likely to fluctuate over several minutes. You are measuring this reaction for 13 hours. Enzyme is likely to become unstable and die over time. You are getting product but the rate is really slow. But good news is that you are getting product.

You should try increasing the enzyme concentration 10-fold to get a faster rate over minutes, not hours .If you don't have enough recombinant protein, guess you will have to purify more. Salt inhibits pretty much every single enzyme so if your purified protein is in 500 mM NaCl and you are 2X diluting it in the assay, you have a lot of salt in the reaction. Dilute your enzyme in the assay by a large factor to reduce salt inhibition.

Also low concentration of enzyme can stick to glass cuvettes and surfaces so in pretty much every single assay ever, investigators used 0.10 mg/mL bovine serum albumin (BSA) in the reaction component. That's really all that BSA does inhibit enzymes from sticking to surfaces, but it is quite useful.

Maybe the reaction will speed up if you increase the pH, but some enzymes actually work better under acidic conditions so you never know. Anyway, you should increase the rate of the reaction, somehow.

Hi there,

I'm a bit puzzled by the spectroscopic monitoring of pNP formation at acidic pH... The major contribution at 405nm comes from the paranitrophenolate... And as the pKa is around 7.5, I'm not sure about what you measure exactly in your assay...

You need to measure the initial reaction rate. If the change in absorbance is constant over a time period and then declines it is due to substrate being limited or product inhibition.

Can decrease enzyme concentration and standardise by changing substrate concentration till you get a constant reaction rate .

Then monitor initIla reaction rate at constant enzyme concentration and saturating substrate concentration.