Question

How to calculate enzyme activity from pnp standard curve?

Answer 1

Enzyme units are expressed in µmol substrate converted per min.

If the question gives enzyme activity in nmol per min, divide by 1000 to convert to µmol.

Then multiply by the volume to get the total number of units.

In summary, the total number of enzyme units 

Answer 2

I need to use Megazyme pNP-oligosaccharides in couple of days. I have established pNP standard curve by taking pNP dilutions 0 to 1 mM (1 µmol/mL) in 20mM tris (pH ~6, optimum pH of my enzyme too). I added 1M sodium carbonate (Na2CO3) to all the samples in the standard range including blank. After mixing well, I took 300 ul of each in flat bottom clear 96 well plate, measured absorbance at 405 nm, 410 nm, and 420 nm on Spectramax plate reader. Yet, I have got best absorbance at 420 nm plotted against known concentrations at X axis with a good equation at 420 nm y = 0.9997x - 0.0321, R² = 0.9998. (so, 0.9997 may be an uncorrected extinction coefficient). From this equation, I can calculate unknown concentration of pNP released during assay. My standard curve equation is persistent by repeating with same plate and some plate and fresh solution. I believe that the equation should give me direct calculation of unknown concentration by putting absorbance (y) in the equation i.e. X (unknown Concentration mM) = Y (Absorbance at 420 nm) + 0.0321/ 0.9997