Enzyme dilution: Dilute the enzyme preparation in the appropriate Assay Buffer (100 mM plus 0.1 mg/mL bovine serum albumin, at optimum pH for the enzyme being tested). An initial 1:100 dilution should be performed in a 100 mL volumetric flask followed by 10-fold serial dilutions to obtain an appropriate enzyme concentration. Pre-incubate the diluted enzyme in a water bath at 40 Celsius for ~5 minutes.
Substrate addition: Add 0.2 mL of pNP-substrate (10 mM) to 8 glass test-tubes (duplicate samples for 4 time points) and incubate in a water bath at 40 Celsius for ~5 minutes.
Enzyme reaction: Add 0.2 mL of enzyme (prepared in Step 4.23) to each of glass test-tubes containing substrate vortex and incubate duplicate samples in a water bath at 40 Celsius for 3, 6, 9 and 12 minutes. At the end of the incubation periods add 3.0 mL 2% tri-sodium orthophosphate to terminate the reaction and immediately vortex vigorously.
Reaction blank: Add 3.0 mL 2% tri-sodium orthophosphate to 2 glass test-tubes then add 0.2 mL of pNP-substrate (10 mM) and vortex then add 0.2 mL of enzyme to both tubes and immediately vortex. Incubate at room temperature.
Measure the absorbance of all samples at 400 nm .
- Zero the spectrophotometer with water.
- Measure the two blank samples (record these values then zero the spectrophotometer against one of them – assuming they are very similar).
- Measure the absorbance of all other samples.
Plot the absorbance versus incubation period for samples and apply the appropriate result(s) to the calculation.
1 unit of activity is the amount of enzyme required to hydrolyse 1 micromole of pNP-substrate to substrate and pNP per min at optimum pH and 40 Celsius.
Calculate activity as follows:
U/mL = (A400 / reaction time in min.) x (total reaction volume in mL / enzyme volume in mL) x (1 / extinction coefficient of pNP) x enzyme dilution factor
U/mL = (A400 / min.) x (3.4 mL / 0.2 mL) x (1 / 18.1) x dilution
A400/min = Absorbance (reaction)/min – Absorbance (blank)/min.
Incubation Time = 10 min.
Total volume in cell = 3.4 mL
Aliquot assayed = 0.2 mL
Extinction coefficient of pNP in 2% trisodium orthophosphate = 18.1
This calculation assumes that the rate of the reaction is linear over the whole 10 minute period for which the change in absorbance was recorded. If this is not the case then use the highest reaction rate recorded covering a period of at least 2 minutes within the 10 minutes recorded.