Chemical Principle

Orthophosphoric Monoester + H₂0 → Alcohol + H₃PO₄

Assay Reagents

Buffers: 0.1M Tris buffer, pH 8.0 and 1.0M Tris buffer, pH 8.0.

Enzyme: Dilute with 0.1M Tris Buffer.  Acceptable dilution: 5-20 µg/ml.

Substrate: 0.001M p-nitrophenylphosphate (P-NPP) in 1.0M Tris, pH 8.0.

 Procedure

1.    Add 2.9 mls of substrate to a Reaction tube and 2.9 mls to a Control test tube.

2.    At time = 0, add 100 µl of the diluted enzyme to Reaction tube and 100 µl Tris to the Control tube.  Mix thoroughly.

3.    Measure and record the optical density at 410 nm (OD 410) every 15 seconds for 3 minutes, or take the end point reading after 3 minutes by stopping the reaction with 100 µl of 5.0 M NaOH.

4.     Use the OD 410 measurement to determine the rate of change in absorbance per minute.

Enzyme Activity Calculations:

One unit of activity is the amount of enzyme to decompose 1 µmole of P-NPP/minute at 25° C.

1.62 x 10⁴ cm^-1 is the molar absorbance of P-NPP.

OD410 / min = ( OD 410 / 3 min – OD 410 Control / 3 minutes ) / 3 minutes

mg enzyme / ml reaction mixture =  [enzyme dilution] / 30

units / mg =  ( OD 410 / min )  /  1.62 x 10 ⁴  ml reaction mixture