An alkaline phosphatase reaction mix contains a buffer at a chosen pH, enzyme, and its substrate, p-nitrophenylphosphate. High pH solutions will slowly hydrolyze the phosphate from the substrate, resulting in non-enzymatic formation of product, p-nitrophenol. Concentrations of alkaline phosphatase reaction mixture in a 1 mL final volume are: 750 uL of 0.075 M buffer, 200 uL of 2.0 mM p-nitrophenylphosphate and 50 uL of alkaline phosphatase (stock concentration 0.125 mg/ml). Reaction is started by adding the appropriate volume of 10 mM p-nitrophenylphosphate which is measured by continuous assay.
How do I calculate IU for p-nitrophenolate, p-nitrophenol, and total product? Assay pH and change in Absorbance/minute are known. I know the equation I need to use is: IU = {(deltaAbs/min)(10^6 umol/mol)(vol of assay in L)} / (Molar absorption coefficient)