A reaction mixture consisting of 500 μL of pNPA at concentrations ranging from
1.2 to 6.0 mM, 400 μL of 13.1 mg mL1 bile acid extract (equivalent to 18.7 mM cholic acid in the total mixture), 500 μL of each PP solution at concentrations ranging from 50 to 300 μg mL1 , and 100 μL of a working solution of protease of 45 U mL1 (one unit is the amount of enzyme required to convert 1 μmol of pNPA) was prepared. All of the solutions were prepared in 50 mM phosphate buffer pH 7.0 containing 20 mM NaCl. The mixture was incubated at 37 1C and the absorbance of the pNP produced was measured at 415 nm every 5 s throughout 60 s.