For pH = 8.0, construct a table like below for each [S]
Abs.410nm 50 µM PNP
0.818
Time (seconds)
D Abs.410nm (0.0 mM PNPP)
D Abs.410nm (0.01 mM PNPP)
D Abs.410nm (0.025 mM PNPP)
D Abs.410nm (0.05 mM PNPP)
D Abs.410nm (0.1 mM PNPP)
D Abs.410nm (0.15 mM PNPP)
0 (without enzyme)
0 0 0 0 0 0
5 0 .002 .005 .003 0.010 0.004 20 0 .006 .013 .008 0.016 0.014 40 0 .012 .017 .022 0.017 0.021 60 0 .019 .023 .032 0.024 0.030 80 0 .026 .031 .042 0.031 0.040 100 0 .033 .037 .051 0.038 0.049 120 0 .039 .044 .061 0.045 0.060 140 0 .044 .050 .070 0.052 0.070 160 0 .050 .057 .079 0.059 0.080 180 0 .055 .063 .088 0.066 0.089
Do the following for both data sets!!
– Construct µM product formed / unit time using the extinction coefficient for PNP and the Beer- Lambert Law and calculate the rate of reaction/alkaline phosphatase activity as either µmole product formed min–1 or nmole product formed min–1 at pH = 8.0.
– Construct a Hyperbolic (saturation) curve of Alkaline Phosphatase activity (µmole min–1 or nmole min–1 product formed vs. [Substrate]) at pH = 8.0.
– Construct a Lineweaver-Burk (double-reciprocal) plot (1/v vs. 1/[S]) with appropriate identification/calculation of kinetic constants (Vmax, Km, kcat) at pH = 8.0.
Determine: Vmax, Km, Kcat, and Enzyme efficiency at pH8
For pH = 7.0, construct a table similar to below for each [S]
Abs.410nm 50 µM PNP
0.678
Time (seconds)
D Abs.410nm (0.0 mM PNPP)
D Abs.410nm (0.01 mM PNPP)
D Abs.410nm (0.025 mM PNPP)
D Abs.410nm (0.05 mM PNPP)
D Abs.410nm (0.1 mM PNPP)
D Abs.410nm (0.15 mM PNPP)
0 (without enzyme)
0 0 0 0 0 0
5 0 .001 .002 .003 0.004 0.004 20 0 .004 .003 .004 0.011 0.014 40 0 .007 .007 .009 0.015 0.021 60 0 .009 .013 .010 0.019 0.025 80 0 .010 .011 .012 0.021 0.030 100 0 .013 .017 .019 0.025 0.039 120 0 .015 .014 .021 0.028 0.040 140 0 .017 .020 .020 0.032 0.045 160 0 .019 .027 .029 0.039 0.050 180 0 .020 .033 .038 0.046 0.059
Do the following for both data sets!!
– Construct a µM product formed / unit time using the extinction coefficient for PNP and the Beer- Lambert Law and calculate the rate of reaction/alkaline phosphatase activity as either µmole product formed min–1 or nmole product formed min–1 at pH = 7.0.
– Construct a Hyperbolic (saturation) curve of Alkaline Phosphatase activity (µmole min–1 or nmole min–1 product formed vs. [Substrate]) at pH = 7.0.
– Construct a Lineweaver-Burk (double-reciprocal) plot (1/v vs. 1/[S]) with appropriate identification/calculation of kinetic constants (Vmax, Km, kcat) at pH = 7.0.
Determine: Vmax, Km, Kcat, and Enzyme efficiency at pH8
