Chemical and Biochemical Engineering 2290A/B Lecture Notes - Alkaline Phosphatase, Reaction Rate, Sodium Hydroxide
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Experiments A and B are 2 experiments performed by mixing alkaline phosphatase enzyme with Pnpp substrate. The reaction was run for 5 minutes for each tube, then stopped by adding NaOH.
A)Changing enzyme concentration
Tube | Reaction Buffer (mL) | 5.4 mM of pNPP substrate (mL) | 0.002 mg/ml of AP enzyme (mL) | 3M of NaOH (mL) | Absorbance | Final Enzyme concentration (mM) | Velocity (umol/min/mL) |
1 | 3.41 | 0.04 | 0.05 | 0.875 | 0.284 | ||
2 | 3.36 | 0.04 | 0.1 | 0.875 | 0.387 | ||
3 | 3.31 | 0.04 | 0.15 | 0.875 | 0.509 | ||
4 | 3.26 | 0.04 | 0.2 | 0.875 | 0.538 | ||
5 | 3.21 | 0.04 | 0.25 | 0.875 | 0.569 | ||
6 | 3.16 | 0.04 | 0.3 | 0.875 | 0.602 | ||
7 | 3.11 | 0.04 | 0.35 | 0.875 | 0.620 | ||
8 | 3.06 | 0.04 | 0.4 | 0.875 | 0.638 |
Calculations for each tube:
1)Calculate final enzyme concentrations in each tube
2)Use beers law to convert absorbance to concentrations (umol). Molar extinction coefficient of p-nitrophenol is 16.2 mM^-1cm^-1. Path length is 1 cm.
3)Calculate velocity (umol/min/mL), make sure to correct the concentration for NaOH addition
B)Changing ph
Tube | Reaction Buffer pH | Reaction Buffer (mL) | 5.4 mM of pNPP substrate (mL) | 0.002 mg/ml of AP enzyme (mL) | 3M of NaOH (mL) | Absorbance | Final substrate concentration (mM) | Velocity (umol/min/mL) |
1 | 7 | 3.39 | 0.04 | 0.07 | 0.875 | 0.194 | ||
2 | 7.5 | 3.39 | 0.04 | 0.07 | 0.875 | 0.244 | ||
3 | 8 | 3.39 | 0.04 | 0.07 | 0.875 | 0.276 | ||
4 | 8.5 | 3.39 | 0.04 | 0.07 | 0.875 | 0.347 | ||
5 | 9 | 3.39 | 0.04 | 0.07 | 0.875 | 0.451 | ||
6 | 10 | 3.39 | 0.04 | 0.07 | 0.875 | 0.292 | ||
7 | 11 | 3.39 | 0.04 | 0.07 | 0.875 | 0.102 | ||
8 | 12 | 3.39 | 0.04 | 0.07 | 0.875 | 0.056 |
Calculations for each tube:
1)Calculate final substrate concentrations
2)Use beers law to convert absorbance to concentrations (umol). Molar extinction coefficient of p-nitrophenol is 16.2 mM^-1cm^-1. Path length is 1 cm.
3)Calculate velocity (umol/min/mL), make sure to correct the concentration for NaOH addition