ml of citrate ml of 25 mM ml of water pH 48 buffer substrate NPP Substrate 1 20

Ml of citrate ml of 25 mm ml of water ph 48 buffer

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ml of citrate ml of 25 mM ml of water pH 4.8 buffer substrate, NPP Substrate 1 2.0 ml 0 2.50 Substrate 2 2.0 ml 0.025 2.475 Substrate 3 2.0 ml 0.035 2.465 Substrate 4 2.0 ml 0.05 2.45 Substrate 5 2.0 ml 0.10 2.40 Substrate 6 2.0 ml 0.20 2.30 Substrate 7 2.0 ml 0.40 2.10 Substrate 8 2.0 ml 0.60 1.90 Substrate 9 2.0 ml 0.80 1.70 3. Using the automated pipettes place into eighteen test tubes 5.0 ml of the 0.1 M NaOH solution. Call these the NaOH tubes. 4. Place the substrate tubes at room temperature. Let the tubes sit for 5 minutes before going to step 5. 5. Note the time when adding 0.5 ml of undiluted enzyme to each substrate tube. In each case gently mix the tubes. Immediately withdraw a 1.0 ml sample of each of the mixtures from the substrate tubes and place it in 5.0 ml of the 0.1 M NaOH. Call this the “zero time” tube. This should be done for each substrate concentration upon mixing. 6. 15 minutes after mixing remove 1.0 ml of the substrate tube mixture and add it to the 5.0 ml of 0.1 M NaOH. Keep track of which substrate solution each of the NaOH tubes represents. This is the “15 minute time” tube. 7. Read the absorbance of each tube at 405 nm in the Spectronic 20. Use 0.1 M NaOH to zero the system.
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18 8. For each concentration of substrate, subtract the A 405 at zero time from A 405 at 15 minutes. Using the standard curve and this difference in A 405 for each substrate, determine the concentration of p-nitrophenolate produced in each tube and plug this value into the following equation to calculate the μ moles p-nitrophenolate produced per hour at each pH. μ M p-nitrophenolate x (6.0 ml/ 1000 ml/L) x (5.0 ml/1.0) x 4 = μ moles p-nitrophenolate /hr 9. Calculate the concentration of substrate in each reaction mixture using the following equation: [S] = 25 mM x ml substrate/5.0 ml 10. Plot the following plots: a. Plot v o ( μ moles p-nitrophenolate /hr ) on the y-axis and [S] on the x-axis. b. Plot 1/v o on the y-axis and 1/[S] on the x-axis. From this plot determine K M and V MAX .
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19 Part F - Effect of Phosphate Inhibitor 1. Use undiluted enzyme in this portion of the experiment. Use 0.05 M ( 50.0 mM ) sodium phosphate (pH 4.8). 2. Set up the substrate tubes according to the following protocol. Be sure to use the substrate labeled as concentrated substrate, 25 mM, not the 2.5 mM NPP that you have been using in some of the previous parts of this experiment. ml of citrate ml of 25 mM ml of water ml of sodium pH 4.8 buffer substrate phosphate Substrate 1 2.0 ml 0 2.30 0.20 Substrate 2 2.0 ml 0.025 2.275 0.20 Substrate 3 2.0 ml 0.035 2.265 0.20 Substrate 4 2.0 ml 0.05 2.25 0.20 Substrate 5 2.0 ml 0.10 2.20 0.20 Substrate 6 2.0 ml 0.20 2.10 0.20 Substrate 7 2.0 ml 0.40 1.90 0.20 Substrate 8 2.0 ml 0.60 1.70 0.20 Substrate 9 2.0 ml 0.80 1.50 0.20 3. Using the automated pipettes place into eighteern test tubes 5.0 ml of the 0.1 M NaOH solution. Call these the NaOH tubes. 4. Place the substrate tubes at room temperature. Let the tubes sit for 5 minutes before going to step 5. 5. Note the time when adding 0.5 ml of undiluted enzyme to each substrate tube. In each case gently mix the tubes. Immediately withdraw a 1.0 ml sample of each of the substrate tube mixtures and place it in 5.0 ml of the 0.1 M NaOH. Call this the “zero time” tube. This should be done for each substrate concentration upon mixing.
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