Reference no: EM131621046
Activity - Effect of temperature on enzyme activity
Materials
Students work in groups of four
Alkaline phosphatase (stored on ice)
Buffer solution pH 10.5
7.5 mM substrate - p-nitrophenyl phosphate (pNPP)
Method
You will be measuring the enzyme activity at five different temperatures (25°C, 30°C, 37°C 45°C and 60°C).
1. For each temperature set up two tubes -10 tubes in all.
| Reagent |
Blank |
Test |
| Buffer pH 10.5 |
1.0 mL |
1.0mL |
| 7.5 mM Substrate (pNPP) |
1.0 mL |
1.0 mL |
2. Pre-incubate the tubes at their respective temperature for 5 minutes, then add 1.0 mL of alkaline phosphatase to each of the tests at 30 second intervals and mix thoroughly.
3. Incubate all tubes for 10 minutes at the respective temperature.
4. Terminate the reaction by adding 1 mL 2 M NaOH to each of the tests, again at 30 second intervals and mix.
5. Add 1 mL 2M NaOH to the blanks and mix.
6. Add 1 mL of alkaline phosphatase to each of the blanks and again mix.
7. Remove all tubes from the water baths, stand for 5 minutes to allow the tubes to cool to room temperature.
8. Set the spectrophotometer to read absorbance at 400nm and zero using water.
9. Read the absorbance of each solution.
Data - Effect of temperature on enzyme activity
| Temp (°C) |
Tests Abs at 400nm |
Blanks Abs at |
Net Abs |
Enzyme Activity (nmoles pNP.min-1.mL-1) |
| 25 |
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| 30 |
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| 37 |
|
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| 45 |
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| 60 |
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|
Results
- Subtract the blank absorbance from the corresponding test absorbance to obtain the net absorbance for each temperature.
- Using the Beer-Lambert Law calculate the enzyme activity at each temperature in nmoles pNP produced per minute per mL of enzyme (as before).
- Plot the data in the form of temperature on the x- axis and enzyme activity on the y-axis. Give the figure a meaningful title and label the axes.
Activity - Effect of substrate concentration on enzyme activity Materials
Materials
Students work in groups of four
Alkaline phosphatase (stored on ice)
Buffer solution pH 10.5
7.5 mM substrate - p-nitrophenyl phosphate (pNPP)
Method
1. Prepare the following 13 tubes.
|
Tests |
Blanks |
| Tube No. |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
13 |
| PH 10.5 Buffer (mL) |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
| 7.5 mM substrate (pNPP) (mL) |
0 |
0.1 |
0.2 |
0.4 |
0.6 |
0.8 |
0.1 |
0.1 |
0.2 |
0.4 |
0.6 |
0.8 |
1 |
| Water (mL) |
1 |
0.9 |
0.8 |
0.6 |
0.4 |
0.2 |
0 |
0.9 |
0.8 |
0.6 |
0.4 |
0.2 |
0 |
2. Pre-incubate the tubes for 5 minutes at 37°C.
3. Add 1 mL alkaline phosphatase to each of the tests (tubes 1-7) at 30 second intervals and mix.
4. Incubate all tubes for 10 minutes at 37°C.
5. Terminate the reaction by adding 1 mL 2 M NaOH to each of the tests (tubes 1-7) again at 30 second intervals and mix.
6. Add 1 mL 2 M NaOH to the blanks (tubes 8-13) and mix.
7. Add 1 mL of alkaline phosphatase to each of the blanks (tubes 8-13) and again mix.
8. Remove all tubes from the water bath.
9. Set the spectrophotometer to read absorbance at 400nm and zero using tube 1.
10. Read the absorbance of each solution.
Data - Effect of substrate concentration [5] on enzyme activity
| Substance concentration mM pNPP (fill in) |
Tests Abs at 400nm |
Blanks Abs at |
Net Abs |
Enzyme Activity (nmoles pNP.min-1.mL-1) |
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Results
- Subtract the blank absorbance from the corresponding test absorbance to obtain the net absorbance for each substrate concentration.
- Using the Beer-Lambert Law calculate the enzyme activity at each substrate
concentration in nmoles pNP produced per minute per mL of enzyme (as before).
- Plot the data in the form of [S] mM on the x- axis and enzyme activity on the y-axis. Give the figure a meaningful title and label the axes.
Hint: To calculate the concentration of substrate [S] for each tube, remember that the [S] is the concentration of substrate in mmolest-1 (mM) in the test mixture at the start of the enzyme incubation. This volume includes all of the reagents except the NaOH - don't forget the enzyme!