BIOLOGY 371
INDEPENDENT RESEARCH
WEEK 9
Determination of Thermal Stability
1. Perform assays according to standard procedure.
2. Incubate assay cuvette in circulating water bath.
3. Vary temperature of assays by adjusting temperature settings of circulating
water bath. Start at 10° C and increase
temperature at 5° C steps. Once an apparent
maximal temperature has been established, fine tune the peak by varying
temperature by 1 - 2 degrees and do triplicate assays.
4. For greater accuracy, determine the temperature in blank cuvette.
5. Plot percent maximum activity or initial velocity vs temperature.
Figure 14. The effects of temperature
on activity of isocitrate dehydrogenase from cauliflower.
Determination of pH Optima
1. Perform assays according to standard procedures,
except substitute phosphate buffers with a range of pH values.
2. Use a single enzyme preparation for all assays.
3. Perform triplicate assays of activity at each pH.
4. If two or more buffers are used, be certain to "over-lap" pH
ranges.
5. Initially, determine pH optima using buffers differing by 0.2 pH unit.
6. "Fine tune" your data by using buffers differing by 0.05 -
0.1 pH unit.
7. Plot percent maximum activity or initial velocity vs pH.
Figure 15. The effects of pH on activity of isocitrate dehydrogenase from
cauliflower.
WORKSHEET
Data Collection Form - pH profile
| pH* of Assay Buffer | Slope @ 0.5 A | Mean Slope | % Max. Activity |
| 6.99 | |||
| 7.08 | |||
| 7023 | |||
| 7.38 | |||
| 7.41 | |||
| 7.54 | |||
| 7.60 | |||
| 7.75 | |||
| 7.87 | |||
| 7.95 | |||
| 8.03 | |||
| 8.23 | |||
| 8.34 | |||
| 8.44 | |||
| 8.54 | |||
| 8.61 | |||
| 8.70 | |||
| 8.78 |
© Copyright 2000 Department of Biology, Davidson
College, Davidson, NC 28036
Send comments, questions, and suggestions to: jowilliamson@davidson.edu