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To investigate the inhibition of enzymes in relation to Catechol Oxidase, specifically PTU.
How does the activity of PTU effect the activity of Catechol Oxidase?
An increase of the concentration of PTU in a solution will decrease the activity of Catechol Oxidase until it can no longer function.
If you increase the concentration of PTU, to a certain extent, then the activity of catechol oxidase will decrease as well.
Procedure
1. The spectrophotometer was adjusted to 540 nm and to read absorbance.
2. The spectrophotometer was blanked using a cuvette filled with water.
3. Three test tubes were prepared according to the table shown just before but the catechol was not added until it was time to start the test.
4.Once Catechol was added to Test Tube 1, a piece of paraffin was used to cover the test tube while its contents were mixed by rolling it.
5. Some of the contents in Test Tube 1 was poured into a cuvette and inserted into the spectrophotometer.
6. A reading of the absorbance was immediately taken and then subsequent readings were taken every 30 seconds for 5 minutes.
7. Steps 4-6 were repeated for Test Tubes 2 and 3.
8. Steps 3-7 were repeated two more times so that there was a total of three trials.
• Independent Variable: Inhibitors (PTU)
• Dependent Variable: the relative amount of light absorbance measured by the spectrophotometer
• Substrate: Catechol
• Enzyme: Catechol Oxidase
Discussion
• Test tubes that contained PTU oxidized the catechol at a slower pace.
• The catechol that was in the test tube without any PTU was oxidized at the fastest rate.
• Both of these corroborate the hypothesis that PTU inhibits the oxidation of catechol.
Ways to improve our experiment:
-use smaller levels of PTU concentration
-invert cuvettes between readings
Further Study:
-Testing different enzyme’s and their interactions with catechol oxidase