Enzyme Lab

This lab tested how enzymes affected a substrate.

Objective: The purpose of this lab was to test our hypothesis on how the enzyme affected the substrate at different increments of time.

Procedure #1

Hypothesis: If the enzyme solution is added to the substrate solution, then the solution will change color.

Materials:Turnip peroxidase 0.1% hydrogen peroxideGuaiacol

Distilled water3 test tubesStop WatchPipettesGraduated cylinderSyringesTest tube rackParafilm

Procedure: Using the one of the three test tubes:

Label the test tube “substrate”add 7 mL of distilled water

add 0.3 mL of 0.1% hydrogen peroxide, add0.2 mL of Guaiacol2. Cover the test tube with the parafilm and gently mix.3. Place the test tube in the test tube rack.4. Repeat steps 1-3 with another test tube. Label this test tube “control”4. Using the last test tube:

Label the test tube “enzyme”add 6.0 mL of distilled wateradd 1.5 mL of peroxidase

5. Cover the test tube with the parafilm and gently mix.6. Place the test tube in the test tube rack.7. Pour the substrate test tube into the enzyme test tube.8. Immediately begin timing the reaction.9. Rotate the test tubes and record the changes in color at 0, 1, 2, 3, 4, and 5 minutes.10. Using the color chart figure out what the change in color was at each time increment.

Independent variable: Enzyme Dependent variable: The color of the solutionControl: “control” test tube with only the substrateConstant: Substrate

Data:Test-tube 0 min 1 min 2 min 3 min 4 min 5 minEnzyme & Substrate

0 1 1 2 2 3

Control (Substrate alone)

0 0 0 0 0 0

Data Analysis:

The test tube with the enzyme and substrate continued the further change color as the time increased. On the other hand the control test tube with just the substrate did not change color and remained clear over the given time period. Other groups in the class received similar results.

You measured the color change at different times. Which time will you use for your later assays? Why?I will use the 3 minute color level 2 for the later procedures because this was the first color change.

When you use this assay to assess factors that change enzyme activity, which components of the assay will you change? Which will you keep constant?I will change the pH of the solution and keep the enzyme constant.

Conclusion:The hypothesis was supported by the experiment. The procedure used was efficient for the

hypothesis tested. Any errors may have resulted from poor measuring of the enzyme and substrate.

Procedure #2

What do you predict will occur if the pH in the reaction changes? How do you justify your predictions?I think by changing the pH in the reaction changes, I think the reaction time will change.

Hypothesis: If the pH is lower then the color change will be less.

Materials:Turnip peroxidase0.1% hydrogen peroxideGuaiacolBuffers with range of pHDistilled (deionized) water12 test tubes and test tube rackTimePipettesGraduated cylinder

Procedure:Make six sets of pairs of substrate and enzyme tubes, using a different pH substrate for each test tubeFor each substrate tube in a pair add:

add 7 mL of distilled water 0.3 mL of hydrogen peroxide 0.2 mL GuaiacolFor each enzyme test tube add:6.0 mL and a specific pH solution1.5 mL of peroxidaseCover each test tube with parafilm and mixCombine the substrate and the enzyme tubes for all six pairsCover with parafilm and gently mixBegin timing the reactionRecord the observation for each test tube, and record the color change at 0 minutes and at 3 minutes

Independent variable: pHDependent variable: The color of the solutionControl: Control from procedure 1Constant: Substrate and enzyme

Data:Test tube number Color after 3 minutes1 (pH 3) 12 (pH 5) 23 (pH 6) 24 (pH 7) 35 (pH 8) 36 (pH 10) greenControl 3

Data Analysis:

The higher the pH was the bigger the color changes were. Other groups in the class had similar results.

In procedure 1, what was the limiting factor of your baseline reaction the enzyme or the substrate? How could you modify the procedure you learned to answer this question?The enzyme; the procedure could be modify to test just the substrate after a time period.

What are three or four factors that vary in the environment in which organisms live? Which of those factors do you think could affect enzyme activity? How would you modify your basic assay to test your hypothesis?Temperature, pH, air quality. Temperature and pH can affect enzyme activity. Procedures 2 and 3 test this hypothesis.

Conclusion:In conclusion our hypothesis was not supported. The lower the pH did not result in a smaller

color change. The technique used many have been flawed, or errors could have resulted from poor measurement and poor timing.

Procedure #3 : Individual Investigation

Hypothesis: If the temperature of the solution is changed, then the enzyme will respond differently at higher and lower temperatures.

Materials: 8 test tubes Hot plate Ice Beakers Turnip peroxidase 0.1% hydrogen peroxide Guaiacol Pipettes Parafilm Timer

Procedure:Create enzyme test tubes following the steps in procedure 1Create substrate test tubes following the steps in procedure 1Pour one enzyme test tube into each substrate test tubeUsing one of the test tube place it in a beaker of iceRecord the color change after 3 minutesUsing another test tube measuring the reaction after 3 minutes (room temperature)Using the third test tube place it in a beaker of hot waterFirst warm the water on the hot plateRecord the color change after 3 minutesUsing the last test tube place it in a beaker of boiling waterBoil the water in a beaker on the hot plateRecord the color change after 3 minutes

Independent variable: TemperatureDependent variable: The color of the solutionControl: room temperature test tubeConstant: Substrate and enzyme

Data:Test At beginning After 3 minutesCold (5° C) 0 4Room Temperature (24° C) 0 4Hot (50° C) 0 4Boiling (94° C) 0 0

Data Analysis:

The experiment results show that the cold, room temperature, and hot solutions all had the same color reaction. The boiling solution did not have a color change, most likely because the enzyme denatured. The other groups in the class did not perform the same procedure, so there are no other results to compare to.

Conclusion:In conclusion, the hypothesis tested was supported. The different temperatures produced

different results in not all, but one temperature. This shows temperature does affect enzyme activity. Any flaws or sources of error in the technique used to test the hypothesis may have resulted from poor measurement of the enzyme and not timing the reaction correctly.