Sangeetha Damiano's Peroxidase Enzyme Lab

Peroxidase Enzyme Lab

Purpose

In this experiment you will investigate the effect of environmental factors on the enzyme hydrogen peroxidase. This enzyme is found in all aerobic (using oxygen) cells and functions to decompose hydrogen peroxide into O2(g) and H2O. The specific environmental factors you will test (as a class) are temperature, pH, substrate concentration, and enzyme concentration. Your team will select one of these factors (variables) to test and report on.

Materials

• 5 to 10 grams of freshly picked bindweed vines with leaves (about 1 handful)
• Digital balance (scale)
• Mortar and pestle
• Distilled water
• 3 100-liter glass or plastic beakers
• 1 mL or 5 mL syringe
• Hydrogen peroxide
• 1 Paper towel square (for filtration)
• Glass test tubes
• Test tube rack or holder
• Small plastic ruler
• Safety glasses

Depending on which environmental factor you choose to investigate, some of the following items will be needed for your experiment:

• Acid solutions with pH values between 2 and 6
• Alkaline solutions with a pH between 8 and 12
• pH test strips
• Ice
• Large plastic beaker (for ice bath)
• Hot water
• Large plastic beaker (for hot water bath)
• Thermometers

Procedure

This part will be determined by the students conducting the experiment. For an overview of the general scientific experimentation and research process, see the flow diagram below. Use the worksheet that follows to write out in detail the hypothesis you are seeking to test, the materials you will use, and the steps you will follow to conduct your experiment.

Enzyme Lab Worksheet

Hypothesis: If the pH level of a substance reacts with the hydrogen peroxidase and produces bubbles, then the higher the concentration of the pH substance, the more of a reaction there will be. The more reactivity it produces will increase the height of the bubbles.

Independent Variable: The independent variable in this experiment are the pH substances (sodium hydroxide ph 12 and hydrochloric acid ph 3,)

Dependent Variable: The dependent variable in this experiment is the height of the bubbles produced over a 80 seconds.

Controlled Variables: The Hydrogen Peroxidase is the control variable.

Justification of hypothesis: We chose this as our hypothesis because the greater intensity of the pH substance might produce more oxygen when it is reacting with the enzyme. The greater oxygen will produce more bubbles.

Materials (Your Team’s Experiment):

1 cc Hydrogen peroxide

10 grams Bindweed vines

30 mL Water

Digital balance

Mortar and Pestle

3 Test Tubes

1 cc sodium hydroxide ph 10

1 cc hydrochloric acid ph 3.5

Procedure:

1. Measure out 10 grams of bindweed leaves on the scale.
2. Use the Mortar and Pestle to crush up the bindweed leaves until a dark green, pulpy paste forms.
3. Add 30 mL of water to the crushed bindweed. Stir and crush until it is thoroughly mixed
4. Use a paper towel to filter the bindweed mixture into a beaker
5. Put 2 cc of the filtered bindweed liquid into a test tube
6. Place 1 cc of the Hydrogen Peroxide into the test tube.
7. Record the height of the foam produced every 5 seconds until foam has reached it’s maximum height.
8. Repeat steps 1 - 7 while combining 1 cc of sodium hydroxide ph 10 in with the bindweed leave paste.
9. Repeat steps 1-7 while combining 1 cc of hydrochloric acid ph 3.5 in with the bindweed leave paste.

Summary: Using the various pH liquids, we will measure the amount of oxygen (in centimeters) produced by putting 1 cubic centimeter of the pH liquid in 2 cubic centimeters full of bindweed liquid.

Data and Results:

(This section should include at least one graph!)

The height of the bubbles produced from the reaction between the enzyme ( Hydrogen Peroxide), the pH substance, and the bindweed liquid is showed in the graph. The height is calculated in centimeters.

Time in seconds	Control  (Hydrogen Peroxide pH 7)	Sodium Hydroxide      (pH 10)	Hydrochloric acid (pH 3.2)
0	3.3	4.4	3.5
5	4	4.5	4.5
10	4.5	5.2	4.9
15	4.6	5.5	5
20	4.8	5.9	5.2
25	5	6	5.2
30	5.2	6.2	5.3
35	5.4	6.4	5.4
40	5.5	6.4	5.4
45	5.6	6.5	5.3
50	5.6	6.5	5.3
55	5.6	6.5	N/A
60	5.7	6.5	N/A
65	5.8	6.4	N/A
70	5.9	6.3	N/A
75	5.8	6.2	N/A
80	5.8	6	N/A

Conclusions: 

My hypothesis for the experiment was that the more acidic or basic the substance, the more of a reaction would occur. However, during the experiment, we found out that the higher concentration of the substance did not produce more of a reaction. Based on our data, the more alkaline the substance was, the more of a reaction occurred. The basic solution, sodium hydroxide, produced the most bubbles because it was reacting with a substance that was slightly acidic. The bindweed liquid, having a pH of approximately 5, would not produce much of a reaction with substances closer to its acidity because there is less to react with.

While we were conducting our experiment, we noticed that the maximum height for the three different pH levels occurred at different times. For the hydrogen peroxidase, the maximum height of the bubbles occurred at 70 seconds. For the hydrochloric acid, the maximum height of the bubbles started at 35 seconds. The rate of reaction for the hydrochloric acid was the fastest because there was not much of a reaction and it did not cover much volume.The rate of reaction for the hydrogen peroxidase was the slowest because there was more of a reaction occurring and it covered more volume in the test tube.

While conducting the experiment, a few questions arose. What would have happened if we used another plant besides bindweed for part of our control? What would the outcome be? Would the sodium hydroxide still react the most? I think the answer to these questions would depend on the pH of control substance you are using. In this experiment, the bindweed we used had a given pH, which ultimately affected the reaction rates of the sodium hydroxide, hydrogen peroxide and hydrochloric acid. If the experiment used a plant with a more basic ph, the acid might probably produce a greater reaction.