Decomposition Rate of H2O2 in an Enzyme Catalyst Reaction

Samantha A. Price
AP Biology
9-16-09
Lab #1
Introduction:
If an enzyme is added to hydrogen peroxide (H2O2) and let to react at different intervals, then the longer

the solution has to react, the less H2O2 will be present, until it is wholly consumed, due to it (H2O2) breaking

down.

A catalyst is a substance that speeds up a reaction by lowering the activation energy (EA) needed to

perform a reaction (Campbell Reece Biology 6th Edition). In this lab we used enzymes, a catalytic protein, to

lower the EA. The lab calls for a 1M solution of H2SO4 (sulfuric acid) to stop the reaction at given intervals (in

seconds). The use of sulfuric acid denatures, changes the configuration, of the enzyme’s activation site (where

the substrate, H2O2, binds to it). This shuts off the reaction allowing for a measurement of consumed H2O2 to be

measured with the aid of KMnO4 (potassium permanganate) that is added to a 5ml sample of the reaction

through a burette. The more KMnO4 used the more H2O2 is present (Class Discussion). This lab shows, or is

meant to show, how a catalysis solution progressively breaks down substrates until most have been broken

down then it slows gradually until it reaches a plateau where all available substraight is used.

Materials and Procedures:

• Burette

• 2% KMnO4

• 1M H2SO4

• H2O

• H2O2

• Enzyme Catalysis Solution

• Glass beaker(s)

• White paper

• Syringes or pipettes (in ml measurements)

• Stand for Burette

Put 10 ml of a 1.5% H2O2 into a clean glass beaker.

2. Add 1 ml of H2O (for base line calculation, control) or an Enzyme Solution (for the actual

experiment)

3. Add 10 ml of H2SO3 (1 M) using extreme care in handling reagents.

4. Mix well.

5. Remove a 5ml sample. Place it into another beaker and assay (test) for the amount of H2O2 as

follows. Place the beaker containing the sample over a piece of white paper. Use a burette, a syringe, or

a 5ml pipette to add KMnO4, a drop at a time, to the solution until a persistent pink or brown color is

obtained. Remember to gently swirl the solution after adding each drop. Check to be sure that you

understand the calibrations on the burette or syringe.

And record readings.

6. Repeat step one and two (being sure to use enzyme catalysis instead of H2O).
 7. Swirl gently for a given amount of time (10, 30, 60, 90, 120, 180, and 360 seconds) repeating

step 6 for each given time.

8. At given time add 10ml of H2SO4 (1M solution)

9. Record data

Results, Data Collection & Analysis:

Base Line Calculation

Final reading of burette 16.7 ml
Initial reading of burette 6.6 ml
Base Line (Final - Initial) 10.1 ml KMnO4

Figure 1 (Determines the amount of H2O2 present in a 1.5% solution)

Time-Course Determination
Time (seconds)
KMnO4 (ml) 10 30 60 90 120 180 360
a) Base Line * 10.1 10.1 10.1 10.1 10.1 10.1 10.1
b) Final Reading 17.4 17.3 17.6 17.7 17.8 17.9 18.2
c) Initial Reading 17.3 17 17.4 17.6 17.7 17.8 18.1
d) Amount of KMnO4 used (b-c) 0.1 0.3 0.2 0.1 0.1 0.1 0.1
e) Amount of H2O2 used (a-d) 10 9.8 9.9 10 10 10 10
*Base line tells how much H2O2 in the initial 5 ml sample. (Figure 1)

Figure 2 (Depicts the amount of H2O2 decomposed)

 Time Intervals (seconds)

Initial 0 to10 10 to 30 30 to 60 60 to 90 90 to 120 120 to 180 180 to 360

Rates (ml
H2O2/sec) 1 -0.001 0.003 0.003 0 0 0

Figure 3 (Portrays the slope of our graph in Figure 5)

Uncatalyzed H2O2 decomposition

a) Final reading of Burette 31.4
b) Initial reading of burette 18.2
c) Amount of KMnO4 titrant (a-b) 13.2
d) Amount of H2O2 spontaneously decomposed (ml baseline- c) -3.1
What percent of the H2O2 spontaneously decomposes in 24 hours [(d/ml baseline) x 100] -31%

Figure 4 (Shows spontaneous decomposition of H2O2)

 Decomposition of H2O2
12
10
Amount of H2O2 used

8
6
4
2
0

10

0
30

60

90

36
12

18

Time (seconds)

Figure 5 (Depicts our results of consumed H2O2)

Figure 6 (What our results should have looked like)

Discussion/ Conclusions:
Major conclusions were hard to draw from our data due to all the errors. Figure 4 shows how much

H2O2 would evaporate (spontaneously decompose) over a 24-hour period. But do to some unknown factor, the

sample of H2O2 didn’t evaporate but it took in Hydrogen and Oxygen from the surrounding atmosphere. Also, as

seen in Figure 2 and Figure 3 there is a negative slope/ less KMnO4 used (less H2O2 used). Another factor come

into play when we see that the catalyses were too strong and very high amounts of H2O2 was consumed even at

the 10-second interval.

Although the data made it hard to draw any clear conclusion, the graph provided in the lab (Figure 6 is

much like the original graph, and is my version of it recreated), shows the desired results. If we had ran the

experiment and had less errors our data would be much like the graph of Figure 6. But, with the data we

collected our hypothesis cannot be confirmed nor deigned. It would be best to run the test again and try to

eliminate all possible errors.

Literature Citation(s):
1. Campbell Reece Biology 6th Edition
2. Class discussion

Questions:
1. Why did our calculations for our Uncatalyzed H2O2 decomposition come out to be a negative %?
a. The % of spontaneous decomposition was a negative due to little evaporation and possible
absorbance of Hydrogen and Oxygen from the atmosphere around the sample.
2. Why was so little KMnO4 used?
a. The catalyses were too strong.
3. What does the sulfuric acid do to the catalase?
a. It denatures the catalase stopping reactions.