Discovering the Effects of pH on Catalase in Purple Cabbage
By Wanda Cornwell Donthnier
December 3. 1999
Table of Contents
Materials and Methods
Catalase is an enzyme that detoxifies hydrogen peroxide, and other similar byproducts, into water and oxygen. An experiment was done to discover the level of pH at which catalase functions most effectively. In this experiment, pieces of purple cabbage and distilled water were liquefied together and combined with various solutions, to which hydrogen peroxide was added, in order to test for reactions at different pH levels. Fizzing, foaming and color change are indications of enzyme activity. Testing seven different variables showed that catalase functions better in a neutral pH than it does in either an acidic or alkaline pH level.
Does the pH of a substance have any effect on how catalase functions in
breaking down toxic chemicals, such as hydrogen peroxide, into oxygen and
Catalase functions differently in different levels of pH.
Acidic or alkaline pH inhibits the enzyme activity of catalase; a neutral pH is an optimum environment for the catalyst to break down peroxide
Purple cabbage was used in the experiment because this plant contains catalase, has a purplish-red color that is sensitive to acid or alkaline solutions, and readily changes color when the neutral pH is disrupted (Carter, 1994).
This study is important because humans, as well as other organisms, have catalase, which helps to protect our cells from damage done by toxic chemicals in our bodies. The catalase in the human body also functions best at a specific pH level (Carter, 1994).
With this in mind, the following objectives were set up for this study:
Gusui Wu and Dilip M. Shah (1995) experimented with the catalase found in potatoes. Their function was to isolate and characterize a subunit of catalase, cDNA (Cat 1). They discovered that the nucleotide sequence of potato catalase is very similar to that of tomato catalase and that the amino acid in potato catalase is similar to tomato, cotton, and maize.
Paula Ellen Burch (1989), for her thesis submitted to Rice University, studied DBA damage and cell lethality (toxicity and death producing amount of poison). Lung mitochondrion is a source of hydrogen peroxide, H2O2. When phagocyte cells (white blood cells that engulf and digest bacteria) are activated, they also produce H2O2. Several kinds of biological damage have been caused by H2O2 in the body including: lens of the eye, cell death in fibroblasts and bacteria, and tumors in Drosophila’s (fruit flies) embryos (3). Catalase catalyzes (breaks down and regulates the chemical reaction, but does not become a part of the reaction) hydrogen peroxide into water and oxygen. 2 H202---à 2H2O2 + O2. Catalase provides a protection against lethality (7, 8).
A group of students at Purdue University (1998) also used the potato to test the optimum temperature for an enzyme and the reaction of catalase as it breaks down H2O2 in cells. They washed, peeled, and cubed a potato. They then place a small potato tissue, 50mL of cold water, and 50mL of crushed ice in a blender and homogenized (broken down into a uniform consistency) it. They strained the mixture through coffee filters and poured it into test tubes. After centrifuging the mixture for 10 minutes at 1300X, they carefully poured the liquid, (catalase solution which had collected on the top of the mixture) into a clean test tube. 50mL of water was added to the catalase solution. The solution was then stored on ice. The research team then tested the effect of pH on enzyme activity by preparing H2O2 solutions at 3, 5, 7, and 9 pH levels by adding appropriate buffers. They placed 5 mL of H2O2 in a fifth, reference tube which had a small magnetic stir bar. Their reference too was a BioBench data acquisition software. H2O2 was added to each of the first four test tubes and then the change in the oxygen pressure was measured and the data was recorded. The conclusion was not published (Zanta, etal 1998).
Catalase was extracted from purple cabbage by tearing the leaves into small pieces, placing them in a blender with approximately and equal amount of distilled water (dH20) and blending until it was liquefied. The mixture was then strained so that only a purple liquid, containing the catalase, was left. The catalase was placed in a 250 mL beaker to be placed in test tubes. Variables in the experiment were created by filling seven test tubes in the following manner: 1) 5 mL dH20 (distilled water) 2) 5mL of the cabbage juice (catalase) 3) 5mL vinegar 4) 5mL ammonia (diluted one part ammonia to 10 parts dH2O) 5) 5mL dH2) and 5mL cabbage juice (catalase) 6) 5mL vinegar and 5 mL cabbage juice (catalase) 7) 5mL ammonia (diluted) and 5 mL cabbage juice (catalase).
As the solutions in test tubes 5-7 were mixed, observations of any color changes and/or reactions were recorded. The last three test tubes were placed in the vortex to thoroughly mix them. There was a slight color change when this was done.
Using pH paper, the pH level of each of the solutions was tested and the results were recorded. One at a time, 5mL of 3% H2O2 (hydrogen peroxide) was added to each tube of solution. As the H2O2 mixed with the solution in each tube, any changes in color or any reaction that occur were carefully observed and recorded. If there was a reaction, a notation was made as to whether it was a weak, strong, or moderate reaction.
The color changes in the solutions are important because they demonstrate the reaction of catalase in acidic or alkaline solutions as opposed to neutral pH. If the liquid fizzes and/or foams after H202 is added, this indicates that the catalase is breaking down hydrogen peroxide. It is important to make careful notations as to how strong the foaming reaction is. Results were charted, compared and evaluated.
|Tube #||pH Level||SOLUTION||COLOR||COLOR WITH H2O2||REACTION WHEN MIXED WITH H202|
|1||6||Distilled Water||Clear||Clear||No reaction, very weak carbonation|
|2||6||Cabbage juice||Purple||Pale purple||Strong Reaction, foamed|
|3||2||Vinegar||Clear||Clear||No reaction, very slight carbonation|
|4||11||Ammonia (diluted)||Clear||Clear||No reaction, no carbonation|
|5||6||dH20 & Cabbage||Purple||Lavender||Heavy fizz and carbonation|
|6||4||Vinegar & Cabbage||Fuchsia||Brighter hot pink||Not much reaction, very weak fizz|
|7||10||Ammonia (diluted)||Green||Yellow||Heavy fizz, moderate foam|
Notice that test tubes 1, 3, and 4 started clear in color and remained clear.
Test tubes 2 and 5 were purple because of the anthocyanins in the cabbage
(Carter, 1995). Tube 2 became a very light purple and tube 6 turned lavender.
Tube 6 started as a fuchsia color and became brighter hot pink when H2O2 was
added. Tube 10 started out green and became yellow.
There was almost not reaction in tubes 1, 3, and 4, with very little reaction in tube 6. Tubes 2, 6, and 10 showed the greatest reaction.
The results of the experiment show that there is a definite difference in the functioning of catalase at various pH levels. When catalase breaks H202 into H2O and O2, oxygen bubbles in the form of foam is produced. The stronger the fizz and the more foam produced indicate that the catalase is functioning at a more efficient level. The stronger reactions were shown when the pH was at a more neutral level of 6, as in test tubes 2 and 5. Each of these contained cabbage juice (catalase). Tube 7, a very alkaline solution with a pH level of 10, had a lot of fizz but only moderate foam, therefore the catalase was not functioning at optimum capacity. Although tube 1 also had a pH level of 6, there was no catalase in the solution so there was no catalyzing effect at all; the peroxide was not broken down. Tube 4, with a pH level of 11 had no catalase to break it down, so it remained highly alkaline. Tubes 3 and 6 had acidic pH levels of 2 and 4, respectively, but the catalase in tube 6 did not react very much, showing that catalase does not function well in acidic conditions. Tube 2 had no catalase, therefore there was no enzyme to break down the peroxide. The conclusion is that catalase functions better in a neutral pH environment.
When the catalase was mixed with an acidic solution (vinegar), the color changed from purple to fuchsia or hot pink. When mixed with an alkaline solution (ammonia), the purple changed to green. The conclusion can be made that when neutral pH solutions are mixed with acidic or alkaline solutions, there will be a color change. By noting the color change, it is easier to predict if a catalase is going to function at its optimum ability. Little color change indicates that the pH will have a high function, while a big change in color indicates that the catalyzing activity will be minimized.
|Birch, Paula Ellen. 1989. DNA Damage and Cell Lethality by Photodynamically Produced
Oxygen. Rice University. Houston, Texas. Pgs 3, 4, 7, 8.
|Carter, J.L. Stein. 1994. Enzyme Activity. Protocol for Biology
Lab I, University of Cincinnati – Clermont College.|
|Wu, Gusui, and Shah, Dilip M. 1995 Isolation and Characterization of
a Potato Catalase cDNA. Monsanto Company. St. Louis, MO.
|Zanta, Carolyn, Gedney, Clark, Gray, Mary, and Labus, Brian. 1998. Properties of Enzymes. Purdue Research Foundation. http://biomedia.bio.purdue.edu/GenBio/GBEnzyme/html/catalase.html|