Every enzyme is selectively specific for the substance in which the reaction is taken place and is most effective over a narrow temperature range. Even though an increase in temperature may accelerate the reaction, (the optimum temperature is 40ºC), enzymes are unstable when heated. The structure of an enzymes can be denatured, by temperature (over 40ºC), pH etc.
Enzymes do not attack living cells, as a rule. This is because of the enzyme’s incapability to pass through the membrane of the living cell. Only when a cell dies, will the enzyme rapidly digest it and break down the protein. This is because the cell membrane becomes permeable when the cell dies, so the enzyme can now pass through the membrane and destroy the protein. The entering of enzymes can also be prohibited by some substrate cells, which contain inhibitors (‘antienzymes’).
Important industrial processes such as alcohol fermentation use enzymes manufactured by yeasts and bacteria in the process. Enzymes are used in treating local inflammation (trypsin is used to remove dead tissue from the wounds), and they are also used in the bleaching of hair.
Catalase:
Catalase is one of the most powerful catalysts known and is claimed that it is the most efficient an enzyme can be. The body makes catalase, which breaks the peroxide down into harmless products. Catalase works quickly and can break down 6,000,000 molecules of peroxide per minute. In the human body, a catalyst is a substance that modifies and especially increases the rate of a reaction without being consumed in the process.
Catalase is a heme (component of hemoglobin.) containing redox enzyme, which is found in high concentrates in a section in cells called peroxisome. Catalase is found in food like a potato and also the liver. This enzyme’s specific job is to remove Hydrogen Peroxide (a poisonous by-product of metabolism), by speeding up it’s decomposition into water and oxygen.
Formula:
Hydrogen peroxide Water + Oxygen
2H2O2 2H2O + O2
The speeding up of the decomposition (reaction) is able to take place because the shape of it’s active site matches the shape of the Hydrogen Peroxide molecule. The correct name given for this type of reaction where a molecule is broken down into smaller pieces is ‘anabolic reaction’.
Catalase effect on Hydrogen Peroxide
Formula:
H2O2 + H2O2 2H2O + O2
The enzyme catalase takes 2 molecules of hydrogen peroxide and changes them into 2 water molecules and a molecule of oxygen gas.
The catalase carries out this reaction by using the iron atom (in the middle of the heme group which it carries around) to help break the bonds in the two molecules of hydrogen peroxide, moving the atoms around to release two molecules of water and a molecule of oxygen gas.
When a dilute solution of hydrogen peroxide is shaken, oxygen bubbles are endorsed. This is not because of the extra energy added to it, but the slight warming of the solution creates a lot more energy.
Potato:
Freshly dug potatoes contain 78% water, 18% starch, 2.2% protein, 1 percent ash, and 0.1% fat. About 75% of the dry weight is carbohydrate. Potatoes are an important source of starch for the manufacture of adhesives and alcohol.
Since the investigation is to track and explain the rate of action of catalase enzyme and hydrogen peroxide, then my dependant variable is the amount of oxygen produced from the reaction against the time.
For this investigation we need accurate apparatus to measure the amount of oxygen produced. We thought through counting the bubbles, if a tube from the air-tight test-tube holding the solution was placed in water, but we came to the conclusion that that may be inaccurate, as the bubbles may be different sizes, and we cannot establish a relative unit for measuring the total amount. So we decided to use an up-turned measuring cylinder, filled with and in a tub of water, with the tube underneath. This way we can accurately measure the oxygen and give recognisable units. (See Page 5 For diagram).
In the experiment I will record the amount of oxygen gas produced every 15 seconds so that I can see if the speed the gas being made changes as the reaction undergoes.
For the catalase effect on hydrogen peroxide investigation it would be best to choose one of these continuous variables to study:
For my experiment my variable is going to be the concentration of hydrogen peroxide (in vol).
Prediction
I predict that as the hydrogen peroxide’s concentration increases, the rate of reaction will go up at a directly proportional rate, until the solution is working at maximum efficiency.
The rate will steadily increase when the concentration (vol) of hydrogen peroxide increases because there will be more chance of collisions (more of the enzyme’s active sites are being used), which means more reactions so the amount of oxygen is produced more quickly. Once the amount of the hydrogen peroxide’s molecules have outnumbered the active sites free, then the rate of the reaction will stop increasing, because the maximum number of reactions are being done, so any extra hydrogen peroxide molecules have to wait until some active sites are made free (when they have finished binding with one substrate and have released a product and are ready to bind with new ones).
Method
Method
- Set up the apparatus as shown above, making sure you take note of the following instructions.
- Accurately measure out 1ml of Potato homogenate (Catalase enzyme) using a 5ml syringe.
- With the bung attached to the syringe, dip the end into the hydrogen peroxide and accurately measure out the required amount (see table above for measurements). This means that when the apparatus is set up air tight, and the syringe is emptied, the space in the tube between the syringe and the bung is full of hydrogen peroxide, not air, as not to affect the results.
- Carefully empty the Catalase into a test tube and firmly replace the plug (airtight).
- Insert the larger syringe containing Hydrogen Peroxide into the small vertical rubber tube attached to the plug. To create an airtight seal, smear vasalene around the rim and any joints.
- Now once the apparatus is set up carefully, you can begin the experiment (it may help to have more than one person helping). All at the same time, start the stop watch and push down the syringe containing the Hydrogen Peroxide (keep the plunger firmly down to prevent gas pressure pushing back up the syringe and waylaying the oxygen). As soon as the syringe is fully emptied, so all the excess air from the tubes is out of the way, (and all the Catalase has mixed in with the Hydrogen Peroxide, quickly slip the U-tube under the upturned measuring cylinder. This is so no oxygen from the syringe is measured in the cylinder- only the reaction.
- Every 15 seconds record the amount of oxygen produced (in ml), for 90 seconds.
- Repeat 1-7 (making sure the test tube is clean) for each experiment- 10vol, 5vol, 2vol (so each variable is recorded twice and then the average can be taken).
Results
(From the graph)
As the time increases, the amount of oxygen produced increases at a smaller rate.
From the graph we can see that from this particular experiment, as the hydrogen peroxide’s concentration increased, the rate of reaction did not go up at a directly proportional rate. As I expected, the results from the 10vol experiment would be approximately double that of the 5vol (2 x more molecules), and the 5vol slowed down faster than the 10vol which was expected (the lower concentration means the less molecules to interact).
From the graph you can see that the rate steadily decreased with time, as expected (the amount of the hydrogen peroxide’s molecules outnumbered the active sites free, the rate of the reaction stopped increasing, because the maximum number of reactions were being done, so any extra hydrogen peroxide molecules had to wait until some active sites were made free). The values from my results table were quite similar, so this experiment was fairly reliable.
Overall conclusion
I measured the volume of the oxygen produced to the nearest ml, and time to the nearest second.
There may have been many errors and inaccuracies in the operation of these experiments that might have contributed or not to the minor errors in the results.
- It was difficult to start the timer (stop watch) exactly and as the hydrogen peroxide was syringed into the test tube because it did not all mix with the catalase at the same time.
- Some Hydrogen peroxide may have got stuck in the tube, thus not the exact amount was mixed with catalase.
- Measuring the chemicals was not exactly accurate with the limited apparatus.
- Some of the hydrogen peroxide may have been flatter than recently used.
- The force of the hydrogen peroxide being thrust into the catalase from the syringe will have been different as it was by inaccurate human ways.
- The apparatus may have not been airtight (letting some oxygen produced escape).
- Oxygen was already in the airtight apparatus before the solution began reacting, so this would obscure results.
- When the u-tube was put under the measuring tube some needed oxygen may have accidentally escaped.
- The potato homogenate was not pure catalase, so the results were not accurate, in comparison to the hydrogen peroxide.
We considered many of these problems before starting the experiment, so after a few dummy runs using the equipment layout as above, we used trial and improvement and we came up with these solutions:
- We put vaseline around any joints, to create an air tight seal.
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We swapped the catalase and the hydrogen peroxide around, so that the H2O2 was in the syringe where as it had a larger volume (less chance of getting stuck in the tube).
- We changed the syringe tube to vertical (less chance of contents getting stuck).
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We used a manageable small amount of catalase to a larger amount of H2O2 (approximately 1:15), which we found that the oxygen production rate was not too fast to measure.
- We used a large measuring cylinder (we found that with even small amounts of solution, there is enough oxygen produced to almost fill a 100ml measuring cylinder).
If I were to do this experiment again, I would consider all these problems more thoroughly. I would try and get access to more accurate measuring equipment and chemicals.
By Rachel Morrell 9Mg
