The reaction rate for most enzyme-controlled reactions varies with the concentration of the available substrate. Increasing the concentration of a substrate can give a corresponding increase in reaction rate, but only when the substrate concentration remains comparatively small. When larger substrate concentrations are used, the reaction rate becomes less dependent upon concentration and tends towards a fixed maximum determined by the amount of enzymes present.
Key Variables
Independent variable: Substrate concentration
Dependent variables: Temperature
Enzyme concentration
pH value
In order for the test to be completed as fairly as possible all the factors apart from substrate concentration must be kept constant.
Temperature
Temperature increases the rate at which the enzyme and substrate molecules meet and therefore the rate at which products are formed. If molecular structure is disrupted, the enzyme cease to function as the active site no longer accommodates the substrate. The enzyme is denatured. To control this variable a water bath of 30 ?C will be used to ensure the temperature of the hydrogen peroxide is kept constant throughout the experiment.
Enzyme Concentration
Provided there is an excess substrate, an increase in enzyme concentration will result in a corresponding increase in the rate of reaction. The celery will be cut up into small equal pieces to ensure the enzyme concentration is not changed. 1.0 g of celery will be used for each experiment.
pH
Any change in pH affects the ionic and hydrogen bonding in an enzyme and alters its shape. Variation either side of pH results in the denaturing of the enzyme and a slower rate of reaction. In this experiment 10cm³ of buffer solution of pH 7.0 will be used to maintain a pH level suited to the enzyme by being equal to the natural environment of the enzyme (celery). This will ensure no variation in pH that might result in an increase or decrease in rate of reaction.
When measuring the volumes of hydrogen peroxide, the measurement should be taken from a 90 degrees angle to avoid parallax error.
Apparatus:
Gas syringe prep kitmeasuring the volume of oxygen gas released.5cm³ measuring syringemeasuring out the hydrogen peroxide.BeakersTo store hydrogen peroxide.Conical flaskPutting in celery and hydrogen peroxide.Bung with 2 delivery tubesWhere gas leaves conical flask and hydrogen peroxide enters.Safety gogglesEye protection.Stop watchMeasuring gas produced every 30 seconds.ThermometerMeasuring temperature at start of each experiment.100cm³ measuring cylinder (x2)Measuring out the different concentrations of H202 and measuring out distilled water.Knife Cutting celery.Chopping BoardPrevent damaging benches.CelerySubstance containing catalase enzyme.Buffer solution (pH 7)Prevent pH value changing.Different concentrations of H202Independent variable substrate concentration.Mortar & Pestle To crush celery into smaller, equal sizes.
Safety:
All biological activity have to be treated with care.
* Wear safety goggles throughout the experiment to protect eyes.
* Tie long hair back to prevent it getting in the way
* Hydrogen peroxide is a toxic substance, if it makes contact with skin wash immediately.
* Take care when using knife to avoid any accidents.
* Never place fingers in mouth or eyes after using chemicals.
* The enzyme catalase will be used in the form of celery. However, the fact that all biological activity have to be treated with care must be taken in to account.
* Concentrated solutions are unstable and the pure liquid may explode violently if heated to a temperature above 100?C.
* If too much pressure buils up then the syringe will come out and break. In order to prevent this, two separate parts of the syringe should be tied together. The bung will be loosend to release pressure.
* Extra care needs to be taken in the laboratory because glass apparatus is being used.
Buffer solution does not keep well due to mould growth.
Once the tests have been carried out do not dispose the experimental substances down the sink. Once the whole experiment is conducted, transfer the contents into an empty beaker. The substance can now be poured down the sink as it is now only water. It is important that the content is not disposed of until certain the experiment is completed because if not it can be dangerous as gas may still be given off and the H2O2 is corrosive.
As a precaution, I have limited my contact with the boiling tubes, as my body heat may raise the temperature; increasing the rate of reaction.
Pilot Study
Prior to conducting my final investigation, a pilot study will be carried out to help me when planning my final experiment & method in order to obtain the best and most accurate results possible.
Aim of the pilot study: To investigate the conditions at which catalase works best.
This procedure will be carried out y reacting catalase, in the form of celery with hydrogen peroxide, which will produce oxygen gas.
Substrate concentration will not be required in the pilot study because it is the key variable that will be investigated in the final experiment.
Temperature: The investigation will be conducted at room temperature because it is the most practical way without the presence of a water bath.
Volume: The volume of H2O2 . required for each test will need to be decided. The rate of reaction will be affected if too much or too little H2O2 is used, therefore I will carry out samples using 2cm³,4cm³,6cm³ & 8cm³ of H2O2 .
This will also benefit me when deciding the volume of buffer solution needed for each experiment.
Time: Each test must be no longer than 4 minutes. This is in order to ensure there is enough time to carry out repeats in a reasonable amount of time. I will record the volume of oxygen gas being produced every 30 seconds to obtain a range of results.
Enzyme Concentration: Catalase is going to be used in the form of celery. The enzyme quantity has to be suitable so that the rate of reaction is not too slow or not too fast. I will measure out different areas of enzymes and decide which is best to use for every test.
pH: I have decided to use the appropriate buffer solution of pH7 because after looking at numerous catalase experiments i have discovered that catalase works at an optimum pH of 7.0.
Results:
Volume of H2O2 .
Controlled variables: Temp: 15?C, Enzyme volume: 1 gram, Substrate concentration: 1.5M, Volume of buffer: 10cm³, pH value: 7.0
Time/secvolume of H2O2 2cm³volume of H2O2 4cm³volume of H2O2 6cm³volume of H2O2 8cm³3022178176036291621904533242712051373034150604138391806652454521075575250240806358562708466636430089677072
All the information I obtained from conducting my pilot study should be very useful because I now have an idea of the best conditions to use for my final experiment.
The results show that the fastest rate of reaction takes place using 6cm³ of H2O2 .
Therefore the total volume of each different concentration required to made in order for enough H2O2 to be available for all the tests, including repeats would be 18cm³.
Evaluation
The previous table shows the final results of oxygen gas produced for the 5 different concentrations (2.5M, 2.0M, 1.5M, 1.0M & 0.5M) of hydrogen peroxide.
From looking at the results and graphs it is clear that as the concentration of hydrogen peroxide is increased, the rate of reaction increases.
Limitations:
To enable the experiment to be carried out as accurate as possible, each test was repeated three times and an average was calculated which made it easier to presenting my data onto a graph with a best fit line.
I followed my method and safety rules to ensure everything was carried out correctly and to avoid any accidents.
I tried my best to keep all the variables apart the concentration of H2O2 constant. Unfortunately, it is impossible to keep all measurements exactly the same with the basic equipment and conditions I had. For example I found it is impossible to precisely measure out exact amounts of hydrogen peroxide each time because the scale of the measuring cylinder shows the measurements to the nearest 1mm³ whereas I needed it to be correct to the nearest mm³. There was also a delay between pouring in the H2O2 and starting the stop watch because only one procedure could be carried out at a time. However this should affect all the results for each individual test I conducted and therefore should not make any significant difference to the overall results.
Further Improvement:
My experiment could be improved in numerous ways. The experiment could be repeated more times to delete any anomalies and to produce a more accurate overall result.
After carrying out my investigation I came to the conclusion that using catalase founded in celery made it difficult to measure precisely the desired amount of catalase. I did however crush the celery into minute equal sizes to ensure the enzyme concentration was as equal as it could be.
For further investigation I believe a source of catalase from yeast would be ore appropriate as it is easier to use and determine the desired amount of yeast catalase by simply measuring it off.
The problem of the delay between pouring in the H2O2, placing the bung securely on the test tube & starting/stopping the stop watch could have been limited by using another person to start & stop the timer when the H2O2 was being poured into the tube.
