I will now carry out a practical to investigate the effect of substrate concentration on the rate of activity of the enzyme catalase.
Below is a list of reasons why I have chosen to investigate substrate concentration instead of the other factors that were mentioned:
- If I were to investigate the effect temperature has, I would find it difficult to keep the temperature constant.
- I decided not to investigate pH because most enzymes only work on a narrow range of pH and keeping these constant would be a struggle and would result as inaccurate outcome.
Justification on the procedures selected:
For my practical I have chosen to investigate the use of potatoes as my source of catalase. I will macerate the potato tissue in a homogeniser to insure that I keep the surface area the same. I will then take this material and weigh 2.0g of it using a weighing scale.
I will use 20-volume solution of hydrogen peroxide as my 100% at 20ml. The reason being for this is that it allows me to take simple readings at a 100%, 75%, 50%, 25% and 0% solution of hydrogen peroxide. I also anticipate that these concentrations will give a clear indication of the effect that substrate concentration has on the activity of an enzyme. I’ve decided to perform for 0% because it will give a clear indication if the practical is going according to plan. If the practical is performing appropriately, the 0% solution should not give off any gas. I also plan to use distilled water to make these solutions complete. For example for 25% solution I will combine a mixture of 15ml of hydrogen peroxide and 5ml of distilled water.
I will measure out 20ml of the substrate solution in a 50ml-measuring cylinder and placed in 100ml conical flask including the potato tissue. The reason I’ve chosen to use a 100ml conical flask is that it will prevent any froth from emitting out.
I also want to prepare my diverse concentrations so that there is a sufficient supply of each concentration for every experiment i.e. the 100% solution will need 80ml so that 20ml can be used for all four experiments. I will also have supplementary hydrogen peroxide if for any case I were accidentally to spill some.
Once the experiment has started I will obtain readings of the gas produced every 20 seconds for 3 minutes using stopwatch. This time is appropriate because it allows me to read the result on the syringe and write them down.
I will obtain readings for the gas produced from the gas syringe, as it is the most precise measurement of gas developing. I have chosen not to use the method of conduct the gas developed from a large glass pipe which is filled with water bubbled through a delivery tube because I believe it is less accurate and is also more difficult to utilize. The gas syringe allows me to acquire readings of the gas produced up to 100cm3.
One requirement to the experiment is that I make sure it has been conducted fairly, and I will do this by initially carrying out a trail experiment and subsequently carrying out 3 repeats and lastly calculating the averages. Furthermore, I will calculate the overall gas developed at different concentrations from all four of the practical since it allows me to create a respectable comparison.
To keep the pH constant, I will use a pH buffer tablet and this pH will have an influence on the rate of reaction.
The experiment will be performing at room temperature, yet it is difficult to maintain a constant temperature given that it is an exothermic reaction. Keeping room temperature is a vital aspect while the experiments in progress and this will be dealt by shutting all windows and doors.
Once all four experiments have been completed, I again determine to calculate averages and overall averages and as a consequence to calculate the rate, at which oxygen is produced at different concentrations, followed by a draw up of graphs.
Preparation of the concentrations:
When adding my potato tissue with different concentrations, I will have to combine them in the following ways four times:
100% - 20ml Hydrogen Peroxide 0ml Distilled water
75% - 15ml Hydrogen Peroxide 5ml Distilled water
50% - 10ml Hydrogen Peroxide 10ml Distilled water
25% - 5ml Hydrogen Peroxide 15ml Distilled water
0% - 0ml Hydrogen Peroxide 20ml Distilled water
Equation:
2H2O0 2H20+O2
Hydrogen Peroxide (Substrate) Water + Oxygen (Products)
Variables:
Dependent variable – Oxygen Produced
Continuo’s variable – Concentration of substrate
Fair testing:
Fair testing allows the experiment to be conducted moderately and giving you accurate results, the following list is of all the fair testing aspects that I will cover:
- Making sure that I weigh 2.0g of potato tissue after it has been homogenised accurately.
- Macerate the potato tissue in a homogeniser to assure that the surface area is kept comparable.
- Making use of the buffer tablets so that the pH is kept constant.
- Accurately measuring out 20ml of hydrogen peroxide and distilled water, as well as pouring out all 100ml of the mixtures.
- Drying the inside of the glass syringe is important, as it may be moist and then closing the syringe entirely. Also the rubber pipe must be all open and the rubber bung must be in reach inside 2 seconds, so there is no excess of gas escaped.
- Making sure that the stopwatch is on 0 seconds and using the correct time and I will do this by asking someone to remind me when 20 seconds have elapsed.
- Making sure that I or no one else shakes or shift the syringe as well as the delivery tube at all times.
- Clean out all flasks that the experiment will be repeated in and dry them.
- Take out any gas left in the gas syringe that had been produced.
Safety measures:
Safety precautions are a serious matter to the practical as a whole and not carrying out any precautions cause danger to myself as well as those around me. The following is a list of the procedures that I will take out:
- Wearing appropriate goggles, apron and safety gloves.
- Work in centre of table with plenty of space.
- Tie any loose ends such as shoelaces and keep bags and coats on the hangers and not in the way.
- Work standing up and with chairs under tables and not in the way.
- Move any irrelevant objects away from the working area.
- Not touch any equipment such as the gas syringe while in progress.
Apparatus:
- 1x Retort stand, boss and clamp.
- 1x Delivery tube.
- 1x Rubber bung
- 1x Stopwatch
- 1x Weighing scale
- 1x Homogeniser
- 1x Tweezers
- 1x Spatula
- 1x 50ml measuring cylinder
- 4x 100ml Conical flasks
- 1x Pair of gloves
- 1x Apron
- 1x Goggles
- 1x Pipette
- 1x Plate
- Hydrogen Peroxide
- Distilled Water
- Potatoes
Method:
- Put all belongings on the hangers and put on safety goggles, apron and gloves. Also clear table and put stools under the tables.
- Gather all equipment and place it on one side of the table.
- Set-up the retort stand along with the boss and clamp and firmly fasten the gas syringe to it, and then attach the delivery tube and rubber bung to the clamp.
- Macerate the potato tissue in the homogeniser for around 10 seconds, allowing it to macerate properly.
- Weigh the macerated potato at exactly 2.0g and place them separately into a plate using the spatula.
- Combine and set up the 100%, 75%, 50%, 25% and 0% of 20-vol hydrogen peroxide and separate them all into a 100ml conical flask and use labels for each solution.
- Begin with pouring out the macerated potato into separate flasks and pour 20ml of the 100 % solution inside the flask and firmly push the rubber bung into the flask, within 2 seconds.
- At this point I will have also started the stopwatch taking readings each 15 seconds for 2 minutes.
- A table will already be made so once 15 seconds have elapsed, I will enter that reading for each concentration. I will also calculate the averages for the concentrations.
- Repeat steps 7 to 9 with the 75% solution.
- Repeat steps 7 to 9 with the 50% solution.
- Repeat steps 7 to 9 with the 25% solution.
- Repeat steps 7 to 9 with the 0% solution.
- Repeat steps 7 to 13 for the next 3 experiments.
- Clean out all objects I use with water and put them away after use.
Analyse of the experiment:
With the results drawn together, the following results show that as the substrate concentration increases so too does the quantity of oxygen gas produced, therefore the rate of reaction increase with the substrate concentration.
By using the 0% solution it showed that my results gathered from the practical were correct as it showed no progress in the development of gas produced. This is a clear sign that the practical was carried out correctly.
The graphs produced on computer also show that each test with the substrate concentration increasing, so too does the rate of reaction.
The last graph shows the variation of all averages of the gas developed at each substrate concentrations and yet again showing that when oxygen increases so does the substrate concentration. The significant aspects of the graphs are that at 100% concentration the gas developed is approximately the double the amount that is developed at 50%. Another implication is that the gas developed at 25% concentration is roughly quarter of the gas developed at 100%.
The science of enzymes shows in my experiment that when the rate of reaction increases as well as the substrate concentration, there is a greater amount of substrate where there is more apparent amount of collisions occurring between the enzymes and the substrate, which results in the enzyme-substrate complex that products are formed from. The gas developed, oxygen, is quicker when substrate is increased. The motive of gas developed at 100% concentration is approximately double the amount of gas produced at 50% concentration is that there is double the amount of substrate molecules vacant for more collisions to occur. The same applies to the reason to the 75% solution, which is three-quarters of the gas produced at 100% and results in less collisions and less gas produced.
Rate of reaction:
In order to calculate the rate of reaction at which oxygen is developed at different substrate concentrations, I will make use of the formula below:
Rate of reaction = Average overall gas produced
Time
The averages of overall gas produced are taken from the tables.
100% Solution – Rate of reaction = 48.32 = 24.16
2
75% Solution – Rate of reaction = 35.47 = 17.73
2
50% Solution – Rate of reaction = 30.03 = 15.01
2
25% Solution – Rate of reaction = 20.94 = 10.47
2
0% Solution – Rate of reaction = 0 = 0
2
The results above illustrate that the rate at which oxygen was developed increases as the substrate concentration increases. This is what I expected before I started the experiment and it also shows I carried it out correctly.
Trail experiment Experiment 1
Experiment 2 Experiment 3
Overall Averages
Average Gas Produced (Trail Experiment)
Average Gas Developed (Experiment 1)
Average Gas Developed (Experiment 2)
Average Gas Developed (Experiment 3)
Average Gas Produced (Overall)
Evaluation:
The results obtained from the experiment shows that as substrate concentration increases so does the rate of reaction, yet there are many aspect that may result in more accurate results. This may include the temperature, apparatus, different potatoes, etc.
If I were to do the experiment again in the future, I would take some further factors into consideration, such as:
- I would attempt to keep the temperature more constant. This is because the room temperature may have varied and could have had an effect on the results.
- I may perhaps use a wider range of concentrations to acquire a wider range of results to investigate and compare.
- Maybe I’d use smaller flasks because this could have an effect on the amount of collisions that occur and the rate of reaction would possibly increase.
- Perhaps I could use a technique of reducing the amount of gas escaping from the syringe because I assume that some may have escaped.
- I could use a gas syringe with a glass pipe, as an alternative instead of the rubber pipe, as some gas may have been lost through it.
- Maybe to use more potatoes in grams because it could increase the amount of gas produced and are more apt.