The affect enzyme concentration has on enzyme activity.

[Type the author name] 11/18/2013

An experiment determining the effect enzyme concentration has on gas production due to the breakdown of hydrogen peroxide by catalase.

The effect enzyme concentration has on enzyme activity.

Aim
To find out how enzyme concentration breaking down a substrate affects gas production.

Introduction
An enzyme is a large molecule made from amino acids and is a globular protein. They are biological catalysts and are vital for life to even exist. They are specific to only break down molecules that can fit in the active site. The way they work is by reducing the activation energy of a reaction so that it happens much easier. The enzyme breaks down larger molecules into smaller molecules using induced fit, which is where the charges on the substrate are attracted to the opposite charges on the enzyme. The enzyme is arranged in such a way that it causes the substrate to undergo stress and distorts the molecule so much that it splits into smaller substrate molecules. Enzyme sizes vary from a 62 to 2500 amino acids long. They are also very large, much larger than the molecule they break down, with usually 2 or 3 amino acids actually breaking the molecule down. The rest of the molecule can be used to attach extra molecules that can speed up the reaction even more.[1] Enzymes are sensitive to heat and pH. The human body has to stay at a constant temperature of about 37C with a pH of about 7. A higher or lower temperature causes the active site to become denatured, resulting in a slower reaction and eventually stopping completely as the enzyme has become too denatured to carry on breaking down substrates. The same thing happens when pH is lowered or raised. An exception to this are the enzymes found in the human stomach, as these must be able to cope with a pH of 1 or 2 to break down proteins and fats within gastric acid. In the experiment, we used a pH solution of 7.3 as this is an optimum pH and also is a known factor while tap water might not be known. Also hydrogen peroxide can react with water while it wont react with the pH solution, meaning all of the hydrogen peroxide is available for the catalase. This ensures that our results arent affected by other reactions and that our results are accurate. The enzyme we used in the experiment was catalase, which is used in cells to break down hydrogen peroxide into oxygen and water through the reaction of 2H2O2 > 2H2O + O2. This reaction is vitally important as hydrogen peroxide is an oxidising agent that would destroy cells very easily. Hydrogen peroxide is a product of converting nutrients into ATP. Because catalase is present, hydrogen peroxide doesnt have the chance to destroy any cells due to enzyme activity. Both of these products can be reused by the body. Experiments on how enzyme concentration affects enzyme activity have been done countless times before and it is a well-known biological fact that enzyme concentration will increase the amount of substrate broken down. Experiments to prove this are done in schools around the world as it clearly shows a feature of enzymes. No official papers have really been published on this simple fact as its a simple and understood experiment.

The effect enzyme concentration has on enzyme activity.

Hypothesis
I believe that the amount of gas produced will climb proportional to the concentration of the enzyme up to a certain point where it will plateau and not increase anymore.

Variables
An independent variable that was changed was the amount of potato discs used as a source of catalase. Using different amounts of discs meant that different amounts of concentration were used to measure how it affected reaction rates. The amount of hydrogen peroxide we reacted with was the same amount for all the samples so that was a control variable as it was kept the same. A variable that changed because of the reaction would have been the amount of gas from the reaction. This is the result that is measured to provide us with data to create a table and graph.

Safety Considerations
Its good practice to never eat anything in the lab as it can be4 contaminated with a variety of bacteria and consuming these can lead to infections. Also, the potato is raw so itll be very hard to digest anyway. Washing our hands after the experiment is the best way to reduce the risk of spreading bacteria. Another safety consideration is the hydrogen peroxide. This is a powerful bleaching agent and any contact on the skin will cause it to bleach the cells. However, if this splashes into the eyes, the damage can be much worse, potentially rendering you blind. We also used pH solution of 7.3 which isnt particularly dangerous, having roughly the same pH as water. While it may not be dangerous, it still can contain a few chemicals that may react within your eyes, potentially causing eye damage. Both of these risks can be removed by wearing goggles. We also used knives to cut the potato into discs. While they werent really sharp, they are still a safety hazard that has to be considered. Basic lab guidelines and adhering to them can easily eliminate this risk. Because we were using test tubes in a water bath, there is a risk of a spillage causing slipping while carrying a test tube, potentially causing it to smash and cut. Easiest way to reduce this is to clear up any spillage immediately to prevent slipping and to clear up any smashed glass in the waste bin.

Apparatus and Justification

Knife -Ruler (30cm) Potato -7.3 pH solution 250ml beaker Test tube x 2 Bung and tube Hydrogen peroxide 5ml pipette 10ml measuring cylinder

The effect enzyme concentration has on enzyme activity.

We used potato as a source of catalase because it has it in high concentrations and means we dont have to measure out specific amounts of enzyme solution. The beaker was used to hold the water bath in with a test tube filled with water upside down. The other test tube had the solution, hydrogen peroxide and potato discs in it. The bung and tube where used to collect as much gas as possible into the water bath test tube to give us an accurate reading of how much gas was produced. The pipette was simply to extract the peroxide and solution out of the bottle into the measuring cylinder, which we measured out 10ml of each solution.

Method
First begin by extracting a cylinder of potato using the potato borer then proceed to cut this up into 1cm thick discs using the ruler. After, use 5 of the discs and add them to a test tube. Collect 10ml of pH solution 7.3 and add to the potato discs in the test tube. Collect 10 ml of hydrogen peroxide, but dont add. Collect a beaker, fill with water and place a filled test tube upside down in the water bath. Make sure that the test tube is still full when upside down, tilt left and right or remove most air. Get the bung and tube and make sure the tube goes all the way into the water bath test tube. Add the hydrogen peroxide and quickly place the bung on the test tube to ensure no gas escapes. After 3 minutes, measure how much gas has collected in the water bath test tube, repeat this experiment for 10, 15, 20, 25 and 30 discs. Inaccuracies may come from different size discs being used so there would be more enzyme present. Also, different parts of the potato might not contain the same concentration of catalase throughout. This means that during a repeat, the concentration might be higher, causing it to react more with the hydrogen peroxide. Another inaccuracy would be from human error and misreading the measuring cylinder due to parallax. This would mean that more substrate would be available and that could affect our results.

Results
Number of discs X 5 10 15 20 25 30 Volume of gas (cm3)
Repeat 1 Repeat 2 Repeat 3 Average

2.0 2.5 3.0 4.0 5.0 5.0

2.5 2.5 3.5 4.0 5.0 5.5

1.5 2.5 3.0 4.5 5.5 6.0

2 2.5 3.16 4.16 5.16 5.5

Graph
Gradient1 = 2.65/16.5 = 0.16 Y=0.17x + 1.05 Gradient2 = 3.5/19.5 = 0.17 Average Gradient = 0.17

The effect enzyme concentration has on enzyme activity.

Using my gradient triangles, I have found out that for every disc added, the volume of gas produced increases by 0.17. However, my gradient doesnt apply to every value and as we approached 30 discs, the gas volume began to plateau, meaning that theres a limit to how much substrate can be dissolved by the enzyme and eventually another factor would need to be increase to continue the increase in gas volume. These factors can include increasing the amount of substrate, increasing the concentration.

Validity
Reliability
I believe that by doing repeats, we attempt to eliminate errors however, because concentration might not be constant across the potato, we might be introducing new errors into our experiment. The only way to do this experiment and get consistent results would be to use an enzyme solution instead of potatoes. Also, we dont know exactly how much of the enzyme was in each potato disc so we dont have very accurate results as 1 disc doesnt tell us an exact value. The only way we could get consistent results would be to first find the average amount of enzyme within a disc and we could then work out exactly how much catalase is needed to break down 10 ml of hydrogen peroxide. I think that were we to repeat the experiment, we would use an enzyme solution instead as this would have a constant concentration. However, for our experiment, I believe our results to be accurate.

Precision
All our results were within 0.5 of each other however we only did 3 repeats so our results might have been all anomalies and a way to get closer to the true value would be to do 5 or 7 repeats so that we can tell for certain whether our results were anomalous. Another way would be to use the line of best fit to determine what the true value could possibly be. Using the equipment we had though, I believe that the results are accurate and could only become more accurate if we used more precise equipment.

Accuracy
The test tubes used for measuring the amount gas produced were only accurate to about 0.5 cm3 so our results are not as accurate as they could be. This means that had the gas been 2.25cm3 we wouldnt be able to tell and our results would have been inaccurate. Our results ranged from 2 to 6.0 which means that the inaccuracy can be from 10% to 25%. Also, the rulers used to measure the potato disc thickness only went to the nearest millimetre. Considering that our discs were only 1cm thick, this gives an error of about 10% which is a huge error and needs to be reduced. However, the measuring cylinders measure to the nearest 0.1ml which gives an inaccuracy of 1% which is minimal. A way to improve this experiment would be to use more accurate equipment such as a gas syringe which is also a much better way of measuring gas produced. Also, using a more accurate measuring device would help to create better discs.

The effect enzyme concentration has on enzyme activity.

Trends and Anomalies
From my graph I can see that as the concentration increases, so does the enzyme activity. However, it starts to plateau at the top. This suggests that an enzyme has a limit to how quickly it can break down a substrate and that another factor would need to be changed such as substrate concentration. One anomaly detected is the result for 30 disks as this has the largest error bars so this would need to be repeated. Also, my results for 5 disks is an anomaly as it has large error bars as well.

Error
The uncertainty of the test tube would be 0.5cm3 due to its most accurate measurement being to the closest 1.0cm3. The uncertainty of the measuring cylinder would be 0.5cm3 as well because the most accurate measurement is also 1.0cm3 Hydrogen Peroxide Measurement (cm3) 10cm3 10cm3 10cm3 10cm3 10cm3 10cm3 Error Percentage (%) Average Gas measurement (cm3) Error Percentage (%) Total error on results

5% 5% 5% 5% 5% 5%

2.00 2.50 3.16 4.16 5.16 5.50

25% 20% 15.8% 12% 9.7% 9.1%

2.00 0.600 2.50 0.625 3.16 0.657 4.16 0.707 5.16 0.758 5.50 0.776

Conclusion
In conclusion, the experiment agrees with the hypothesis however, its hard to tell on the graph whether it truly plateaus out or if the rate of reaction just slightly decreases. This is a limitation of the experiment and a way to improve this would be to use higher concentrations so that the results can carry on and we can determine whether the experiment truly plateaus or just decreases. We could have used a control to make sure that it was the catalase in the potato that was breaking down the hydrogen peroxide and not something else. To do this we could have used other vegetables which dont contain catalase just as a control.

The effect enzyme concentration has on enzyme activity.

Limitations
Using potato disks limited us with getting exact values because we didnt know how much catalase was in each potato disk. A way to improve this would be to use a catalase alginate beads or a known amount of catalase so we get more accurate results. Also, this would allow us to use more concentrations and see whether it truly plateaus. We could also use less hydrogen peroxide so there isnt as much solution for the enzyme to break down and so if we used the same concentrations of catalase we would see the plateau much earlier and this would allow us to determine whether it truly plateaus.

Bibliography
[1] General information about Enzymes
http://en.wikipedia.org/wiki/Enzyme

[2] General information about Catalase

http://en.wikipedia.org/wiki/Catalase

[3] General information about Hydrogen Peroxide

http://en.wikipedia.org/wiki/Hydrogen_peroxide