Investigating The Rate Of Enzyme Activity With Varying Enzyme Concentration.
Investigating The Rate Of Enzyme Activity With Varying Enzyme Concentration.
Introduction.
In this experiment I hope to find out the effects of enzyme concentration on the rate of enzyme activity. I will do this by watching the varying levels of reactivity whilst experimenting with liver (which contains catalase) and hydrogen peroxide that will react to give off oxygen.
Enzymes are biological catalysts that work according to the lock and key hypothesis. Enzymes contain an active site where the reaction of the two substrates takes place; the shape of this active site determines how effective the enzyme is. The higher the temperature the more collisions between the enzyme and substrates so the more reactions can take place. If the enzyme becomes too hot it will become denatured and the active site will be mutated and ineffective. Also, pH has an effect on the effectiveness of an enzyme. An enzyme needs to be in the right environment (pH) or it again becomes denatured and useless.
Before I carried out the final experiment I did preliminary work in which I tried various ways of measuring the oxygen given off from the reaction. The first problem I came across was the matter of taking liver samples. It was far too difficult to accurately cut the liver, as it is too slippery. Instead I prepared a stock solution (I will detail this later) and reacted varying concentrations of the solution with hydrogen peroxide in a boiling tube and measured the frothy progress up the tube, I reacted 10ml of the stock solution with 5ml of hydrogen peroxide. After a few measurements it was clear that this was inaccurate as the results were so varied so I devised a new method of measuring the oxygen output. This time I caught all the oxygen produced and ran it under water and into a measuring cylinder so I found out exactly how much water was produced (detailed later).
What do you predict will happen?
I believe that the more concentrated the enzyme is the more enzyme activity there will be. The less concentrated the enzyme is the less enzyme activity there will be. I believe that if the enzyme concentration is doubled then the enzyme activity should double.
For my all or nothing test I believe that by adding an enzyme to the substrate then it will cause a reaction. Without the enzyme the hydrogen peroxide will have too high an activation energy to react. It will not contain enough energy to react. By adding an enzyme the activation energy needed for a reaction is lowered.
Use scientific understanding to explain your prediction.
I believe that the higher the enzyme concentration the more the substrates will react as there is more chance of a collision. There is more chance of a collision between the substrates and enzymes if there are more enzymes to collide with. The opposite also holds true, as the less of the enzyme there is the less collisions there are so the less reactions there are. I believe that if the enzyme concentration is doubled the rate of reactivity will be doubled, because if there are twice as many enzymes ...
This is a preview of the whole essay
Use scientific understanding to explain your prediction.
I believe that the higher the enzyme concentration the more the substrates will react as there is more chance of a collision. There is more chance of a collision between the substrates and enzymes if there are more enzymes to collide with. The opposite also holds true, as the less of the enzyme there is the less collisions there are so the less reactions there are. I believe that if the enzyme concentration is doubled the rate of reactivity will be doubled, because if there are twice as many enzymes the substrates will surely be twice as likely to collide with the enzymes.
There are two theories as to how enzymes operate. They both explain it in terms of shape. The first is the lock and key hypothesis in which all substrates and enzymes have a certain shape. In this the active site of the enzyme fits exactly to the shape of the substrate. The substrate/s fit exactly in the active site, which brings them closer together and causes them to react. This idea of set shapes offers an explanation for denaturisation.
The other theory tries to explain why some enzymes can react with more than one substrate. In this theory the enzyme has no set shape and simply morphs around the substrate/s so it fits exactly then twists them together or apart. Unfortunately this has no explanation for denaturisation.
Fig 1.
What measurements do you intend to take?
Once the reaction is under way I intend to measure the amount of oxygen produced when the hydrogen peroxide reacts using the catalase (in the liver). I also intend to vary the concentration of the solution I am using in my reaction so I can monitor the effect of enzyme concentration on the rate of reaction.
Describe your procedure.
Firstly I prepared a stock solution. This entails grinding up a small piece of liver with a pumice and mortar then adding it to 150mls of water, it was then left to dissolve in the water. This solution was then filtered into a conical flask. This left a stock solution which was of a set concentration and I could easily make the other concentrations of the solution accurately.
Fig 2.
I then poured 10mls of the first concentration into a very large conical flask. In this I placed a test tube holding 5ml of hydrogen peroxide. I then placed a bung in the flask with a tube leading from the flask into a water bath containing a measuring cylinder filled with water. The tube led into this. To start the reaction the conical flask was tipped upside down with the tube held shut. This mixed the solution with the hydrogen peroxide and any oxygen produced was drawn through the tube into the measuring cylinder, the level of the water was forced down to allow room for the gas and from this change I could tell the volume of oxygen produced. I took the reading from the measuring cylinder after 30 seconds and wrote this down. For each different concentration and repeat experiment, the apparatus was washed and set up again.
Fig 3.
For my first experiment I poured 10mls of the stock solution into the test tube and then lined this up with a ruler. I then added 5mls of the hydrogen peroxide and waited 30 seconds and then measured how far up the tube the froth had reached. This was recorded and the tubes were washed.
Fig 4.
What factors do you need to control or vary in this experiment?
I need to control the amount of hydrogen peroxide in each experiment. This must always be the same, I have chosen to use 5mls every time. The amount of stock solution I use will always be 10mls, this is also a constant. I will vary the concentration of said stock solution, by diluting the original concentration as detailed above. This is an independent variable. I will also measure the amount of oxygen released by the reaction. This is a dependent variable as it is dependant on the concentration of catalase. I also need to maintain a constant temperature for all my experiments so this doesn't affect it. I will use the same size tubes and flasks each time so this has no effect on the reaction.
How have you made your experiment more accurate?
The original experiment was very inaccurate and involved measuring the height of the froth produced when the hydrogen peroxide reacted. This was very inaccurate as much of the oxygen produced would not form in the froth and as the reactions became less vigorous the froth had time to recede before more was produced. So instead I decided to measure the amount of oxygen produced by displacing the air in a flask with it. The more oxygen produced the more air was displaced into the measuring cylinder. This is more accurate as it measures all the oxygen produced. I also washed all the equipment in between each test and used the most accurate equipment I could get my hands on. The stock solution I made also made my experiment more accurate as it meant I could use the exact concentration I wanted to, and used the exact same concentration in each repeat.
Present your results in a suitable form.
Fig 5.
These are my results from my accurate experiment, using my finely tuned more accurate equipment.
These are my results from my more inaccurate test using merely a ruler and measuring the froth and multiplying that by pi and the radius of the boiling tube squared.
st time
2nd time
3rd time
With Enzyme
Without Enzyme
These are the results I got from my all or nothing test done using my finely tuned accurate equipment.
Explain simply what your results show.
Simply, my results show that as the enzyme concentration was decreased, the rate of reaction also decreased. As the enzyme concentration was increased the rate of reaction also increased. From my all or nothing test it is clear that by adding an enzyme this enabled a reaction. Because there was no reaction without the enzyme, this proves that the enzyme is the instigator of the reaction.
Explain scientifically what your results show.
The reason that the rate of reaction went up when the enzyme concentration was increased was due to their being more enzymes. Enzymes are biological catalysts that work on the lock and key hypothesis. By taking substrates into their active site and bringing them closer together it allows the reaction to take place. They do this by lowering the necessary activation energy by providing an "alternate route" for the chemical reaction to take.
Fig 6.
i.e. A + B --> C + D without a catalyst.
A + B + catalyst --> Acatalyst + B --> C + D + catalyst. With a catalyst.
In the all or nothing test, the test with no catalase present, the reaction did not take place, this was probably due to the fact that the substrates did not have enough energy to react. By adding the enzyme, the activation energy was lowered.
Fig 7.
So, by increasing the enzyme concentration there are more enzymes, because there are more enzymes there will be more active sites. If there are more active sites there will be more collisions between the substrate (hydrogen peroxide) and the active site of the enzyme. The more collisions there are, the more successful reactions there will be, so the faster the reaction will be.
Safety and fair test.
I made sure that my test was fair by always using the same equipment. This meant that after every experiment I had to wash every piece of equipment bar the water bath. I always used the same amount of the chemicals, this helped ensure a fair test. I always used 5ml of hydrogen peroxide and 10ml of the stock solution. I measured using the same flasks and stopwatch so that it was the same (fair test) for each experiment. it is not a very dangerous test but I made sure there was no injury by standing away from the experiment whilst it was reacting and wearing safety goggles at all times. I believe my test to have been as fair as the laboratory conditions allowed using the most accurate equipment to hand. I even made my own piece of equipment to measure all the oxygen released, as I believed the original measuring the froth method was too inaccurate.
How Accurate are your results and did you get any anomalous results?
My results are very accurate thanks mainly to the way I collected the oxygen given off. I have all ready detailed how I did this. Although I did not receive any anomalous results, I did not receive the results I expected to. When I doubled the concentration of my stock solution I expected to double the rate of reaction. I think this may have been due to some of the oxygen that was produced getting caught in the tube or some of the hydrogen peroxide not reacting, or some of the stock solution not reaching the hydrogen peroxide.
How could the experiment be made more accurate?
By using the equipment I did I made sure that all of the oxygen produced was definitely collected. Unfortunately, by using the equipment like I did, it meant that there was a chance that not all of the hydrogen peroxide would react. This was because when the flask was tipped not all of the hydrogen peroxide definitely came out of the test tube on the inside, I believe this was the cause of any inaccuracies in my results. To remedy this I would think it would be better to take a smaller test tube and attach it to the bottom of the flask, or use a small shelf attached to the side of the flask. This way it would make sure all the hydrogen peroxide mixed with all the stock solution.
Fig 8.
David Harrison 11A
Fig 1. The two enzyme hypotheses.
Fig 2. Preparing stock solutions.
Fig 3. My final experiment.
Fig 4. My preliminary experiment.
Fig 5. My results.
Fig 6. A catalyst and non-catalyst reaction, the alternate route.
Fig 7. A catalyst and non-catalyst reaction, the lowered activation energy.
Fig 8. How to make my experiment more accurate.
