Variables
In order to ensure a successful experiment and so that accurate results can be recorded we need to include variables in the experiment. To observe and measure the effect of different factors on the rate of enzymatic reaction you can vary one or more of the following: a) substrate concentration; b) enzyme concentration; c) pH; d) temperature; and e) inhibition. In this investigation, the variables that affect the activity of the enzyme
Catalase should be considered and controlled as to not disrupt the success of the experiment.
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Temperature – As the temperature of a chemical reaction rises, molecules move faster (they gain kinetic energy) and collide into each other more frequently and with more intensity. In an enzyme controlled reaction, such as the decomposition of hydrogen peroxide, the rate at which the enzyme and substrate molecules meet increases and therefore the rate at which the products are formed also increases. With increasing temperature however, the hydrogen and ionic bonds which hold the enzyme molecules in place and if the molecular structure is disrupted, the enzyme will cease to function as the active site will no longer accommodate the substrate. This means that the enzyme has been denatured.
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pH – Any change in the pH of a solution affects the ionic and hydrogen bonding in an enzyme and so alters its shape and structure. Each different enzyme has an optimum pH at which its active site best fits the substrate. Variation either side of the pH scale results in the denaturation of the enzyme and a slower rate of reaction.
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Substrate concentration – When there is an excess of enzyme molecules, and an increase in the substrate concentration there is a corresponding increase in the rate of reaction. If there are sufficient substrate molecules to occupy all of the enzymes’ active sites – the rate of reaction is unaffected by further increases in substrate concentration as the enzymes are unable to break down the greater quantity of substrate.
To control the substrate concentration, identical quantities of the substrate were used for each reading.
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Inhibition – Inhibitors compete with the substrate for the active sites of the enzyme – these are known as competitive inhibitors, they can also attach themselves to the enzyme away from the active site – this action alters the shape of active site so that the substrate is unable to occupy it and the enzyme cannot function properly. The type of molecule which attaches itself away from the enzyme’s active site – causing it to not function properly anymore is known as a non-competitive inhibitor. Inhibitors therefore slow the rate of reaction. As none are to be added to this investigation, there will be no effect from inhibition.
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Enzyme concentration - The amount of enzyme available to process the substrate can control an enzyme catalytic reaction. By carrying out this experiment we can investigate how doubling the amount of enzyme affects the enzyme catalysis rate, amount of substrate, and amounts of reaction products. Provided there is an excess in substrate, an increase in the enzyme concentration will lead to a corresponding increase in the rate of reaction. When the substrate is in short supply (i.e. it is in limited quantity) an increase in the enzyme concentration has no effect.
Apparatus
- 250ml conical flask and beakers.
- 40ml Hydrogen peroxide (start off with 10vol and then the concentration will be varied thereafter)
- Tongs
- Distilled water
- Liver (to be cut up into fine pieces which will weigh 1 gram each)
- A razor blade (Scalpel)
- Ruler
- Stopwatch
- A bung
- 2 5ml syringes
- Rubber tubing
- Graduated measuring cylinder
- Pestle and mortar
- Safety Goggles
- Ceramic tile.
- Gas Syringe
Method
To test how the concentration of Hydrogen peroxide affects the rate of reaction you will first need to set up the apparatus by following the procedures below:
1. Using the cork borer and scalpel, take a sample of liver and cut this piece into cm intervals using the scalpel and plastic rule. Once the liver is finely cut up it must be weighed precisely to measure one gram
2. Fill the beaker with approximately 40cm3 of the pre-made measured solution of Hydrogen Peroxide and using the graduated measuring cylinder. The hydrogen peroxide should be diluted with the appropriate amounts of H2O in order to get the correct Vol.
3. Immerse the gas syringe with the attached bung into the conical flask and drop the piece of liver into the hydrogen peroxide solution, care and precision must be taken as the stopwatch should be started as soon as the liver is in the solution.
4. Measure the volume of oxygen given off every 10 seconds and then alter the concentration of the Hydrogen peroxide.
5. Repeat the procedure with the other Hydrogen Peroxide solutions with the different concentrations (e.g. 10vol, 9vol, 8vol etc...). Be aware not to contaminate each solution with one another, this will cause inaccurate results.
6. Repeat all the tests at least three times (Time dependant) so that an average can be obtained. Repeating the experiments several times will help to produce better and more accurate results.
To ensure this experiment is completed as fairly as possible, all the variables except for the concentration of Hydrogen Peroxide must be kept the same for all experiments. Variables that must not be altered include: Temperature, Catalase Concentration, dimensions of liver, air pressure and humidity.
Predictions
I predict that as the substrate concentration increases, the rate of reaction will go up at a directionally proportional rate until the solution becomes saturated with the substrate Hydrogen Peroxide. When this saturation point is reached, then adding extra substrate will make no difference.
The rate steadily increases when more substrate is added because more of the active sites of the enzyme are being used which results in more reactions so the amount of Oxygen released in a given time is higher. Once the amount of substrate molecules added exceeds the number of active sites available then the rate of reaction will no longer go up. This is due to the maximum number of reactions being done at once, so any extra substrate molecules have to wait until some of the active sites become available.
.Interpretation
When the concentration of Hydrogen Peroxide is increased, the rate of reaction increases at a directionally proportional rate until the concentration of Hydrogen Peroxide reaches about 10vol. If the concentration were doubled, I would expect the amount of Oxygen released to be a figure twice as much.
From 10vol to 1vol shows a directionally proportional decrease in reactivity rate, after 10vol the rate of reaction slows down. At this point virtually all active sites are occupied making the active sites saturated with Hydrogen Peroxide. With an increase the concentration of Hydrogen Peroxide, the number of active sites increases, hence, makes a more violent reaction (Quicker).
The theoretical maximum rate of reaction is when all the sites are being used but in reality this theoretical maximum is never reached due to the fact that not all active sites are being used at the same time. The substrate molecules need time to join onto the enzyme and to leave it so the maximum rate achieved is always slightly below the theoretical maximum. The time taken to fit into and leave the active site is the limiting factor in the rate of reaction.
Safety
Laboratory coats can be worn during the investigation to prevent chemicals from spoiling clothes. Care was also taken whilst handling the chemicals as hydrogen peroxide is corrosive and very toxic (harmful to the eyes and skin). Whilst using the razor blades, care was also taken to hold them by the handle and not the blade to prevent an accident occurring.
