If the substrate concentration is kept constant, but the enzyme concentration is increases the initial rate of reaction will increase. There is more of a chance of a successful collision between the substrate and the active site. The overall volume of products produced will be the same. If the enzyme concentration is kept constant but the substrate concentration increases, the rate will change. At the beginning there are more substrate particles so more collisions will take place, but after the initial rate, it plateaus as the active site will be occupied by the substrates, and so have to wait to react with the active site. It does not slow it down, but stops the rate from increasing. The maximum rate of reaction is called V max.
Variables
Dependant. The variables I will control are temperature and enzyme concentration, in the form of a potato; instead an animal tissue could be used such as liver; pH, which will be controlled using a pH buffer. I need to control these variables so that I can identify what factor changes the rate and volume of oxygen produced. Enzyme concentration needs to be controlled because if it increases there will be more active sites available for the substrate to bind to increasing the rate of reaction, and if the enzyme concentration decreases there will be less active site for the substrate to bind to reducing the rate of reaction. I also have investigated how many potato slices are best in producing the oxygen. I repeated the experiment at 30°in a buffer solution of neutral. I repeated the experiment with 2, 4, 6, 8 and 10 potato slices.
2 slices
4 slices
6 slices
8 slices
10 slices
The ph needs to be neutral, this is why a pH buffer is used, as it goes more acidic the concentration of hydrogen ions increase, these react with the R-groups of amino acids affecting the way they bond. A ph which is extremely different can cause the denature of enzymes. To look at the effect of pH on the gas produced I set up the water displacement apparatus, I ran the experiment with ten discs of potatoes soaked for 5 minutes in the desired pH buffer solution to equilibrate it, before adding the hydrogen peroxide. I carried out the experiments at pH 4, 7 and 9, alkali, neutral and acidic respectively.
ph 7
28/210 cm³/s
ph 4
3/210 cm³/s
ph 9
13/210 cm³/s
As there is more oxygen produced at pH 7 I have chosen to buffer my experiment at this ph value, as I will get the most oxygen produced as the enzyme is working most efficiently here. I worked out the rates of reaction by drawing a graph and finding the gradient.
The temperature needs to be controlled because as temperature increases the molecules will have more kinetic energy so more collisions will take place between active sites and substrates, so the rate of reaction will increase producing oxygen more rapidly. But if temperatures decrease the rate of reaction will decrease as there is less kinetic energy therefore fewer collisions between substrate and active site will take place. To look at the effect of temperature on the gas produced I carried out the same experiment as before using a Ph 7 buffer. I used 10 potato slices for this experiment.
20°C
30°C
40°C
55°C
The temperature that produces the most oxygen in the quickest time is 40°C. But this temperature is difficult to maintain. As the second quickest temperature for the production of gas is 30°C, and this is around room temperature s o will be easiest to maintain. As the temperature increases the particles have more kinetic energy and so more collisions will occur increasing the rate of reactions. This will occur only up to a certain point where the enzymes begin to be denatured. The collisions have sufficient energy (activation energy) to break existing bonds and form new bonds. For this to occur the kinetic energy must be at least as high as the activation energy
Independent. The variable I am investigating, and therefore varying is substrate concentration, in the form of hydrogen peroxide.
I have decided to vary the substrate concentration to see if the rate of reaction increases as substrate concentration increases.
I will vary the substrate concentration by diluting hydrogen peroxide with distilled water to give five different concentrations. I will have concentrations of 20%, 40%, 60%, 80% and 100%, 100% being non-diluted hydrogen peroxide. I will make up the solutions of 20 cm³ as given below:
Apparatus
- Cork borer; used to make a cylinder, the use of this implement ensured that it always remained the same size.
- Potato, acting as the catalyse
- Tile
- Knife
- Ruler
- Gas syringe to collect oxygen
- Clamp to hold the gas syringe
- Conical flask
- Hydrogen peroxide, about 20 cm³ to cover the potatoes.
- Dropping funnel, this is tapped to let the hydrogen peroxide drop onto the potatoes in an efficient manner, so that the bung can be placed on the flask as quickly as possible so that no oxygen is lost.
- Stop watch, so measure the intervals at which to measure volume of gas collected.
Prediction
As the substrate concentration increases so does the rate of reaction. This is because the more substrate molecules there are there more likely it is that the enzyme’s active site can bind to a substrate. However there will become a point where the rate of reaction cannot increase any further as all active site are bound with an active site and there is surplus substrate waiting to be bound with an active site, as shown in the graph. When there is a decreases substrate concentration the initial rate of reaction decrease so it takes longer to reach Vmax, this is because there ate less substrate to bind to the active site; oxygen will therefore be produced at a slower rate. When the substrate concentration increases oxygen will be produced at a faster rate as there are anymore substrate to bind to the active site, therefore Vmax is reached much quicker the initial rate of reaction will be increased.
All active site are filled
Rate of
Reaction Enzyme active site being filled
Substrate concentration
Method
- Put potato into flask.
- Drop hydrogen peroxide onto it; using the dropping funnel start the clock.
- When you drop it in, you have to loosen the bung, open the tap, close the tap, and then put the bung back on tightly.
- then record the volume of oxygen produced using a gas syringe every ten seconds
- this method has to be repeated for each of the five substrate concentrations, each concentration should be repeated three times
Strategy
The most oxygen is produced with more catalyse, so I will use ten discs. To get a faster rate of reaction will cut each disc into quarters so there is more surface area of catalyse available to break down the hydrogen peroxide. The more enzyme concentration the faster the oxygen will be produced. A gas syringe is used to measure the volume of oxygen as it is easier to read off, if I used a cylinder it has to be turned upside down, and so the scale when measuring oxygen is the wrong way round, this way it is easier to make mistakes. The stop clock is very accurate, to within 0.1 seconds so that the oxygen can be recorded at more equal time intervals. A water bath is used to pit the conical flask in which has the hydrogen peroxide and potato in, so that the reaction can take place at a constant temperature. The water bath is electronically controlled to within 1º.
There are several ways of collecting the oxygen gas. A very basic way is to count the numbered of bubbles produced, which is the result of an evolution of gas. But this is not very accurate as each bubble could contain a different volume of oxygen. I think a more effective way of doing this is to use a gas syringe. As the bottom the measuring cylinder has no scale on it, and so is sometimes difficult to record the volume of gas. In this way a gas syringe is much more accurate. Also to make the experiments more reliable I will repeat the experiment three times, so that the anomalies can be filtered out. I have decided from my preliminary work to collect the volume of gas collected every 10 seconds; these intervals will be accurate as I am using a stopwatch. I will be able to depend more on my results if I repeat the experiment and I collect the same results.
Risk Assessment
When making cylinders with the cork borer be sure not to put your hand on the other side of the potato as the borer may cut tour hand as it comes through the other side.
When slicing the clingers using a knife on the tile the potatoes will be slippery so be careful at all times, holding the cylinder firmly so that it does no slip, otherwise you may cut yourself. And when not using the knife, put it out of the way at the back of the bench.
Hydrogen peroxide is corrosive. It is dangerous if swallowed, it causes serious internal damage due to the release of oxygen. If swallowed the mouth should be washed out with water. Cuts on hands should be covered as it will make open wounds sting. Eye protection should always be worn so it will not come into contact with the surface of the eye.
