H3; I believe that the rate of reaction will show that as the concentration is multiplied by four, the amount of gas given off will have doubled. This theory is expressed by the equation 4y=2q where y represents the concentration and q represents the amount of gas given off.
Ho= no significant difference in the rate of reaction will be shown as the substrate concentration increases.
PRELIMINARY WORK;
As preparation for the investigation, I carried out a test practical in order to ascertain what quantities of the substrate and enzyme source to use, and what sort of the results were to be expected. It also allowed me to determine what apparatus would be the most accurate.
TYPES OF APPARATUS AVAILABLE;
1. Using liver as a source of Catalase, place in a boiling tube with H2O2. This is temperature controlled in a water bath yet the oxygen gas volume given off is only measured by counting bubbles. This is inaccurate and unreliable. The bubbles could vary in size and it is very easy to loose count. It was also very hard to cut the liver into equal portions or 1cm3.
2. The Catalase source is again liver, making this experiment partially unreliable to start with. The substrate and enzyme temperatures are still controlled in a water bath. However this time the volume of gas given off is measured by being collected in an upturned measuring cylinder. Here, it is difficult to read since the measurements are upside down. Also the accuracy of the measuring cylinder is poor.
3. The method of the volume of gas given off is the accurate here with it being collected in a gas syringe, which has an error value of +/- 0.5. Everything else is the same as previous experiments.
4. The is very inaccurate since the froth bubbles over the top of the buiret. Also we are unable to tell the height of the froth. Sometimes the bubbles were much larger. Therefore this is a very inaccurate form of measurement. However this time the source of Catalase is pureed liver. This means that it is much more accurate when measuring 1cm3. However by crushing the liver you cannot be sure how much of the 1cm3 is Catalase or liver tissue.
5. Here, various vegetables and fruit were used as the source of Catalase. These were much easier to cut exact cubes from, therefore controlling the surface areas and size much more precisely.
Therefore from the choices available l chose set-up number 3 since this was the most accurate apparatus with the Catalase source or potato from number 5. I then went on to use the chosen piece of apparatus for my preliminary work. The results from this experiment are shown in the following table.
Results from Preliminary Work;
Gas given off (cm3)
Time (min) 1 2 3 4 5
Concentration (M)
1 3.5 7 9 11.5 N/A
2 5 7 9 N/A N/A
4 3.5 126 179 2,112 14.5
APPARATUS LIST;
Distilled water
Glass rod
Side-arm boiling tube
Bung
Glass beaker (500 cm3)
Boss and clamp stand
White tile
Scalpel
Callipers
Thermometer
Stop clock
Goggles
Cork borer (1c diameter)
Gas syringe
Potato (Catalase)
Hydrogen Peroxide (4m)
Syringes (10 ml, 20 ml)
Ruler
Graduated pipette and filler
DIAGRAM OF APPARATUS
METHOD;
Prepare the various molarities of Hydrogen Peroxide, following the instructions as shown in the table below, to produce concentrations of 0.5M, 1.0M, 1.5M, 2.0M, 2.5M, 3.0M 3.5M, and 4.0M. Then prepare a control of distilled water, to use as a comparison.
From the potato, use the cork borer to cut out tubes of potato of 1cm3 in diameter. On the white tile, using the ruler to measure and the callipers to check size, measure out five 1cm3 cylinders. This is your source of Catalase. Set up the apparatus as illustrated by the diagram. Clamp the gas syringe sufficiently so that it is secure, but not so tight that it affects the movement of the plunger. Check that the plunger is fully inserted.
Prepare a water bath of 25oC. Record the temperature, and keep it constant by adding either hot or cold water as required.
Place the side-arm boiling tube into the water bath, use a clamp stand to stabilise it and connect the rubber hose to the gas syringe, again ensuring that the plunger is fully inserted.
Add the 20cm3 of the respective solution concentration to the side-arm boiling tube and heat it to the same temperature of the water bath
Add five potato cylinders, insert the bung and start the stop clock. Seal the joins between the bung and rubber hose by squirting water in the connections.
Record the amount of gas given off every 30 seconds, subsequently for five minutes.
Repeat this process five times for each concentration. Collate class data and present in a table, outlining anomalous results.
INSTRUCTIONS FOR CONCENTRATIONS OF SUBSTRATE;
Molarity (M) Volume of H202 4M (cm3) Volume of Distilled water (cm3)
4 20 0
3.5 17.5 2.5
3 15 5
2.5 12.5 7.5
2 10 10
1.5 7.5 12.5
1 5 15
0.5 2.5 17.5
0 0 20
VARIABLES TABLE;
Variable Why? How?
Temperature; controlled As the temperature increases, enzyme activity increases until the active site is destroyed and the enzyme denatures. At this point the enzyme will no longer have any effect on the substrate. Therefore the temperature is controlled in order to keep the enzyme at its optimum temperature, the point at which it acts fastest on the substrate. This was controlled by placing the solution in a water bath which is heater to the same temperature for every repeat and concentration. Therefore in each experiment the Catalase is subjected to the same external environmental conditions, therefore controlling the speed at which the enzyme works. The enzyme itself could not be heater due to the form in which it is found. The water bath was maintained by adding hot or cold water as required.
pH; uncontrolled Although the pH of the solution will effect the action of an enzyme since each enzyme has its specific optimum pH, of which either side it will gradually denature or slow down, this variable was not controlled since the use of a buffer interferes with the reaction. If this could have been controlled l would have used a buffer.
Molarity of H2O2 Solution; uncontrolled, independent variable. This was varied in order to ascertain how the concentration of the substrate effects the action of the enzyme on its substrate.
Concentration of Enzyme; controlled. If the concentration of the enzyme was not controlled the volume of Catalase each molarity of H2O2 was subjected to would be different and therefore the results would be unfair. Since the more volume of the enzyme present, the faster the rate of reaction since there are more active sites to act on the substrate and break it down into products quicker. This was controlled by cutting potato cylinder with a cork borer, then measuring out five 1cm3 cylinders with callipers, a ruler and a scalpel. This was, both the volume and surface area was controlled.
Source of Catalase: controlled Depending on the variety of potato, the amount of Catalase present in each cell may vary. This would then have meant that although the size and surface area of the potato cylinder had been controlled the volume of the Catalase had not. By using the same breed and crop of potato it can be assumed that they have all been subjected to the same conditions and are, therefore, similar in the volume of Catalase they contain per cell.
Gas given off; dependant variable This cannot be controlled since this is the dependant variable
