The volume of buffer and the use of it
The pH buffer volumes were constant to the results obtained from the pilot test throughout the experiment. This helped in keeping the results as accurate as possible.
Temperature
Temperature was kept constant throughout the entire experiment because as the temperature increases the kinetic energy increases causing the enzyme and the substrate to collide more often. This results in more enzyme-substrate complex to be formed therefore speeding up the reaction. If the temperature increases more the atoms vibrate and it causes the breakage of the bonds and forces that hold the enzyme’s shape in its specific shape. This would alter the active site’s shape and the substrate would not be able to fit in to the active site (the enzyme is denatured). So to control the constancy of the temperature the experiment was carried out at room temperature.
Volume & concentration of the substrate
The volume & concentration of the Hydrogen peroxide was also kept constant because, altering the volume or concentration of the substrate would affect the rate of reaction to a certain range. This is because increase in substrate atoms would result in more substrate enzyme collisions, which results in more enzyme-substrate complex. As the volume of the substrate increases or made more concentrated, it comes to a point were all the enzyme active sites are occupied by the substrate making the rate at which products are being produced constant. To keep the volume constant hydrogen peroxide was measured in a syringe with the volume that was obtained from the pilot test.
Type of potato
It was important to use the same type of potato in each experiment otherwise different types of potatoes would result in totally different readings. This is because the potatoes would have different concentrations of enzyme or different cell wall thickness therefore effecting the release of oxygen. There would also be the difference of age which can effect the experiment. The three types of potatoes that was used in the experiment are Desiree, Maries Piper and Estima.
Hypothesis: -
At low pH the enzyme will produce the least oxygen but as pH increases the production of oxygen will also increase. At pH 7 the experiment will have its highest value of the results (potato catalase has an optimum pH of 7) and this value decreases with continues increase of pH. High pH values would result in no results because the enzyme would be denatured.
Pilot works
Pilot 1
The first pilot work that was done was the test for the way in which the gas was going to be collected. Everything else was kept constant that is the potato size and the volume and concentration of both the hydrogen peroxide and the pH buffer solutions. pH 4 was used at 6ml and the volume of hydrogen peroxide was 4ml.
Water bath method
Gas syringe method
Looking at the results one can see that the best way to collect the gas is with a gas syringe.
Pilot 2
The second pilot that was done was the volume of hydrogen peroxide that needed for the experiment. The volume and the concentration of the pH buffer and the size of the potato was kept constant throughout the pilot test.
Here the results show see that the best volume of hydrogen peroxide was 8ml because it releases the largest volume of oxygen.
Pilot 3
The third and last pilot test that was done was that of the volume of pH solutions that required for the main experiment. The volume of hydrogen peroxide obtained from pilot 2 was used and kept constant. The size of the potato was also kept constant.
4ml of pH was the best volume because it produced the most oxygen
Apparatus need
Water basin
Test tubes
Test tube bungs
Delivery tubes
Measuring cylinder
Syringes 10ml and 5ml
Digital stopwatch
Beakers 50ml
Beehive
Gas syringe
pH solutions pH (pH2-pH4 –pH 6 –pH 7 -pH 9)
Hydrogen peroxide 20 volumes
Three types of potatoes Desiree, Maries Piper and Estima
Cutter and ruler
Health and safety rules
When working in the lab one must always be extremely careful. When performing this experiment there were several requirements that needed to be insured in order to comply with the health and safety rules. They were the careful handling of the knife used to peel the potatoes and the cube cutter. Insure that the pH and the hydrogen peroxide solutions did not get into contact with skin and eyes because they are both irritants and corrosive. They are also harmful to swallow. Lab coat and gloves were worn.
Method
All the apparatus, solutions and potatoes needed were placed on the table far from the edges to avoid accidents. The apparatus were set properly as required. The water basin was filled with water as well as the measuring cylinder and it placed up side down on the beehive in the water basin. It wais necessary to make sure that no air bubble was trapped in the cylinder or under the beehive and the measuring cylinder was placed in the middle of the beehive to make sure that the oxygen coming from the test tube went directly into the measuring cylinder. The cylinder was secured properly with a clamp and the delivery tube coming from the test tube was placed properly under the beehive. It was made sure that all the gas goes in to the beehive and non-escaped due to leakages in the tube.
It was very important that the measuring cylinder was accurately measured before and after the experiment and that made sure no air was trapped in the cylinder or the delivery tube throughout the experiment.
After setting the apparatus up the experiment could be started. 1cm3 of potato that was 6cm in length was placed in a test tube and then 3ml of the buffer solution was added. When this was done it was made sure that none of the buffer solution came into contact with skin or eyes. Put the stopper back on the test tube. 4ml of hydrogen peroxide was then injected into the test tube. It was important to take note of the pressure used to inject the contents into the test tube otherwise this would affect the rate of the reaction. If it was done slowly it would slow the reaction because the enzyme would not be in contact with much substrate but if it was done quickly the enzyme would be in contact with as much substrates as possible. As soon as the solution was in to the test tube the stop- watch was started, to time the reaction. Every reaction was timed 5 minutes (300 seconds). The produced oxygen was measured as a result of the water that was pushed down the cylinder by its production. This pilot test was repeated three times to see whether it was the best method to collect the oxygen gas.
For the experiment using the gas syringe to collect the gas, the rubber tubing was inserted into the syringe first before it was clamped safely. This rubber tubing connected the syringe to the test tube. The exact same method was carried as that of the water bath when placing the contents into the test tube. This pilot test was also repeated three times to see which way was better for collecting the oxygen gas produced, between the water bath method and gas syringe method.
The method that was chosen was used to carry out the rest of the experiment.
Table of Results
Oxygen produced in different types of potato with 8ml of hydrogen peroxide and 4ml of pH solution. Time was kept constant to 5 minutes per experiment.
Raw Data
Calculated Data
Analysing the evidence and drawing the conclusions: -
According to the results table and the average production of oxygen in different pH conditions it shows that the experiment complies with the hypothesis. The results show that the potato catalase enzyme works its best at pH 7. This is because the enzyme potato catalase has its optimum pH at pH7. At lower pH (i.e. acidic pH) solutions there are lots of hydrogen ions free. These ions change the shape of the active site therefore preventing the substrate to bind with the enzyme. When this happens the amount of products produced is decreased if not stopped completely as a result of the enzyme denaturing. With high pH (i.e. basic) solutions there is lots of hydroxyl ion free in this solution. This too also changes the active site of the enzyme making it produce less products and eventually resulting in denaturation of the enzyme if the pH is increased even more. There is an anomalous. The Maries Piper potato gives an odd result in that pH2 has a higher oxygen gas production then pH4. The values are also fairly low compared to that of the other two potatoes. This could be a result of the soil in which it was grown. When soil becomes acidic alkalis such as calcium hydroxide or calcium carbonate are used to neutralise the soil. If excess alkali is deposited into the soil it becomes basic and the vegetation could be contaminated with it.
Evaluation: -
The experiment is fairly accurate. The only method that contributes to in accuracy is the pressure at which the hydrogen peroxide is injected into the test tube. This is because during the course of the experiment the ones hand gets tired and sore therefore stops when injecting the contents into the test tube. The is not completely accurate at 5 minutes. There were always a few seconds that went with it. If the seconds seemed close to another minute the experiment was repeated to be as accurate as possible. During the course of the experiment the hydrogen peroxide might have been decomposed by light energy. The best thing to have done is to have kept it in a black container.
Bibliography
http://www.ekcsk12.org/science/lelab/localenzymelabpartone.html
http://www.ekcsk12.org/science/lelab/localenzymelabpHstudy.htm
Longman’s AS Biology chapter 4, page 101-104
