How pH effects enzyme Catalase in potato cells

The experiment data will be taken 3 times at the pH levels 3-9. The data to be recorded is  the time it takes to produce (oxygen and water) to rise the ink in the U-shaped tube 5cm.

Equipment:

Beaker (20mlx3)-to measure and contain the solution

Syringe (10ml) x8

21+Test tubes-for experimentation

U-shaped tube

Coloured ink

Metal stand and grips

Pen markers-to mark the test tubes

Stop watch

pH buffer 3-9        

hydrogen peroxide (vol 10)

tube rack

potato

razor blade

ceramic white tile

goggles- some of the pH solution are potentially harmful to the eyes

thermometer

forceps

Experiment apparatus Figure 1

Method:

1. Cut up the potato in to small discs the same size (radius: 0.5cm, thickness:2mm)
2. Prepare the experiment set by injecting the ink, make sure there is no air bubbles, measure 5cm from the ink at water level and mark as the recording point. Mark the Test tube as experiment No.1.pH3.
3. Place 5 discs of potato in the test tube.
4. Measure 10ml of pH3 and add to test tube.
5. Quickly inject 10ml of hydrogen peroxide
6. Plug the rubber cork attached to the U-shaped tube on to the test tube
7. Let the catalase reactions begin for 15 seconds. Start timing once the peg is attached to the rubber tube to isolate the experiment products.
8. Repeat this method 3 times at each pH level at all the levels from pH3 to pH9. Record the results in the table prepared below.
9. note down the temperature at different times of experimentation, also note down any additional notes of the factors beyond control during the experiment.

The data to collect from experiment include the time for the ink in the U-shaped tube to reach 5cm, and also the temperature(as explained before). Each set of data will be experimented at the same time and be taken 3 times. Additional note will be taken down during the practical if there is to be any factors at the time that have the potential to influence the results. Anomalous data which is found during experimentation with no scientific way of explanation will still be noted down but repeated to replace the anomalous data.

Data collection table

The results will be analysed after the experiment. Anomalies will be identified, explained with the notes and temperature readings, then excluded from the final result which is the average of each test in the 3 experiments. The result will be plot on a graph to show if it supports my hypothesis or not.

Graph to show the rate of catalase at different pH levels:

Data recorded from the experiment:

As seen in the table, I have not taken any data of the experiment for pH3, this is because it was not possible for the catalase to react and produce enough product for the ink to rise up to 5cm. I had repeated this test for pH3 several times before I reached this decision to not record any data. Toward the end of my experiment, it was almost 3hours from when I cut and prepared the potato discs, and the quality and freshness compared to the potato used on the first few tests, are not exactly the same, but condition of the potato was still usable, so I decided to precede. As for the potato pieces floating to the surface, it was unpredicted but to solve the problem of the pieces becoming partially exposed to the surface, and effect the rate of reaction, I gently shook the test tube, and the pieces floated down immediately. The reason for them to float up it because of the oxygen catalase produced stuck to the surface of the potato and caused them to be lifted up to the surface of the solution. The temperature remained the same throughout the experiment, so it will not have caused any influence on my data.

There were several anomalies I found during experimentation, which was an advantage for I was able to correct and redo the tests, instead of lose and exclude them from my results. Therefore I have no anomalus data in my results table, now I will calculate the average time of each test and calculate the rate of catalase.

Using this information, I will construct a graph to show the rate of catalase reaction at different pH levels to find if there is a trend in the pattern of data.

As seen in the graph (next page) the rate of reaction rises up to a peak of the rate 0.18 at pH 7, then start to decline. Without any irregular fluctuations.

Conclusion

My graph shows the rate starting from 0.00 at pH3 rising up to 0.18 at pH7, which is the optimum rate, after pH 7, the rate declines. The result of this experiment supports my prediction, so I will reject my null hypothesis and accept my hypothesis of the rate of reaction increasing rapidly until it’s optimum point pH7, and then decrease.

Enzymes usually only work within a small range of pH. According to all scientific studies, enzymes work their optimum best at pH levels o around 7.2 – 7.4 condition. In different conditions, their functioning becomes less efficient, due to the effect acid and alkaline has on the enzyme structure:

“pH can make and break intra- and intermolecular bonds, changing the shape of the enzyme by destroying the bonds holding its structure, denaturing the binding site and therefore, its effectiveness.”

 In extreme changes it will inactivate the enzymes so the substrates are unable to bind to the active site. This will stop the enzymes from working (denature) ending in the death of cells, and also to stop producing oxygen and water. Which explains the low rate of respiration at too low and too high pH levels, including pH3 where the rate was so low I was not able to obtain any results.

Various scientific studies also show, amino acids in potatoes do help out by neutralizing acidic conditions by acting as buffers. Although they do not make a significant amount of impact, their effect can be taken into consideration if the rate of catalase in the low pH ranges are slightly higher than the higher pH levels. In my results, it shows the differences between the rates of pH6 and pH8 as only +0.01, and the difference between pH 5 and pH9 as +0.03. This contrasts with the theory of amino acids neutralizing acidic conditions. But because the difference is only minimal, I have to accept I have either made a mistake in my experiment and overseen any negative influences, or the contribution of amino acids to the rate of catalase is not significant enough to make a difference in the data.

There was no need for me to use the temperature data, as it had remained the same throughout the entire session of experimentation, anomalous data was recognised and corrected during data collection.

The experiment I carried out was suitable as the results and the graph showed no irregular pattern, seeing that my aim and prediction were proved correct, as what I predicted was accurate. I had predicted the 3 main factors that have the potential of influencing the data. My method was easy to follow, the data table was simple and effective, especially the “notes” came in useful when processing the data, and for this evaluation. To recognise anomalous data during the experimentation, allowed me to be able to repeat the tests and gain satisfactory results, this is beneficial to the final results by its accuracy, compared to just excluding the anomalous data.

However there are a few limitations in my experiment that might have influenced the results:

If the rubber plug was not air tight, or the peg did not seal off the air completely, it’s affect on the data could be totally unpredictable. And there was no way to tell how this was to affect the data. The only way to solve this is to guarantee the apparatus is air tight, and fitted in the same in each test.

The ink indicator in the U-shaped tube may have evaporated during the course of the experiment. It’s being slightly less than the original level causes the time for it to reach the originally marked 5cm a longer time, which results in the rate of catalase being calculated lower than it should be. Potentially affecting the last sets of data (pH8, pH9). But its influence is only minimal, and not enough to change my conclusion.

The problem with the potatoes discs floating up to the surface I found during the experiment and noted down, it the most likely factor to have caused any impact on the data. I failed to predict the oxygen as a product of catalase would stick to the surface of the potato and cause it to float up and expose out of the solution, I realised immediately that the exposed areas will no carry out catalase activity, which will slow down the time and then the rate will be lower than it should be. At the time of experiment, I was able to temporarily solve this problem by gently shaking the tube, which allowed the air bubbles on the surface of the potato to break free and the potato disc could sink down in to the solution. This method may be affective but not scientific enough to ensure the accuracy of the data collected. A more reliable way to solve this problem is to cut the potato discs thicker, and decrease the surface area to volume ratio, so that the weight of the potato will not be lifted by oxygen bubbles produced on the surface. So instead of 0.2cm thickness, it can be increased to 0.5cm. and instead of 5 discs of potato per tube, only 2-3 will be needed for the same amount of solution as before.

References:

John Walker. Principles and Techniques of Biochemistry and Molecular Biology. Cambridge University Press; 7 edition (31 Jan 2010) Page 568.

Arthur T Johnson. Biological Process Engineering: An Analogical Approach to Fluid Flow, Heat Transfer, and Mass Transfer Applied to Biological Systems. WileyBlackwell (5 Jan 1999)