Enzymes are vitally important for every living organism, because without them the reactions within the cells would occur to slow to sustain life. However there are many factors which affect the rate of enzymes reactions. One of them is pH, which influence on the enzyme activity will be the subject of this investigation. Catalase of potato tuber cells will be the enzyme used in the experiment.
What is the influence of different values of pH on the activity of catalase in potato tuber cells?
Independent: potato discs treatment (placing them into buffer solutions of different pH values, mixed with hydrogen peroxide)Dependent: the activity of the catalase, reflected by the rate at which oxygen is evolved, measured at every time the pH is changed.
Controlled: whether the potato discs were chemically threaten, whether the catalase and hydrogen peroxide concentration was the same in every sample ( whether the potato discs were accurately cut and whether they all came from the same potato, whether the same fresh hydrogen peroxide was used in all samples) and whether the temperature remained the same during the whole experiment.
Different pH values influence the activity of catalase. It was obtained from the literature, that the range of pH values, at which catalase works is approximately 4 to 9, with an optimum pH of 7. Bearing this in mind the following hypothesis has been made:If the potato discs are placed into a test tube with hydrogen peroxide and a buffer solution at pH approximately 7, then the activity of catalase will be the greatest. If the pH value is less or more than 7 the activity of the enzyme will be smaller.
An enzyme is a biological catalyst. This means that it’s a substance, produced in living organisms, which speeds up a certain reaction, because it lowers the activation energy. Activation energy is the amount of energy, which is needed for the reaction to start.
Catalase is one of the fastest acting enzymes known, which speeds up the decomposition of hydrogen peroxide, liberating oxygen gas and releasing energy as shown below:2H2O2 2H2O + O2Hydrogen peroxide is a toxic by-product of metabolism in certain pant and animal cells, and in a reaction catalyzed by catalase, it is efficiently decomposed into harmless substances- water and oxygen.
Buffer solution is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. It has the property that the pH of the solution changes very little when a small amount of acid or base is added to it. Buffer solutions are used as a means of keeping pH at a nearly constant value in a wide variety of chemical applications.
A pH is the measure of the acidity or alkalinity of a solution (see figure 1). An acidic solution has many hydrogen ions (H+) and a pH below 7. An alkaline, or basic, solution has very few hydrogen ions and a pH above 7. A neutral solution has a pH of 7.
The optimum pH of an enzyme, is the value of pH at which the enzyme has its maximum efficiency.
Figure 1. Increasing alkalinity and acidity on pH scale.
In this experiment, potato discs in solutions of known pH act on hydrogen peroxide, and the rate of oxygen evolved is measured. This reflects the activity of the catalase in the potato.
The essential formulae for data analysis were:arithmetic mean:Standard deviation:Standard error:wheres is the sample standard deviationn is the size (number of items) of the sample.
scalpelcork borerpetri dishrulerboiling tubesupport standmanometer tube with rubber bung(approximately 2mm diameter)beaker (10ml)pipetteclipclockpotatohydrogen peroxide (50 ml)citric acid phosphate buffersat pH : 3,4,5,6,7,8 (100 ml each)tap water
Adding hydrogen peroxide to the samples should be carried out carefully, as it is corrosive and may burn the skin or clothing.
1.With a cork borer a cylinder of potato tuber tissue of about 1cm in diameter and at least 6cm long was cut. The ruler was placed beside the cylinder and it was cut into 60 discs 1mm thick. As the discs were cut, they were immediately placed under water in petri dish.
2.For measuring the rate of oxygen evolved, the technique shown in Fig. 2 was used. First the boiling tube was placed in a support stand and the manometer tube was filled with water (but only to a level, which was marked on the tube). Then the boiling tube was filled with 5 ml of buffer solution at pH 3. Carefully 10 potato discs were added. Then, 5ml of hydrogen peroxide was added to the boiling tube and it was carefully mixed.
3.As soon as the hydrogen peroxide was added, the rubber bung was sticked into the boiling tube and the clip at the top of the boiling tube was closed in a way, that it provided an airtight seal (see Fig.2).
4.As the reaction began and the oxygen was produced the water, which was in manometer was pushed down the left hand side of the manometer tube. Time how long did it take for the fluid to rise through a distance of 5 cm in the right hand side of the tube was measured. The result was recorded.
5.As soon as the water has reached the marked level in the right hand side, the clip at the top of the boiling tube was opened so that the manometer water returned to its original position.
6.Points 3-5 of the procedure were repeated 3 times more and the results were recorded. The average reading was worked out and recorded.
7.After 4 measurements the rubber bung was removed and the tube was washed out.
8.Five further tests were carried out, each with a fresh set of 10 potato discs. The same procedure was followed but with buffer solutions 4,5,6,7 and 8 in turn. At every time a clean beaker was used to measure 5 ml of buffer and hydrogen peroxide. The data was recorded.
9.The place of work was cleaned up.
10.The data analysis was carried out.
Figure 2. Technique for measuring the rate of evolution of oxygen from hydrogen peroxide in the presence of living tissue.
RESULTS DATA COLLECTION
The row data, which was collected during the experiment, is shown in Table 1.
3.Finally it was checked, whether 68% of time values are in a range of standard deviation:e.g. for buffer solution at 3 pH:x + S = 199.5 + 7.43 = 206.93x- S = 199.5- 7.43= 192.07Three out of four time values (75%) are in the range of standard deviation.
Similar was done with time values of other buffer solutions. (see Table 2)Table 2. Values of mean, standard deviation and standard error of times at which oxygen was evolved from buffer solutions from 3 to 8 pH.
Value of pH of buffer solution3 pH4 pH5 pH6 pH7 pH8 pHMean of times (s) [+/- 0.5 s]199.5165157154.2559.2560.5Standard deviation (3sf)7.4313.027.7815.666.577.50Standard error (3sf)3.726.513.897.833.293.75Whether 68% of values are in a range of SDYes (75%)No (50%)No (50%)Yes (75%)Yes (75%)No (50%)Graph 1. Rate of decomposition of hydrogen peroxide catalyzed by catalase at different pH values.
Graph 2. Mean values of time at which oxygen was evolved during decomposition of hydrogen peroxide catalyzed by catalase in manometer tube at different pH values.
CONCLUSION AND EVALUATIONThe data support the alternate hypothesis that the value of pH at which catalase works the most efficiently is approximately 7 (see Graph 1.). When the pH is lowered from 7 to 3 the efficiency of catalase decreases more than a half. Also, when the pH is greater than 7 the efficiency of the enzyme begins to decrease. Unfortunately the range of pH at which the activity was investigated was very narrow, so it can be only assumed that the data on the graphs are normally distributed (because it can be predicted that on the first graph the rate of reaction after 8 pH will continue decreasing, and on the second graph the mean value of time at which oxygen is evolved will continue increasing after 8 pH).
Looking at the error bars on the second graph it can be concluded the mean values of time at 4 pH and 6 pH have the smallest statistical fit to the data. Therefore the most inappropriate measurements of time at 4 and 6 pH could be excluded from the calculations (see Table 1). Excluding some measurements from calculations might be also done for 5 and 8 pH values, because with these measurements only 50%, instead of 68%, of the results are in a range of standard deviation (see Table 2.).
The possible explanation of the fact, that the data support the hypothesis, may be: almost all known animals use catalase almost in every organ, particularly high concentrations occurring in the liver, where the pH is approximately 7, hence this is also the value of optimum pH of catalase. When the pH is lowered the catalase’s activity decreases, because the enzyme becomes denaturated – its structure is changed which results in the loss (usually permanent) of its biological properties. At the molecular level, the tertiary structure of the catalase is altered but the peptide bonds between amino acids are left intact. The denaturation involves the disruption of covalent interactions between amino acid chains, non-covalent dipole-dipole interactions between polar amino acid side chains and the surrounding solvent, and finally the disruption of Van der Waals interactions between non polar amino acid side chains. The denaturation occurs also when the pH is greater than 7 and it increases.
Catalase is also universal among plants, in which the optimum pH for the enzyme is similar to the pH in animals. That is why it may be concluded that the optimum pH for potato catalase is 7. It may be misleading, because it is a known fact that potatoes are grown in soil of pH approximately 6, which means it is significantly lower than the optimum pH of catalase. But it was obtained from the literature, that many plant cells at the beginning of their existence grow in an environment of a lower pH than the optimum pH of their enzymes. That is why it assumed that optimum pH of potato catalase is approximately 7, although the potatoes grow in a slightly acidic environment.
The whole experiment could be carried out thanks to using buffer solutions. A buffer solution is one which resists changes in pH when small quantities of an acid or an alkali are added to it. It was valuable to use buffer solutions because thanks to them the pH didn’t change, when the hydrogen peroxide was added to it.
Although the data support the hypothesis, some improvements to the experiment can be implemented. First of all, in order to check, whether the buffer solutions at different pH are made correctly, before the experiment the pHs should be examined with an electrical pH meter. The most accurate clock of all available should be used in order to obtain the most accurate data. It is crucial, that the manometer tube and rubber bung are airtight, hence the equipment used should be new and foolproof. Finally it would be beneficial to do more than only 4 measurements at every pH and also a wider range of pH should be investigated, because with more results statistics calculations would be more precise.
Neglecting these things may be a source of inaccuracy in the experiment. But it can also be caused by other possible experimental errors. These are:systematic errors: the accuracy of the clock measurements was only to the whole seconds, scales on the beakers were minimally differentrandom errors: using potato discs from different potato species, inaccurate washing of the tube before filling it with another buffer solution, inaccurate cutting of the discs and the time of human reaction (when it comes to starting and stopping the clock).
Using the same apparatus the volume of the gas evolved per unit time can be measured. In order to do it, the scale on the manometer tube should be labeled. Then the experimental time period should be determined (for example 5 seconds).Finally, using the same procedure as in the experiment described the evolved gas volume should be read on the scale as the determined time of measurement ended.
Catalase can decompose also other substances such as formaldehyde, formic acid, phenols and alcohols. Using these substances as a substrates for the catalase and the same apparatus some further experiments can be carried out. It is also possible, that using the same apparatus, the activity of potato catalase can be investigating by changing the temperature of the samples, or adding different amounts of hydrogen peroxide to different samples, or placing in a tube different numbers of potato discs. But it is important to remember that these procedures cannot be carried out at the same time- only one option (variable) can be changed and at the same time the others should be controlled and remained unchanged.
materials from the biology classesTaylor, Green, Stout: Biological Science 1, Cambridge University Presshttp://www.chemguide.co.uk/physical/acidbaseeqia/buffers.htmlhttp://en.wikipedia.org/wiki/Denaturation_(biochemistry)http://en.wikipedia.org/wiki/Enzyme_kinetics#Chemical_mechanismhttp://en.wikipedia.org/wiki/Catalase