Investigating the influence of pH on the activity of potato catalase

Biology Coursework.

Investigating the influence of pH on the activity of POTATO CATALASE

Conclusion.

Enzymes are globular proteins which can be denatured at extreme pH levels

The active site is a region of the protein/enzyme such as a cleft or crevice in the tertiary structure of the enzyme, often constituting less than 5% of the surface area. The active site can be negatively charged to attract for example a positively charged amino group (basic side chains) and vice versa, namely positively charged to attract for example a carboxylic group.

As the hydrogen ion concentration decreases the pH number increases and a solution becomes more alkaline / basic. Amino acids are amphoteric having both a basic (amine) group and acidic (carboxylic acid) group). In water, the carboxylic acid partially dissociates becoming negatively charged and similarly the amine group accepts a hydrogen ion becoming positively charged. In this state, an amino acid becomes a zwitterion which enables amino acids to act as buffers. When the hydrogen ion concentration of a solution of an amino acid increases, protons are attracted to the negatively charged carboxyl group and therefore the overall charge becomes positive due to the amine group also being positively charged. Alternatively, reducing the hydrogen ion concentration of the solution means the amine group donates hydrogen atoms (ions??) making the overall charge negative. Therefore, changing the hydrogen ion concentration of a solution, namely the pH value, alters the charge of amino acids, which in turn interact with any charges on the R groups (of the substrate??). This leads to changes in the ionic charges and eventually causes ionic bonds to break.

All enzymes have a 3D tertiary structure.

The function of a protein or an enzyme depends on its particular characteristics, in particular the shape of the protein / enzyme, which is determined by its bonds. If the bonds are altered or broken, the shape of the protein / enzyme changes, the characteristics change and the protein / enzyme function changes. The protein / enzyme has been denatured.

All proteins have tertiary structure which depend on the sequence of amino acids and R groups (determined by) in the primary structure of the polypeptide chain. This is because the bonding brings about tertiary structure and interactions between the R groups determines the compact 3D shape of the globular proteins. Different R group sequences cause a different sequence of folds which may change the shape of the active site and consequently the function of the enzyme, as substrates will no longer be able to fit into the active site.

One type of bond which holds the tertiary structure of the enzyme are ionic bonds between oppositely charged R groups. Altering the hydrogen ion concentration of a solution, effects the charges and these bonds may be broken. If this happens the shape of the enzyme will change and especially that of the active site so that the substrate can no longer fit. The pH will also alter the charge of the active site which can also disrupt the electrical fit of the active site and charged substrate. All enzymes have an optimum pH which they catalyse a reaction at the maximum rate. At this pH, the shape of the active site and the charges within are ideal for fitting the substrate and catalysing the reaction.

Effect of pH can cause irreversible denaturation at extreme values of pH.

The data shows a trend between the rate of reaction and the pH value. As the pH level increases from pH5 to pH8, the rate of the reaction (i.e. the number of bubbles produced over a three minute period) increases from an average bubble count of 73 at pH5 to 132.9 at pH 8. This trend is caused because as the hydrogen ion concentration decreases, the solution becomes more alkaline creating conditions similar to the normal environment of potato catalase. The hydrogen ion concentration (pH level), has the effect on the enzyme potato catalase in that it alters the charges on the acidic and basic groups of the protein, altering the ionic charges causes the ionic bonds to break. These ionic bonds hold the specific shape of the enzyme (and particularly its active site). The active site is the region of the enzyme where all the reactions take place. Therefore, breaking these bonds means that the shape of the substrate, hydrogen peroxide is no longer complementary to the altered shape of the active site on the enzyme. As the hydrogen peroxide molecules no longer fit in the active site, no more potato catalase and hydrogen peroxide molecules can combine to form enzyme – substrate complexes, which would have formed enzyme – product complexes resulting in the inhibition of the release of the products, in this case water and oxygen. This accounts for the decrease in rate of reaction observed by the decrease in bubble count.

Another trend that I can conclude from the line graph is that at a pH level above pH8, the rate of reaction also begins to decrease. This …………

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Another trend that I can conclude from the line graph is that at a pH level above pH8, the rate of reaction also begins to decrease. This …………

I conclude from the graph that the hydrogen ion concentration of pH8 produces the maximum number of bubbles over a three-minute interval and is therefore the optimum pH level, producing the maximum rate of reaction and maximum turn over rate. (This approximate pH value may not be exact as the level of accuracy is only measured in integers). At approximately pH8, the conditions are optimum for the enzyme potato catalase, to decompose hydrogen peroxide into its products, namely water and oxygen. The experiment using a buffer solution of pH8, produces the greatest number of water and oxygen molecules per minute from the substrate hydrogen peroxide and therefore, can be said to have the greatest turn over rate. A general trend I conclude from my line graph is that above or below the optimum pH8 value, the rate of reactivity decreases.

The decrease in the rate of reaction can be explained using the ‘lock and key’ theory, where the substrate, in this case hydrogen peroxide is the key, whose shape is complimentary to the enzyme potato catalyse, which is the lock. As the shape of the active site of the enzyme potato catalase has been altered, by the breaking of various ionic bonds, the substrate hydrogen peroxide can no longer fit into the active site, thereby decreasing the effectiveness of the enzyme potato catalase as there is a decrease in the number of catalase – hydrogen peroxide (enzyme – substrate) complexes formed. This inhibits the formation of catalase – oxygen/water (enzyme – product) complexes, which obviously reduces the production of oxygen and water (products).

This explanation however, is not totally satisfactory of enzyme activity as it relies on the unlikely event of randomly moving substrate molecule entering the active site in the right orientation, like trying to throw a key into the lock, with one’s eyes shut. The induced fit theory is more appropriate as it does not rely on such precise contact being made between the hydrogen peroxide molecules and the active site of potato catalase, but suggests that the enzyme can take up the most effective catalytic shape after binding with the substrate. The shape of the enzyme moulds the substrate as a glove moulds to the hand wearing it.

All enzymes have specific 3D shapes to their polypeptide chains, known as their tertiary structure. The tertiary structure depends on the primary structure (the number and sequence of amino acids), as the bonds between the R groups attached to the amino acids holding the tertiary structure can only form if the correct amino acids are at specific points along the polypeptide chain. Catalase has a specific globular structure where every alpha helical coil and beta-pleated sheet (secondary structure) is important. The shape is held by various bonds, including ionic bonds, therefore, changing the pH level alters the hydrogen ion concentration effecting primarily the bonds formed between oppositely charged R groups, ionic charges.

Another explanation is that deviations of the pH value away from the optimum because the centre of the active site takes the form of charged ions, some values of pH are inhibitory because they cause these ions to re-associate. This therefore, causes changes in the electric charges in the active site and even on the substrate. The optimum pH level is where the rate of reaction and turnover rate area at their greatest. This is because the shape of the active site of potato catalase is complimentary to the shape on the hydrogen peroxide molecule and the charges within are ideal for catalysing the reaction.

Extremely low pH levels i.e. pH2 may have caused the potato catalase enzyme to be denatured as the extreme hydrogen ion concentration will cause all ionic bonds to break changing the tertiary structure of the enzyme and therefore its function as an enzyme. The rate of reaction would be decreased to almost zero.

Evaluation

STATE ANOMALIES

After evaluating the results it is possible to see that there are anomalies. These appear in the pH5 experiment, where an unusually low bubble count of 62 bubbles was recorded and in a repetition of the experiment at pH5, resulted in a bubble count of 80 during a three minute interval, which was unusually high for the trend of results gained. Another anomaly was in the experiment at pH8 where a very low count of 125 bubbles was recorded. In plotting the line graph, I ignored these anomalies in the calculations of the mean bubble count for the individual pH levels because they would distort the average answer.

These anomalies could have been caused by a number of different factors due to the design of the method or the practical interpretation by the individual scientists. The errors that could have been caused by the design of the method are factors, such as the size and number of discs, osmosis in the petri dish containing water, partial breakdown of the hydrogen peroxide, loss of oxygen, amount of agitation and the varying temperature at which the experiment was performed.

The method explains that cut discs should be used, which creates a large surface area of potato increasing the rate of the reaction, as there is a larger enzyme concentration exposed. As a result the percentage error increases, as the same degree of inaccuracy in a shorter period of time has a larger effect and percentage error than the same degree of inaccuracy in a large period of time due to the formula:

Percentage error = error o X 100

exact value

To overcome this, a smaller surface area should be used and therefore, I believe a suitable improvement is to use a cylinder of potato, 1cm in length. A number of discs in one test tube have the ability to overlap or become stuck on the inner surface of the test tube and therefore, decrease the surface area, which decreases the rate of reaction. The potato cylinders or discs will already be accurately cut with a pre-determined sized cork borer and therefore, have the same circumference length. Error may occur in the accuracy of the length of the potato cylinders and therefore, the mass of the cylinders will change. A higher mass of potato will increase the rate of reaction, as there will be a larger surface area and therefore, a higher enzyme concentration. Another modification would be to weigh the 1cm cylinders so that they are all the same mass. The most accurate method of calculating the weight of the potato cylinder would be by difference, i.e. the weight of the conical flask should be recorded, the potato chip added and the new weight recorded. To calculate the weight of the potato chip, the weight of the flask should be subtracted from the weight of the flask and potato chip. This eliminates any error.

Another source of error due to the method is that osmosis will have occurred to a greater extent on the discs that remained in the water in the petri dish for a longer period of time causing the potato chips to have a larger mass, as the water molecules move from a higher to a lower, concentration of water. The 1mm discs have a much larger surface area to volume ratio, compared to a 1cm cylinder, therefore osmosis will be at a greater rate due to Fick’s Law, which states that increasing the surface area, increases osmosis. During different experiments and repetitions, different potato cylinders would have been taken from different areas of the same potato or from different potatoes altogether, which means the concentration of the enzyme catalase may vary. Therefore an improvement would be to cut a 5cm cylinder from one potato allowing all pH levels to be tested with the same concentration of the enzyme.

Light contains enough heat energy to begin decomposing hydrogen peroxide into water and oxygen. The hydrogen peroxide solution was kept in a glass beaker and therefore, due to the time factor, a higher percentage of the hydrogen peroxide would have broken down when performing the pH9 experiment than the pH5 experiment. The hydrogen peroxide solution would therefore, be more dilute in the sample used in the pH9 experiment causing a slower rate of reaction, as there is a decrease in substrate concentration. This premature decomposing of hydrogen peroxide will be relatively insignificant but I believe a suitable improvement would be to keep the hydrogen peroxide in a black bottle throughout the experiment.

The design of the method in recording the rate of the reaction is relatively inaccurate as it limits the results to discrete data only, as the bubbles produced are counted. The bubbles are produced over 10 second intervals and therefore, when the 3 minute time period is over a bubble may be in the middle of being produced or just about to emerge but does not get counted. Another error is that the bubbles produced will be of different sizes and volumes and therefore as they are not standard this method is inaccurate. An improvement would by to modify the equipment used to measure continuous rather than discrete data. This could be done by inverting a measuring cylinder in water to accurately record the decrease in water level due to oxygen being delivered. This is due to the inaccuracies of the individual people who record the number of bubbles as they may lose count or become distracted.

Oxygen dissolves in water. This may mean that the bubble count measured is actually less because a small percentage of oxgen would dissolve directly into the water rather than form a buble. An improvement may be to choose another inert solvent , one in which oxygen does not dissolve e.g. petrol may give a more accurate reading. Using water would be a systematic error because the bubble count would be lowered for each experiment.

Alternatively, a conical flask with a side arm could be used for the reaction to take place in and attach a gas syringe to the side arm in order to collect and measure accurately the volume of oxygen being produced. However, this reaction may not be vigorous enough to produce a great enough force to overcome the pressure and resistance of the gas plunger and may cause a systematic error in that the volume of oxygen recorded may be slightly less than the actual volume. This modification also eliminates the error due to the loss of oxygen, which would have been produced before the bung is placed on. This error is not systematic but random, as the volume of oxygen lost will change with each experiment and repetition. Therefore it is essential to eliminate and control the error by using an air tight system. The original method would have allowed oxygen produced by the reaction to escape after the potato chips had been added and the test tube sealed with the bung. An alternative method to both mentioned previously would be to allow the oxygen to pass through a delivery tube into an inverted measuring cylinder and measure the decrease in water level. This method would eliminate the resistance of the pressure but would still lose some of the oxygen when attaching the bung. A separating funnel could be attached through a hole in the bung sealing the conical flask in order to deliver the buffered hydrogen peroxide and start the reaction. The hydrogen peroxide would not be affected by mixing it with the buffer, therefore these two solutions should be mixed thoroughly and both poured into the separating funnel to be added simultaneously.

When ready, the tap could be turned and the buffered hydrogen peroxide solution released into the conical flask, then the tap closed to make the system air tight as the reaction will start immediately, with no loss of oxygen. This modification would produce the effect of a greater mean value for the result originally obtained.

The method states to agitate the contents of the test tube gently, which limits the reliability of the results due to the error in the practical interpretation by the scientists in carrying out the experiment. Individual people may agitate their test tube contents with a different force and length of time, which gives rise to another random error. Additionally, one individual person may agitate their test tube contents differently in each experiment. One improvement to eliminate this error would be not to agitate the contents at all, as a cylinder of potato will be used instead of discs which may have to be shaken to free them from one another and the side of the test tube. Also, due to the modified method there will be no need to shake the contents, as the buffer and hydrogen peroxide solutions will have been previously mixed thoroughly. However, it is essential to agitate the contents gently because as the reaction takes place, water will be produced and accumulate on the surface area of the potato cylinder, as this is where the enzyme is exposed and this may block other hydrogen peroxide molecules being decomposed. I believe the solution must be shaken constantly and to the same degree of accuracy. To do this the conical flask must be placed on an electronic magnetic stirring device or electronic shaking device.

The experiment was carried out at room temperature. Catalysts and enzymes are affected by temperature in the relationship that increasing the temperature increases the heat energy, which is transferred to molecules as kinetic energy, increasing in movement of particles. This in turn causes more collisions between molecules, increasing the rate of the reaction as there is a greater chance of successful collisions as the particles have more energy to overcome the activation energy level.

The opening of a door or window would have caused a draft and a decrease in temperature, as the ambient temperature (20°C) was far greater than the outside temperature (approximately 10°C). Also, experiments performed next to a window will have a slower rate of reaction and therefore a lower bubble count compared to experiments conducted in the middle of the classroom. Some experiments may have been performed directly under a light, increasing the temperature and therefore the rate of reaction. The design of the method did not control this confounding factor therefore, an improvement to the experiment would be to perform it in a temperature and light controlled environment, such as a water bath.

The anomalies may also have been caused by the practical interpretation by the scientist performing the experiment e.g. in the cutting and measuring of the discs, by accidentally adding the wrong number, slight variations in the thoroughness of the rinsing of the equipment used or inaccuracies in counting the number of bubbles produced.

Two of the anomalies were obtained from the same experiment, indicating one individual, suggesting error due to the practical interpretation of the method by this particular scientist. Two of these anomalies, namely the 65 bubble count in the pH5 experiment and the bubble count of 125 at pH8 were both much lower than expected, indicating a slower rate of reaction than expected. This may be due to the scientist cutting the potato chips too thinly causing a smaller surface area and a smaller enzyme concentration. The scientist may have measured 1mm but cut a wedge shape e.g. from 1mm at the top to 0.1mm at the bottom. Alternatively, the scientist may have miss-counted the number of discs placed in the test tube causing a slower rate of reaction, e.g. if only 9 discs were placed in the tube.

The extremely high result namely 80 bubble count at pH5 may be due to a larger disc being cut increasing the surface area and enzyme concentration. However, cutting one disc larger than the others would not have as great an effect therefore more discs could have accidentally been put into the test tube. Another possibility is that the scientist may have begun cutting the disc with the scalpel but decided this length was under 1mm, so cut a longer disc leaving a dent in the surface. Also, when transferring the discs to the test tube the scientist may have pinched the discs too tightly with the tweezers. These features would both increase the surface area, which would have caused an increase in the rate of reaction and a possible reason as to why an unusually high bubble count was recorded. An improvement would therefore be to use larger discs e.g. using a cylinder of 1cm in length. Therefore using only one disc, eliminates the error of miss-counting and overlapping. In addition, the percentage error of cutting 1mm length would decrease as if the measurement was 0.1mm in length this would cause a smaller percentage error on the 1cm cylinder than on the 1mm disc.

Another random error may have occurred from rinsing the equipment. The water residue remaining in the test tube may have been sufficient to dilute the hydrogen peroxide buffered solution causing a decrease in rate of reaction in the two unusually low results. The anomaly in the experiment at pH5 with a bubble count of 80, may have been due to the equipment not being washed thoroughly enough causing the substrate and buffered solution to remain in the tube, increasing the hydrogen peroxide concentration which in turn increases the rate of reaction because there are more substrate molecules to be converted into products, producing a larger volume of oxygen. An improvement would be to ensure the equipment was washed with the same degree of thoroughness or to use new equipment for each experiment. However, a more economical way would be to rinse and thoroughly dry the equipment in order to remove remaining residue. In conclusion the anomalous results could have been caused by any number of combinations of these factors. I predict that the modifications and improvements I have suggested, would produce a more accurate and reliable set of results.