Investigate the effect of changing substrate concentration on the rate of the reaction between catalase and hydrogen peroxide.

Aim : To investigate the effect of changing substrate concentration on the rate of the reaction between catalase and hydrogen peroxide.

Introduction :

Enzymes are proteins and biological catalysts, produced by living things. They have high specificity, i.e., they only catalyse one particular reaction; every biological process has its own enzyme designed specially for it. Enzymes are not changed when they perform their function, which means that the enzyme molecule can be used over and over again.

Each enzyme has a unique shape. The active site of an enzyme is the region where it fixes itself with the reactant. The reactant on which the enzyme acts is called the substrate. This is why an enzyme’s profile is so important; it can only work if the shape of its active site is perfectly correct to interact with the substrate. This is called the lock and key mechanism.

Hydrogen peroxide is a by-product formed during metabolic reactions in cells. It is a toxic and has to be gotten rid of as quickly as possible. For this, catalase is a tremendously useful enzyme. Catalase is present in every living cell of the human body. It carries out a very vital reaction :-

Hydrogen peroxide Water + Oxygen

2H2O2 H2O + O2

For this particular reaction, catalase can be obtained from potatoes, which may be added to the hydrogen peroxide. The rate of reaction can be calculated very effectively by measuring the amount of oxygen that gets produced. A simple and accurate method of doing this would be to count the number of oxygen bubbles produced during a set time in the reaction.

Key Factors :

Enzyme activity is mainly affected by temperature and pH value. At high temperatures and extremes of pH, an enzyme changes shape and can no longer work. This is called denaturing. They discontinue working because the shape of the enzyme molecule changes and so does the shape of the active site. Thus, the lock and key mechanism cannot take place.

The optimum temperature of an enzyme is the temperature at which the rate of reaction is at its highest and can increase no further. This is the most suitable temperature an enzyme can work at. Most enzymes have an optimum temperature around 40°C. Similarly, the optimum pH for an enzyme is the pH at which the reaction rate is greatest and can go up no further. The enzyme works best at that pH. Most enzymes have an optimum pH around pH7.

Rate of reaction will also be affected by surface area. The larger the surface area of a substance, the faster the rate of reaction. This is because there are more particles per cm3. Therefore, there will be more collisions every second between the substrate molecules and the enzymes.

Volume is another key factor that can influences the rate of the reaction between hydrogen peroxide and catalyse. Greater volume means more particles and more collisions every second. More collisions signify a faster reaction rate. Also, since, the number of particles is greater, the amount of product formed will be greater.

For this specific reaction, two other essential factors that need to be considered are the enzyme and substrate concentration.

The higher the enzyme concentration, the faster the rate of reaction because if extra enzymes are present, the substrate will be able to get catalysed faster. More enzymes per cm3 will lead to more collisions between the substrate molecules and enzyme molecules, every second. Thus, a faster rate of reaction.

Likewise, the higher the substrate concentration, the faster the rate of reaction as the presence of more reactant will lead to more collisions between it and the enzymes. The enzymes are all the more likely to collide with the substrate molecules if more are present in very cm3. Also, additional product will be formed as the initial amount of reactant is greater.

The aspect that I have chosen to investigate in this experiment is the concentration of the substrate, i.e., the hydrogen peroxide. I shall keep changing this factor as it will help me explore how the rate of reaction changes with changing concentration of hydrogen peroxide.

The factors that I will need to control are volume and surface area of substrate and enzyme, as well as temperature and the concentration of the enzyme. It would be best to carry out this chemical reaction at the optimum temperature of catalase, as then, we can focus entirely on how the concentration of hydrogen peroxide limits the rate of reaction. It can also be carried out in a beaker full of water at room temperature as then, the temperature would remain constant. Fluctuation in temperature during different parts in the experiment would lead to an unfair test.

Potato, or enzyme concentration also needs to be controlled as this will help us investigate how the different amount of substrate (hydrogen peroxide) for the same amount of catalase (in potato) causes change in the reaction rate.

Prediction :

Based on scientific theory, I can predict that a high substrate concentration will lead to a faster rate of reaction than a lower substrate concentration. As the concentration is initially increased, the rate of reaction will increase because of the presence of more particles in every cm3. There will be more collisions every second and the reaction will proceed quicker.

Then again, the concentration can only be augmented to a certain extent. Above this limit, the reaction will be able to go no faster. This is because, as the number of substrate particles builds up, the number of collisions between them and the catalase molecules increases. But soon, all the enzymes present will be engaged in catalysing the substrate molecules and none will be left to catalyse the extra reactant. The lock and key mechanism can no longer take place effectively as one enzyme molecule cannot interact with more than a specific amount of substrate molecules at a time. We can say that the enzymes are then limiting the reaction rate since there is a lot of substrate, but not enough of enzymes to catalyse it.

If I start from a low substrate concentration and keep increasing it, my results’ graph will have the following profile –

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If I start from a low substrate concentration and keep increasing it, my results’ graph will have the following profile –

RATE -

OF -

REACTION -

SUBSTRATE CONCENTRATION

The higher the substrate concentration, the greater the rate of reaction, until the substrate concentration starts exceeding the enzyme concentration. Then the enzymes start limiting the reaction rate.
Hence, the pattern of my results increases initially until a certain point where the reaction rate will stop increasing and remain the same no matter how much higher the substrate concentration gets.

Controlling key factors :

I plan to control each key aspect of this experiment as far as I can, using possible equipment and appropriate procedures.

Enzyme concentration - The amount of potato should be kept the same each time. I will do this by using a cylindrical cutter to slice the same length of potato pieces each time. Hence, the mass of these pieces is what should be considered most importantly and should be the same for all parts of the experiment.
Temperature - I shall keep the temperature of the hydrogen peroxide stable by carrying out my experiments in a beaker full of water at room temperature. Thus, the test tube, surrounded by water from the same source, will have the same temperature each time.
Ph - The pH of the hydrogen peroxide is a constant and will be the same every time it is used in the experiment. The potato also has a set pH and so, the reaction will not be affected. Water has a pH around 7 (neutral) and its addition to the hydrogen peroxide will not cause much change.
Surface area - I have decided to use a specific mass of potato each time and this will be mashed. I will mash the potatoes using a pestle and mortar. The surface are will be more or less the same each time. Also, the surface area of the potato will be at its greatest all throughout the experiment (as it is mashed) so that I can acquire the best possible rate of reaction.
Volume - The same volume of hydrogen peroxide will be used each time. A measuring cylinder will be used to measure out the amount of hydrogen peroxide and the water to dilute it. The amount of potato will also be the same. The potato’s mass will be kept the same each time by measuring it on weighing scales.

Producing accurate and reliable results :

To make sure that my results are as dependable as possible, I will have to control my key factors as precisely as I can, except the substrate concentration that I am investigating.

I will try to use a different test tube to carry out the reaction each time, so that there can be no contamination. I may pour fresh water into the beaker each time so that any change in temperature during one reaction cannot affect the rest of the experiment.

All the same, my results might not be absolutely accurate and may not generate an entirely fair test. Firstly, although counting bubbles is straightforward and reasonably accurate, it might not be a very dependable course of acquiring results. The size of the bubbles may be different each time so that the amount of oxygen being produced is altered.

The surface area of the potato could be different for each different reaction as some of it may have been pulverized in a discrete way. Also, after the potato has been mashed, a certain percentage of it is lost when being transferred from the pestle & mortar to the test tube.

Eyesight can be a hindrance in counting the number of bubbles produced in a cylinder full of a transparent fluid like water. Consequently, the amount of bubbles counted can only be called a precise approximation of the amount of oxygen released since it is possible to miss a few bubbles in the process of counting.

Preliminary work :

At the beginning of my preliminary experimentation, I used whole cylindrical pieces of potato. Then, I tried the reaction with mashed potato and recorded that the number of bubbles produced was more even though the mass of the potato being used was the same. So as not to let the surface area of the potato become an impediment to the reaction rate, I decided to carry out the remaining reactions with mashed potato. I will use mashed potato for my final investigation too.

The volume of hydrogen peroxide that I began my investigation with was 50 ml, but this turned out to be too much because after the addition of the potato, the boiling tube began to overflow. As a consequence of this, I decided to use 20 ml of hydrogen peroxide solution and it filled the tube perfectly, even after the adjunct of the potato.

Subsequently, I observed that 2 grams of potato produced more oxygen bubbles than just 1 gram of potato. 2 grams of potato produced the bubbles at a steady rate. I premeditated that using more potato than that would increase the reaction rate considerably, and it would be hard to keep track of the production of bubbles.

While timing, I perceived that nothing much happened during the first 30 seconds of the reaction. Thus I resolved that I would start to time the production of oxygen bubbles after the first the 30 seconds of the reaction. For the remainder of the experiment, I waited for the reaction to get going in the first half minute and then counted the bubbles produced for the next one minute.

The following were the results I got for different concentrations of hydrogen peroxide :-

My results from my preliminary work are the aspects that will influence my final investigation the most. Now I know what concentrations, amounts and timings produce what sort of results. I know that specific concentrations of hydrogen peroxide and amounts of catalase in potato will boost my reaction rate in certain ways. I also know what will limit the rate of reaction.

After considering the results from my preliminary work, I have decided to use the following : -

Amount of potato - 2 grams

Volume of Hydrogen Peroxide - 20 ml

Range of Concentration - Six different concentrations between 10% and 90%

Method :

Since the apparatus for my preliminary work was most suited to this particular experiment, I have decided to use the same for my final investigation.

Apparatus - (1) Boss, clamp and stand

(2) Measuring cylinders

(3) Test tubes

(4) Beakers

(5) Cylindrical potato cutter

(6) Pestle and Mortar

(7) Rubber cork

(8) Rubber tubing (to carry the oxygen released)

(9) Stopwatch

(10) Potato

(11) Hydrogen Peroxide

(12) Water

Setup -

Instructions -

Take a potato, and using a cylindrical cutter, cut out the number of pieces necessary.
Weigh the amount of potato required and mash it using a pestle & mortar. Set aside.
To prepare the above setup, fill in a beaker with water from a tap or a bowl previously filled with water at room temperature. The level of water should be such that the test tube can be submerged in it, without it having to overflow.
Fill up another beaker with water from the same source and then fill a measuring cylinder with the water from the beaker. Invert the cylinder immediately into the beaker, allowing no water to escape or overflow from the vessel.
Insert one end of the rubber tubing into the measuring cylinder, making sure that no water escapes. The other end of the tubing should be attached to the rubber cork that will be used to plug the boiling tube during the course of the reaction.
Clamp the required boiling tube onto the extension from the stand and immerse into the relative beaker.
In a measuring cylinder, measure out the required quantity of hydrogen peroxide.
Pour out the volume of water needed to dilute the hydrogen peroxide, in another measuring cylinder. Do not use the same measuring cylinder for the hydrogen peroxide and water as it will cause pre-integration of both substances.
Dilute the hydrogen peroxide in the boiling tube.
Add the potato to the test tube and plug the tube instantaneously. Start the timing with this addition.
Observe how not much change occurs during the first 30 seconds of the reaction. Hence, do not record any changes that do occur.
After the first 30 seconds, record the production of oxygen bubbles on the upper surface of water in the inverted measuring cylinder.
Count the number of bubbles created for one minute. This is because, when the production of oxygen first begins, it is slow. Soon, the bubbles start increasing in number rapidly and over a minute, it can become difficult to get an accurate calculation.
Vary the substrate concentration from 10% to 90%, altering it about six times.
After each reaction, either wash & dry the test tube thoroughly, or use a new test tube.

Safety Precautions -

Wear safety goggles at all times.
Do not misuse any equipment, especially knives, cylindrical cutters and scalpels. Keep such equipment away from the rest of the equipment and from areas of extreme activity.
Do not allow any chemicals, like hydrogen peroxide, to make contact with skin, eyes and any other body areas. Rinse well with cool water if such contact occurs.
Discard potatoes used for the investigation at the end.
DO NOT drink any water used for the experiment.
Do not eat or drink near ongoing experiments.
Do not allow mixing of any substances unless specifically mentioned.

RESULTS :

ANALYSIS :

It can be observed that my results mostly followed a specific trend. However, there were certain parts of this experiment that created anomalous results, where the pattern created didn’t fit in with the rest of the results.

It is noticeable that during the second and third attempts, the number of bubbles produced with a concentration of 12 vols. was greater than the number of bubbles produced with a concentration of 16 vols. The rate of reaction followed a basic pattern until 16 vols., when it dropped atypically.

A logical explanation that can be given to this phenomenon is that, as the substrate concentration kept increasing, the enzymes had a better chance of colliding with the particles. There were more collisions per second because of the presence of extra particles per cm3, leading to a faster rate of reaction - until a particular point in the concentration (16 vols.) was reached. This was where the substrate concentration had become so high that the number of enzymes present was insufficient to catalyse it. Because these enzymes began limiting the rate of reaction, the number of oxygen bubbles being produced, dropped. The lock and key mechanism could no longer take place because of the inability of an enzyme to catalyse more than a limited number of molecules at a time.

The rate of reaction is high at the beginning of the experiment. For example, between 0 & 4 vols., 4 & 8 vols. and 8 &12 vols., the reaction rate increases by almost 30 bubbles per minute. However, the difference in the reaction rate between 12 & 16 vols. and 16 & 20 vols. is very small. These were the stages in the reaction where the enzymes had begun to limit the rate of reaction. The substrate concentration in 12, 16 and 20 vols. was extremely high and each time, limitation in the reaction’s progress was similar. This explains why there was little difference in the amount of oxygen bubbles released each time.

My graph displays a straight line of best fit. The graph begins with an initial reaction rate of 5.25 bubbles per minute. It goes on in a straight and steady pattern until 16 vols., where it begins to level off. It is probably somewhere around this concentration that the substrate particles began exceeding the number of enzymes and above this concentration, the rate of reaction could go no higher. The lock and key mechanism could no longer take place because there were too many reactant particles for the enzymes to fit themselves with correctly and for the reaction to proceed as normal.

There are phases where my results match my prediction and phases where they do not. As predicted, the rate of reaction increases with increasing concentration of the substrate. However, there came a certain concentration where the rate dropped and then increased again at a higher concentration. I had not foreseen this when planning my investigation and these results do not agree with my prediction. Then again, this change happened in only two out of the three attempts made at our experiment and so, it may not be very reliable.

The following part of my results completely disagrees with my prediction :–

Attempt 2 –

Concentration Bubbles produced

12 80

16 76

20 91

attempt 3 –

Concentration Bubbles produced

12 93

16 63

20 98

Additionally, during attempt 3, I observed that the same amounts of oxygen bubbles were produced with 8 and 16 vols. of hydrogen peroxide. This also diverges from my prediction as I had forecasted an increase in the rate of reaction with increasing substrate concentration. This reaction rate does not fit in with my results because it increases steadily up to 16 vols. and then falls back to the same as that with 8 vols. It also suggests that the rate of reaction obtained with 12 vols. of hydrogen peroxide was anomalous.

With the complete concentration of hydrogen peroxide at 20 vols., the rate of reaction was at its highest and could go up no further. At this point, the number of enzymes limited the reaction rate because there were not enough present to catalyse the volume of reactant. I recorded that with 20 vols. of hydrogen peroxide, the number of oxygen bubbles produced was relatively close every time. This aspect of my results links with my prediction as I had foreseen that above a particular concentration, the reaction rate would stop increasing because the collision theory would not be able to take place, i.e., there were too many substrate molecules for all of the enzymes to collide with. The lock and key mechanism also could not occur to its fullest extent because all the enzymes were already engaged in catalysing the substrate molecules present.

EVALUATION :

In my feeling, the biggest drawback of my method was the technique of procuring results. I think that counting bubbles is what may have chiefly caused some parts of my results to be different from the rest.

In the higher concentrations of hydrogen peroxide, the number of bubbles produced was often difficult to keep track of. Around 4 – 5 bubbles were formed at a time and as the reaction proceeded rapidly, it became problematic to observe the strict quantity of oxygen bubbles being released. Also, all the bubbles were of different sizes, so it can only be presumed how much oxygen was actually produced.

A better method of acquiring results would have been by using a gas syringe or by measuring the amount of water being displaced. Instead of using an inverted test tube, it would be wiser to use an inverted measuring cylinder. In this fashion, we could account for the tangible quantity of oxygen that was produced in one minute as it could easily be calculated within a measuring cylinder. A gas syringe could have been used similarly, though to greater extents and a more practical outcome. The reaction could have been carried out in a conical flask with a side-arm flask, attaching it to a gas syringe that would have directly measured the amount of oxygen being given off.

Moreover, the surface area of the potato could have caused some of the incongruities . After being cut, mashed and then transferred to the test tube, a reasonable amount of potato must have been lost. Although we tried our level best to remove all the potato after pulverising it with the pestle & mortar, some amount of it was inevitably left behind, causing the amount used in the reaction to be different from the 2 grams we started out with. It is possible that the mass of potato used for each reaction was slightly different every time.

A more feasible method would have been to cut up enough potato at the beginning of the experiment and pound it. It could then have been measured out before each of the reactions, so that not too much would be lost when it would be transferred from the weighing scales to the test tube.

In my thinking, the rubber tubing was another element that caused some inaccuracy of results. The rubber tubing frequently slipped outside the inverted test tube when it should have been inside so that it could produce the bubbles with the oxygen it was carrying. We often had to discontinue a particular experiment because of this. Also, the tubing was sometimes inserted too high up into the test tube and in the middle of the reaction, we had to rearrange the apparatus so as to keep the tubing immersed in the water and keep up the production of oxygen bubbles. The tubing habitually got kinked and the production of oxygen bubbles would cease. When this kink was adjusted, a large number of oxygen bubbles would suddenly be released into the test tube so that it became difficult to keep up the count.

There were a small number of anomalous points in my results. For instance, in the very first attempt of our experiment, when the concentration of the hydrogen peroxide was 4 vols., the number of oxygen bubbles produced was ten, whereas, for the rest of the attempts, the amount of oxygen released was dissimilar to this. Another anomalous point was in Attempt 3, where the quantity of bubbles produced with 12 vols. of hydrogen peroxide was very different from my remaining attempts with this concentration.

The cause of these anomalous results could have been the volume of either hydrogen peroxide or the water used to dilute it. It is possible that we measured the wrong amount of either of these substances during the reactions mentioned. It is also possible that the liquid may not have been transferred completely from the measuring cylinder to the boiling tube.

There was another uncharacteristic result during Attempt 3 where 8 vols. and 16 vols. of hydrogen peroxide produced the same quantity of oxygen bubbles. This may have been caused because of a mistake in the counting of bubbles or again, because of a misjudgement about the volume of hydrogen peroxide or water.

I feel that our results are more unreliable than reliable. Firstly, this is highlighted by the fact they go against my prediction at several points. They are not very accurate as can be seen from two critical parts of my investigation where the rate of reaction rises again after falling for a particular concentration. This is highly aberrant because it is only possible for a reaction rate to stop increasing, not drop altogether. Even after this sudden fall, it happened to increase again – and this was displayed not once, but twice, during two different attempts at this experiment.

Furthermore, the fact that my results are not completely reliable, is demonstrated in the results from our third attempt, when the amount of oxygen being produced for 8 vols. and 16 vols. of hydrogen peroxide, is the same. This cannot be correct because one of the concentrations is twice the other and so, the amount of oxygen being produced in one case, should be, more or less, double the amount being produced in the other. The greater the amount of reactant, the greater the amount of product formed. But this part of my results goes completely against this theory.

I subsequently conclude that my results cannot be considered either accurate or reliable, because not only have they gone against my prediction, they are also opposing scientifically proven theories and phenomenon.

To extend this reconnaissance, I would first and foremost, choose to explore a wider range of concentrations. This would give me a broader view of the processes that take place during the course of the reaction and get me a more extensive array of results. I would also prefer to undertake a separate option for acquiring results as counting bubbles of oxygen turned out to be a rather chancy procedure.

There may even be better source for obtaining the catalase. Cutting up the potato, weighing it, mashing it and then transferring it to the boiling tube, took very long. In this process, a certain amount of potato was lost so that the amount we carried out the reaction with was not the same 2 grams we began with.

Catalase is also present in liver and this could be a substitute for potato in a further task. If this is done, it will also help us in comparing the amount of catalase in potato and liver and judge which one would be a better ingredient for an appropriate enzyme concentration.

If I had to do this research again with a different method, I would, most importantly, use different apparatus. Firstly, I would use a gas syringe to measure the amount of oxygen being produced, instead of just counting bubbles, which in my opinion, did not prove to be very accurate. This may also prevent further hassle with the rubber tubing.

Furthermore, I feel that the experiment will be more successful if carried out at the optimum temperature of catalase. In this way, the reaction rate will not be limited by low temperature.

As a final point, I feel that extending the investigation would generate better results because it would give me a chance to view better circumstances for the experiment to be carried out in, as well as to explore, in depth, the reason for all the anomalies and atypical ingredients of this initial research.