To investigate how concentration of the enzyme catalase in celery extract affects the rate of reaction with hydrogen peroxide

Aminul Hoque

Aim: To investigate how concentration of the enzyme catalase in celery extract affects the rate of reaction with hydrogen peroxide

Reaction:

2 H2O2 (aq) → 2 H2O (l) + O2 (g)

Scientific Knowledge:

Enzymes are protein molecules which can be defined as biological catalysts. A catalyst is a molecule which speeds up a chemical reaction. Nearly every metabolic reaction which takes place within a living organism is catalysed by an enzyme.

Catalase is present in all living cells. Enzymes are globular proteins and like all globular proteins, are coiled into a three-dimensional shape with hydrophilic side chains, ensuring solubility.

Enzymes also contain an active site (usually a cleft or depression) to which a substrate molecule can bind to. The shape of the active site is complementary to the shape of the substrate molecule and each enzyme is substrate specific, meaning each type of enzyme will only act on only one type of substrate molecule.

Like all catalysts, enzymes work by lowering the activation energy (ΔG‡) for a reaction, thus dramatically accelerating the rate of the reaction.

Catalase breaks the chemical hydrogen peroxide down to water and oxygen. Catalase is found in all cells and protects them from this dangerous waste chemical. I will use the catalase found in celery extract for this investigation.

The substrate (hydrogen peroxide) and the catalase molecules are continuously on the move. Every so often they will collide so that the substrate molecule(s) fits into the enzyme’s active site.

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How the enzymes work can be explained using the “lock and key” hypothesis, substrate approaches the active site and fits exactly in to it. No other types of substrate have this ability. Products are formed as a result. The enzyme is ready to be used in another reaction.

Each type of enzyme has its own specific optimum conditions under which it works best.

Enzymes work best when they have a high enough substrate concentration for the reaction they catalyse. If there is too little substrate available, the rate of the reaction is slowed and as a result cannot increase any further.

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Material from - http://www.columbia.edu/cu/biology/courses/c2005/lectures/lec06_05.html

When the enzyme and substrate are first mixed, there is a large number of substrate molecules. At any moment, virtually every enzyme molecule has a substrate in its active site. The rate at which the reaction occurs will depend on how many enzyme molecules there are, and the speed at which the enzyme can convert the substrate in to product, release it, and then bind with another substrate molecule. As more substrate is converted in to product, there are fewer and fewer substrate molecules to bind with the enzymes. As less substrate molecules are left, the reaction gets slower and slower until it gradually stops as all the substrate has been converted in to product.

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The pH must be correct for each enzyme, because if the conditions are too alkaline or acidic then the activity of the enzyme is affected. This happens because the enzyme’s shape, especially the active site, is changed. It becomes denatured, and cannot hold the substrate molecule.

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Temperature is a key factor too. If it is cold there will be little or no kinetic energy and so the enzyme molecules will move around too slowly to meet the substrate molecules, so the reaction rate is slowed. Likewise, if it is too warm they do not work properly either. This is because the extra heat energy shakes them around so much that the active sites change shape so, just like with pH, the enzyme molecules are denatured, and cannot hold the substrate.

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In order for particles to react, they need kinetic energy. Reactant must collide, but only a certain fraction of the total collisions, the effective collisions, cause the transformation of reactant molecules into products. This is due to the fact that only a fraction of the molecules have sufficient energy and the right orientation at the moment of impact to break the existing bonds and form new bonds. The minimal amount of energy needed so that the molecule is transformed is called activation energy. In a chemical reaction, catalysts lower this.

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REFERENCES:

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Biology 1, authors Mary Jones, Richard Fosebery, Dennis Taylor;

Method Outline:

In this experiment I will measure the amount of oxygen released when hydrogen peroxide reacts with the enzyme catalase found in celery extract. I aim to see how concentration of enzyme affects rate of reaction. I shall use 15ml of Hydrogen Peroxide and vary the concentration of celery extract by making dilutions from a 10ml base. Concentration of the enzyme is the only variable. All other variables (temperature, pH) will be attempted to be kept constant.

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REFERENCES:

; ;

Biology 1, authors Mary Jones, Richard Fosebery, Dennis Taylor;

Method Outline:

Hypothesis:

I predict, as the catalase concentration increases, so will the rate of reaction. This is due to more active sites of enzyme molecules being available for substrate molecules to bind instantaneously. The collision theory reinforces this idea as more reactant collisions take place when there is a greater concentration, :. more product formed.

I will measure the volume of oxygen released in cm³. Eventually as all the substrate is used up, the reaction will come to a slow. The initial rate of reaction will increase as concentration does. The initial rate of a reaction is the instantaneous rate at the start of the reaction. It can be measured by doubling the amount of oxygen released after 30 seconds for each reaction.

Variables that will be kept constant

Temperature; all the experiments shall be done on the same day so that the room temperature does not change very much, however extraneous variables are impossible to control. Temperature changes can cause enzymes to work at a slower or faster rate as the reaction will have more or less energy to increase or decrease number of collisions. Increase in temperature will provide more kinetic energy for the molecules, making them react more frequently thus, increasing the rate of reaction.

Substrate concentration; for each experiment 15ml of hydrogen peroxide will be used. Changing the substrate concentration each time will result in the enzyme having more or less substrates to bind with. Increase in substrate would result in greater rate of reaction, and greater initial rate of reaction only if there is an increase in enzyme concentration too.

pH; in order to maintain a constant pH, a few drops of a pH buffer solution shall be added to the celery extract each time an experiment is conducted. This will keep the pH neutral for each experiment. With the absence of this, there may be a difference in the acid to alkali ratio of each substance, affecting rates of reaction.

The volume of substrate and enzyme will be kept constant. I will also stir the celery extract each time before I use it so celery is of even consistency & maintain equal amounts of enzyme molecules.

Independent variable: The variable in the investigation is the enzyme concentration (celery extract concentration)

Fair test:

To make this I fair test I will ensure that all the variables are kept constant apart from my chosen variable, if I do not keep the variables constant this could give me many inaccurate results. To keep the substrate concentration the same I will measure it with a 20ml syringe to make sure that the measurements are accurate. I also must not get the syringes mixed up as they will mix with other substances, which may again, cause anomalous results. Temperature is another variable that will have to be kept the same. In order to achieve this, the experiment will have to be conducted on the same day as the room temperature may change due to the weather. The pH of the celery extract is another variable that will have to be kept constant, to do this I will add 1ml of pH 7 buffer to keep reactants neutral. I will also have to stir the celery extract well before I conduct each experiment, as this will maintain equal distribution of enzyme molecules. Also I will have to look at the stopwatch closely to ensure each experiment has the same amount of time.

Pilot Study

I conducted a pilot study to see what volume of celery extract and hydrogen peroxide would be convenient to use in my experiment, and in turn, what apparatus will be most appropriate. Pilot study also helps to minimise any methodological errors.

CONDITION ONE: 5ml celery / 5ml of Hydrogen Peroxide

COMMENTS: Too little oxygen produced

CONDITION TWO: 5ml celery / 10ml of Hydrogen Peroxide

COMMENTS: Slightly better but still too little oxygen produced

CONDITION THREE: 10ml celery / 15ml of Hydrogen Peroxide

COMMENTS: Sufficient amount of oxygen produced after 30 seconds. I shall use these concentrations for my final experiment.

Now I must test the various concentrations of celery with my chosen concentration of hydrogen peroxide.

CONDITION FOUR: 2ml celery : 8ml distilled water / 15ml of Hydrogen Peroxide

CONDITION FIVE: 4ml celery : 6ml distilled water / 15ml of Hydrogen Peroxide

I have chosen to use a test tube rather than a conical flask, although the conical flask would prevent overflowing, a test tube is more convenient and easier to use in successive experiments.

Apparatus

Gas Syringe
20ml syringe
2 x 10ml syringe
Needle
3 test tube
Clamp stand
2 x Boss
Distilled water
Hydrogen peroxide 20 vols
Celery extract
pH buffer
Rubber tubing
Blu-Tack
Stopwatch
Vaseline

The apparatus has been chosen carefully to ensure accuracy. I am using a gas syringe as it more accurate than the alternative way of collecting gas while the tube is submerged in water. In a gas syringe you can measure the gas to 0.5ml of a number, which is relatively accurate. I am using a needle so when I inject the hydrogen peroxide no gas will escape after the instantaneous reaction of catalase with hydrogen peroxide. The needle will be secured with Blu-Tack and held firmly down so no gas can escape. Vaseline was spread on the inside neck of the test tube to ensure the rubber bung was sealed tightly. Two sizes of syringes will be used, 10ml and 20ml. In all, 3 syringes will used, one for each solution (hydrogen peroxide, catalase extract, distilled water) this ensures there is no premature reaction taking place due to mixing of solutions with the syringe. They volumes of solution will be read at eye-level with the black line.

Independent variable range: The range of the variable in the investigation will span 5 concentrations, 0.2 ml, 0.4 ml, 0.6 ml, 0.8ml and 1.0 ml. (effectively 20%, 40%, 60%, 80% and 100%)

Fig 1.1 – Table to show percentage of celery concentrations

I experimented with both the concentrations of the celery extract and hydrogen peroxide. I aimed to get a relatively high amount of oxygen released at 100% concentration of celery extract.

Method:

I will carry out an experiment using the apparatus shown below.

It will be setup as below:

Set up the clamp, boss and stand, placing the test tube and gas syringe firmly in the clamp
Place a rubber bung with tubing on the head of the test tube and connect to the gas syringe
Check for any air locks
Measure the amount of celery extract needed (15ml)
Measure the volume of hydrogen peroxide
Load the separate syringes with their separate solutions and place the celery extract solution in the test tube + add a few drops of pH buffer
Hold the syringe firmly and press the stopwatch as soon as the hydrogen peroxide is injected and reacts with the celery extract.
Record the volume of gas produced after every 30 seconds for a total of 180 seconds

Due to measurements being made to one decimal place, my mean value of the amount of oxygen produced shall also be to one decimal place. I shall repeat the experiment three times each for each concentration and take the mean average value. To get this, I add all the values up and divide by how many there are, in this case, three. Taking averages ensures reliability, depending on how close your results are. The gas syringe allows me to record data at a high level of precision, to 0.5cm³. As a result, my experiment will be more accurate. After calculating the mean, I will plot two graphs. One will show how concentration of catalase affects rate of reaction; the other shall show how the initial rate of reaction is affected. In order to calculate the initial rate of reaction, you must find the amount of oxygen produced after 30 seconds and then double it. For example if after 30 seconds 29cm³ of gas was released, the initial rate of reaction for that particular concentration would be 58cm³.

Safety:

Wear goggles when conducting an experiment.
Wash your hands should you get hydrogen peroxide on it.
Wear an apron as the hydrogen peroxide can stain your clothes.
Clear up any spillages immediately, hydrogen peroxide is corrosive
Should a test tube break, sweep up broken glass safely and quickly

Fig 2.1 - A table to show how concentration of the enzyme catalase in celery extract affects the volume of oxygen given off

RESULTS TABLE: Time taken / seconds

Fig 2.2 - A table to show how concentration of the enzyme catalase in celery extract affects the initial rate of reaction with hydrogen peroxide

Analysis

The graph shows that as the enzyme concentration is increased so is the volume of gas produced. From the graph you can see there is an increase in the rate of reaction. This is due to more enzyme molecules for the substrate molecules bind with. The substrate molecules collide more frequently and faster when there is an increase in enzyme concentration as more active sites are available. The more enzyme molecules there are the more collision there will be with the substrate molecules. At the start of the reaction the enzyme molecules and substrate molecules collide instantaneously, and after the product has been formed, the free active sites seek substrate molecules to bind with them. As the reaction continues, the reaction slows down gradually, as there are not enough substrate molecules to keep up a rapid rate of reaction. This happens until there are no more substrate molecules to collide into and there are no more substrate molecules to turn into products. At this stage, the reaction will stop as there will not be any reaction-taking place. All the substrate has been converted to product, no more oxygen is given off.

The graph shows a trend, as the concentration level increases, so does the rate of reaction. When the substrate first reacts with the catalase there is a rapid increase in rate of reaction, more gas is produced. This is because all the substrate molecules are competing for an active site therefore there a more reactions taking place during the first 30 or so seconds, which will cause more gas to be released and therefore more product made. Then the reaction starts to slow down and the gas produced is decreasing slowly because there are less substrate molecules to collide into active site because they have already been turned into a product.

At 100% concentration of enzyme in the celery extract solution, the substrate and enzyme react rapidly up until 30 seconds and then slowly becomes less rapid and more gradual as the 180 seconds elapse. This is because as it was a concentrated solution, more active sites were available, more substrate binded instantaneously and more product was formed in the first 30 seconds.

At 80% concentration of enzyme in celery extract solution, less active sites than in the 100% concentration are available so less gas is evolved, still greater than 60% concentration, nevertheless.

At 60% concentration of enzyme, the volume of gas released was in between the 80%concentration and 40& concentration. The volume of oxygen produced was due to the number of active sites available to make products.

At 40% and 20% enzyme concentration, the volume of gas released is very gradual. The collisions are slow and the substrate molecules are colliding into the enzyme molecules at slow speed hence the low rate of reaction.

At the beginning of the reaction there is a rapid rate of reaction. The reaction becomes less and less rapid over time and stops. Eventually all reactions at all concentrations will level off at the same point.

The greater the enzyme concentration, the steeper the gradient of the line of the graph. This means it has a greater initial rate of reaction.

The initial rate of reaction graph shows that the initial rates of reaction have increased linearly. The reaction rate is directly proportional to the enzyme concentration. The more enzyme molecules present, the more active sites will present for the substrate molecules to slot into. If there is enough substrate to bind with them, the initial rate of reaction will increase proportional to the enzyme concentration.

The conclusion of this experiment agrees with my hypothesis in that the rate of reaction increased directly with concentration of enzyme. The trends that the graph follows are very similar to those achieved in the pilot study. Scientific knowledge from research and previous knowledge can explain this in terms of the collision theory.

Evaluation

The method used to investigate the effect of enzyme concentration on rate of reaction was appropriate to the aim as it allowed me to control the variables, take measurements, and make relevant conclusions. The experiment showed that the greater the concentration of enzyme, the greater the rate of reaction. However the experiment did contain sources of error and could have been improved.

There is one possible anomalous result in my experiment although it still fits in line of the trend of the graph. At 100% concentration at 180 seconds the amount of gas released is perhaps a bit too much. Should it have been a bit lower it would have followed the course of the rest of the reactions. Anomaly was possibly due to insertion of too much catalase, which would increase the rate of reaction.

There was an extraneous factor which could have influenced the reaction which was the temperature. Although the experiment was done in same day, the temperature was not exactly constant throughout. An increase in temperature results in increase in rate of reaction, due to more kinetic energy present, collisions are more frequent. Decrease in temperature will have the opposite effect and cause a decrease in rate of reaction. An improvement would be to use a water bath, where temperature can be maintained at a constant level. This way, results will not be subject to changes in temperature, and more accurate results can be achieved.

Major sources of error:

Perhaps the most major source of error was the gas syringe sticking. Especially after the 100% concentration when there was an overflow of reacted solution into the gas syringe via the rubber tubing. The gas syringe was dried vigorously using tissue and a hairdryer. It was a relatively lengthy process in an attempt to reduce possible errors in readings but I still believe this was the cause of most inaccuracies compounded in this experiment.

Validity of results:

The results of my experiment and the graph it produced, in comparison to standardised textbook graphs, shows my experiment was valid enough. Despite sources of error, which were relatively small, accuracy was maintained at a maximum level achievable in a classroom, as it was possible to control most of the variables.

Reliability of results:

As the experiment was repeated 3 times for each concentration, I would consider the results reliable. A mean average was taken of the three results for each concentration, of which in general, was not more than 2.0cm³ within each other. To achieve this consistency implies reliability. Only one possible anomalous result was obtained.