Independent Variable:
In this investigation the independent variable, the condition to be changed, is the concentration of the yeast solution. The range to be used is 0, 2, 4, 6, 8 and 10%. These concentrations were calculated using the table below:
Dependant Variable:
In my investigation I will measure the volume of oxygen given off in 20 seconds for each concentration of yeast solution. The start point for the taking of this reading will be identified by when the hydrogen peroxide is added to the yeast solution in the boiling tube.
Biology Coursework
Sharon Dulai
CATALASE –Experiment to investigate the effect of varying the concentration of the enzyme Catalase between a reaction with hydrogen peroxide.
AIM:
The aim of this experiment is to investigate and examine if the concentration of the enzyme Catalase affects the rate of reaction of the decomposition of the substrate Hydrogen Peroxide (H2O2), and if so how.
INTRODUCTION:
Enzyme molecules are proteins that act as biological catalysts, so they are not themselves used up when converting substrate molecules. On every enzyme molecule is an active site where the substrate molecules are joined to the enzyme molecule, and converted to product molecules. Every enzyme is specifically shaped to fit the substrate upon which it works, and therefore they do not work in conditions that denature the molecules, for example very high temperatures. This combined structure of the enzyme and substrate is called the enzyme-substrate complex.
When the enzyme catalyses a reaction, the substrate molecule then splits into two or more molecules however, it could also catalyse the joining together of two molecules. When there is an interaction between the R groups of the enzyme and the substrate atoms, this can cause breaking or encourage the break of the bonds of the substrate molecule. This breaks to form one, two or maybe more products. The products then leave the active site, however the enzyme remains unchanged and is able to receive another substrate molecule. Enzymes are important to life as enzymes catalyse nearly every metabolic reaction that occurs within living organisms.
Catalase catalyses the following reaction:
2H2O2 2H2O+O2
Hydrogen Water + Oxygen
Peroxide
Enzymes such as Catalase are protein molecules, which are found in living cells. They are used to speed up specific reaction within the cell. It is found in tissues where it speeds up the decomposition of hydrogen peroxide into water and oxygen. Speed of action of an enzyme is expressed as its turnover number. This is the number of substrate molecules which one molecule of the enzyme turns into products per minute. Catalase has a turnover number of 6 million and is one of the fastest enzymes. Catalase is a hemoprotein. It uses H2O2 as a substrate as well as a hydrogen acceptor. They are all very specific as each enzyme just performs one particular reaction. Catalase is an enzyme found in food such as potato and the liver. Catalase speeds up the decomposition of Hydrogen Peroxide into water and oxygen It is used for removing Hydrogen Peroxide from cells. Hydrogen Peroxide is the poisonous by-product of metabolism. Oxygen is, of course, a strong oxidizer. Furthermore, most metabolisms in the presence of atmospheric oxygen leads to the production of hydrogen peroxide. It is the peroxide that kills. It is able to speed up the decomposition of Hydrogen peroxide because the shape of its active site matches the shape of the Hydrogen peroxide molecule. This type of reaction where a molecule is broken down into smaller pieces is called an Anabolic Reaction. The optimum pH at which Catalase can work is 7.0.Cu 2+ can inhibit the reaction. Freezing can also cause inactivation, and Catalase can also be inactivated by sunlight under aerobic conditions. Catalase is one of the most catalysts known. The reactions it catalyses are crucial to life. The reactions it catalyses are crucial to life. Catalase catalyses conversion of H2O2 to oxidise toxins including: Phenols, formic acid, formal dehyde and alcohols. Catalase can be stored at 2-8 o c; preparations are stable for 12 months when stored at 5 o c.
Uses of Catalase:
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In tissues: 2H2O2 2H2O+O2
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Bacteria can reduce diatomic oxygen to hydrogen peroxide or super oxide, both of these molecules are toxic to bacteria, some bacteria can possess a defence mechanism, which can minimize the harm done by the two compounds. The resistant bacteria use two enzymes to catalyse the conversion of H2O2 and super oxide back into diatomic oxygen and water. One enzyme is Catalase.
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Catalase is of interest commercially whenever H2O2 is used.
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It is used in the dairy industry to dispose of H2O2 used in milk pasteurisation prior to cheese making.
Tests for Catalase and its presence:
The action of Catalase can be demonstrated by placing a small sample of liver into a beaker containing hydrogen peroxide. If it fizzes, this indicates that oxygen is being given off; it is a dramatic demonstration of an enzyme in action. Finely divided platinum or Iron filings also speed up the decomposition of hydrogen peroxide but nothing like as effectively as a piece of liver as it is rich in catalases. This enzyme achieves greater activation energy than can be brought about by inorganic catalysts.
An enzyme is a protein molecule that speeds up chemical reactions in all living things. Without enzymes, these reactions would occur too slowly or not at all, and no life would be possible. All living cells make enzymes, but enzymes are not alive. Enzyme molecules function by altering other molecules. Enzymes combine with the altered molecules to form a complex molecular structure in which chemical reactions take place. The enzyme, which remains unchanged, then separates from the product of the reaction. Therefore, an enzyme is a sort of biological catalyst. Those enzymes identified now number more than 700.
Enzymes are classified into several broad categories, such as hydrolytic, oxidising, and reducing, depending on the type of reaction they control. Hydrolytic enzymes accelerate reactions in which a substance is broken down into simpler compounds through reaction with water molecules. Oxidising enzymes, known as oxidises, accelerate oxidation reactions; reducing enzymes speed up reduction reactions, in which oxygen is removed.
Catalase is present in the peroxisomes (micro body organelles that house various oxidation reactions in which toxic peroxides are generated as side products) of nearly all-aerobic cells. It serves to protect the cell from the toxic effects of hydrogen peroxide by catalysing its decomposition into molecular oxygen and water without the production of free radicals (An atom or a group of atoms with an unpaired electron. Radicals are unusually reactive and are capable of causing a wide range of biological damage).
Identification of Variables::
In the investigation, the variables that affect the activity of the enzyme Catalase were considered and controlled in order to prevent them from disrupting the success of the experiment.
The variables that could have been used in the experiment to investigate the effects they have on the rate of reaction between hydrogen peroxide and catalase, but were my control variables were:
Temperature of the solutions- as the temperature increases, molecules move faster (kinetic theory). The rate of reaction would most likely double with each 100c rise in temperature, however this will only happen up to a certain point. In an enzyme-catalysed reaction, such as the decomposition of hydrogen peroxide, this increases the rate at which the enzyme and substrate molecules meet and collide and therefore the rate at which the products are formed. As the temperature continues to rise, however, the hydrogen and ionic bonds, which hold the enzyme molecules in shape, are broken. If the molecular structure is disrupted, the enzyme ceases to function as the active site no longer accommodates the substrate. The enzyme is therefore then denatured.
To control this variable, the temperature was maintained at a fairly constant level that allowed the enzyme to work effectively (room temperature, approximately 23ºC). This can be achieved by using a test tube rack and tongs to handle the apparatus so that the heat from hands do not affect the Catalase. A water bath could also be used in order to keep the temperature of the solutions constant. This temperature would be suitable to work with as it is below body temperature therefore the reaction rates will go slightly lower therefore it will be easier to record the amounts of gas being produced during each reaction.
- pH - Any change in pH affects the ionic and hydrogen bonding in an enzyme and so alters it shape. Each enzyme has an optimum pH at which its active site best fits the substrate. Variation either side of pH results in denaturation of the enzyme and a slower rate of reaction, as it would be difficult for the substrate molecule to bind with the active site of the enzyme. In this experiment, the pH was can be kept constant by using a pH 7 buffer, selected to maintain a pH level suited to the enzyme by being equal to the natural environment of the enzyme. However the pH level should remain the same level for each experiment, as the same solutions will be added for each one.
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Substrate Concentration - When there is an excess of enzyme molecules, an increase in the substrate concentration, produces a corresponding increase in the rate of reaction. If there are sufficient substrate molecules to occupy all of the enzymes active sites, the rate of reaction is unaffected by further increases in substrate concentration as the enzymes are unable to break down the greater quantity of substrate.
To control the substrate concentration, identical quantities of the substrate should be used for each reading. To ensure that this is measured precisely, 1cm 3 syringes were used to accurately measure to exact quantities. The hydrogen peroxide will be obtained from the same source for each experiment, so any errors that may occur will not affect the constancy of the results. A 20vol solution of hydrogen peroxide will be used throughout.
The concentration of substrate at which the reaction rate is half of its maximum is characteristic for each combination of enzyme and substrate. It is called Michaelis’ constant.
- Inhibition - Inhibitors compete with the substrate for the active sites of the enzyme (competitive inhibitors) or attach themselves to the enzyme, altering the shape of the active site so that the substrate is unable to occupy it and the enzyme cannot function (non-competitive inhibitors). Inhibitors therefore slow the rate of reaction. They should not have affect on this investigation, however, as none were added.
- Enzyme cofactors - cofactors are none protein substances which influence the functioning of enzymes. They include activators that are essential for the activation of some enzymes. Coenzymes also influence the functioning of enzymes although are not bonded to the enzyme.
Unless enzyme cofactors were present the Catalase, they were not included in this investigation and therefore would not have affected the rate of reaction and the results of this experiment.
- Pressure- altering the pressure of the solutions would affect the rate at which they react. For example, if the pressure was increased then the substrate and enzyme molecules would be forced together, therefore the rate of reaction will increases as the molecules will be encouraged to collide with each other, however this will only happen up to a certain point as the molecules can only be induced to collide a certain amount, any point beyond this will have no affect on the reaction rate. In my experiment the conditions in which I carry out the experiment will be kept the same, therefore the pressure should not alter and will remain constant.
The variable will be: Enzyme Concentration - Provided there is an excess substrate, an increase in enzyme concentration will lead to a corresponding increase in rate of reaction. Where the substrate is in short supply (i.e. it is limiting) an increase in enzyme concentration has no effect and the rate of reaction will no longer increase. I will vary the enzyme concentration by altering the volume of water added to the Catalase, in the reaction (by dilution of the hydrogen peroxide). DIAGRAM OF APPARARATUS:
Once all the equipment had been collected it was assembled. The Hoffman’s clamp was attached tightly to the clamp stand to make sure that the equipment would be held securely. The test tube was then placed in the claw of the clamp then tightened. A bung, which had two holes in it, was then placed in the top of the test tube. These two holes were for the connection that would need to be made to the delivery tube, and one hole for the hypodermic needle through which the hydrogen peroxide would be inserted into the test tube. The plastic container was then filled to the top with water. The measuring cylinder was then held in the water at an angle so that it would get filled up with water more easily. Once the measuring cylinder had completely filled up with water it was turned upside down in the water to keep the water content inside and to prevent any gas seeping in. This water filled measuring cylinder was our aid to measure accurately the amount of gas that was being produced during the reaction between the Catalase and the hydrogen peroxide. Once the measuring cylinder had been prepared, the delivery tube was connected from the bung then placed underneath the open end of the measuring cylinder that was in the water container. Once the apparatus had been set up, the experiment was ready to be carried out. Firstly 2cm3 of yeast solution, which was at a concentration level of 10%, was measured using a 5cm3 syringe. A 5cm3 syringe was specifically used so that the yeast could be measured more precisely to a higher degree of accuracy. The 2cm3 of yeast was then placed in the test tube. 2cm3 of hydrogen peroxide (20vol) was also measured with a 5cm3 syringe, again for accuracy. This was then squeezed through the hypodermic needle into the test tube along with the yeast. As soon as all of the hydrogen peroxide (2cm3of it) had been added into the test tube with the yeast, the stopwatch was started in order to record how much gas was being produced in the reaction at certain periods of time. 5-second intervals were used when timing the reaction rate and recording how much gas was used at each interval during the reaction.
Time intervals of five seconds were used in order to find out how much gas had been produced during the reaction at each time, in order to determine what would be the best time to record the volume of gas produced for each concentration of the yeast solution in the actual experiment. However, when 2cm3 of hydrogen peroxide was used with 2cm3 of yeast, the rate of reaction was so quick that a suitable time at which the volume could be recorded for each experiment was not able to be determined, as the reaction had already completed by the time the 5 second intervals of gas volume recording was ready to begin. Therefore, as 2cm3 of hydrogen peroxide was obviously too large a quantity to add to the yeast, we used 1cm3 of the hydrogen peroxide instead assuming this would slow down the reaction rate.
Table of results from the reaction between 2cm3 of Hydrogen
Peroxide and 2cm3 of yeast:
TIME IN SECONDS VOLUME OF GAS PRODUCED (cm3)
0 0
5 4
10 4
15 4
20 4
25 4
30 4
As you can see from the results that from 0-5 second the rate of reaction had already completed, as there was no further change in the volume of gas produced after 5 seconds. 1cm3 of hydrogen peroxide was therefore used in the second experiment.
When 1cm3 of hydrogen peroxide was used, the rate of reaction went at a slow enough pace for a suitable time to determined as to when to record the volume of gas produced during the reaction in the final experiment. The amount of gas produced could be recorded at 5 second intervals as the reaction rate was at a much more convenient pace.
Table of results from the reaction between 1cm3 of hydrogen peroxide and 2cm3 of yeast:
TIME IN SECONDS VOLUME OF GAS PRODUCED (cm3) VOLUME OF GAS
With yeast concentration 10% yeast conc.n (5%)
5 4 2
10 10 5
15 14 8
20 15 11
25 15 12.5
30 15 14
The results above show how the amount of gas produced during the reaction changed with increasing time. This change in the volume of hydrogen peroxide enabled the recording of gas produced in the reaction, to be done more accurately. This ratio of hydrogen peroxide to yeast solution (1:2) will be used in the actual experiment as it proved to be rather successful. Whilst using the plastic container, it appeared not to be suitable as a container through which the volume of gas being produced would be recorded, as it was difficult to see through the it, this would then result in inaccurate readings off the measuring cylinder. Therefore another container would need to be used in order to make sure the most accurate possible results would be obtained. Instead of the plastic container a 500cm3 glass beaker was used instead so that it would be easier to see the amount of oxygen being produced, and so that more accurate readings could be obtained. Another way to ensure that accurate readings off the measuring cylinder were obtained was by reading off the measuring cylinder at eye level.
When the reaction with 1cm3 of hydrogen peroxide was carried out, the time at which the volume of gas produced with each concentration of yeast solution would be recorded was determined. This will be 20 seconds, so when the actual experiment is carried out, for each concentration of yeast solution, the amount of gas produced during the reaction will be recorded at 20second. The reason for this is that when the highest concentration was tested (10%) the reaction was complete at 20second. This is an ideal time, because if we recorded it at an earlier time, then the lower concentrated solutions may not have began their reaction yet therefore no gas will have been produced, and if we did it at a later time, the higher concentrated solution would have finished their reactions so the recorded results would be unfair because of this.
The following concentration ranges will be used I the experiment:
Concentration of yeast Yeast solution (cm3) Distilled water
10% 2 0
8% 1.6 0.4
6% 1.2 0.8
4% 0.8 1.2
2% 0.4 1.6
0%(control) 0 2
My dependant variable will be the measurement of gas that is being produced by each concentration of yeast solution when reacting with the hydrogen peroxide after 20seconds into the reaction. The reaction will be timed from as soon as the hydrogen peroxide is added to the yeast solution.
Prediction:
Prediction/hypothesis:
Hydrogen peroxide will breakdown to oxygen and water in the presence of Catalase. I predict that the reaction will increase with increasing enzyme concentration when molecules of hydrogen peroxide are freely available. However this will only happen up to a certain concentration level because when molecules are in short supply (if the yeast solution is too concentrated) the increase in rate of reaction is limited and will have little effect. As the concentration of the yeast solution is increased, the reaction will go up at a directionally proportional rate until the solution becomes saturated with Catalase, and each time there is a doubling in the concentration of enzyme added this will most likely result in the rate of reaction doubling until a maximum volume is reached. When this saturation point is reached, then adding extra will not make a difference.
Scientific Theories
In order to support my prediction I would like to refer to scientific theories, which have led me to come to my prediction. Firstly I would like to explain the structure of the enzyme so the theories can be understood more clearly.
Enzymes control almost all cellular reactions. They are each responsible for control of a single reaction and are thus responsible for control of metabolism. Enzymes function as catalysts, which are substances that speed up reactions without actually entering into the reaction. They are used over and over and a single enzyme molecule may mediate thousands of reactions in a single second. Enzymes operate on reactants, which are known as substrates, and convert them into products. The reaction may require energy or it may release energy. The enzyme is unaffected by the reaction.
Enzymes are globular proteins, each with a specific structure (native conformation), function, distribution of electrical charges, and surface geometry whose specificity depends on their tertiary structure. The tertiary structure determines the three-dimensional shape. In their globular structure, one or more polypeptide chains fold, bringing together a small number of amino acids which form the active site: the location on the enzyme where the substrate binds and the reaction takes place. Enzyme and substrate fail to bind if their shapes do not match exactly. This ensures that the enzyme does not participate in the wrong reaction. The enzyme itself is unaffected by the reaction. When the products have been released, the enzyme is ready to bind with a new substrate. This process is the lock and key hypothesis. In order for chemical reactions to take place, the reactants must be bought together. If this process of collision is speeded up then it will speed up the reaction rate. “One of the characteristics of enzymes is that they increases the chances of molecular collisions by drawing the reactants together. They do this by what is called the lock and key mechanism”
The active site of an enzyme is the region that binds the substrate and contributes the amino acid residues that directly participate in the making and breaking of chemical bonds. The amino acid residues are called the catalytic groups. Enzymes differ widely in structure, function & mode of catalysis so active sites vary, but possible to make some generalizations. The cycle of induced fit enzyme action starts with the active site open and substrate (hydrogen peroxide) free in solution. The substrate approaches the enzyme and then binds at the active site. Substrate binding causes a change in the shape of the enzyme such that the catalytic parts of the enzyme are brought to bear on the substrate. The chemical reaction occurs and the two products are left in the active site. If hydrogen peroxide is the substrate, then the two products will be oxygen and water. The two products leave the active site and the enzyme returns to its original active site open configuration. An actual enzyme catalysed reaction occurs so quickly that enzymes can process as many as millions of substrate molecules in one second. Enzymes are thought to operate on a geometric principle. The tertiary and quaternary structures of an enzyme render it exactly appropriate to bind closely with molecules of the substrate it is designed to utilize. Enzymes change shape slightly when the substrate enters, a process known as induced fit.
The changed shape lowers the energy of activation by either stressing an existing bond or correctly orienting two molecules to favour a reaction. The enzyme holds the substrate molecules in exactly the right position relative to each other to facilitate the reaction due to geometric and electrical configuration.
A + B + ENZYME -------> AB + ENZYME
Substrates ---------> product
The reaction occurs and the new product molecule leaves the enzyme due to diffusion gradients, or to new repulsive electrical forces or the shape changes in either the enzyme or the product. New substrate molecules move into position. This change of enzyme moves me onto my next point of the induced fit hypothesis (Koshland 1959). This hypothesis suggested that the enzyme structure was more flexible than it was once thought to be. When the enzyme joins with the substrate, the substrate induces a chnge in the structure of the enzyme changing its conformation. This therefore then puts pressure onto the bonds, which are holding the substrate molecules together making it easier for the bonds to be broken and new bonds to be made, this reduces the activation energy.
````The active site is lined with hydrophobic R groups, which bind with the substrate. The binding is brought about when the groups on the substrate and complementary ones on the active site and attracted. The substrates are held in the correct position, then any of the charged groups in the substrate become more reactive as they are no longer surrounded by water, which is shed as the substrate enters the active site. The binding of the enzyme and substrate move (pushes, twists) the reacting groups and changes the shape of the active site and the enzyme. As the substrate then enters it causes a rearrangement of the molecule and catalytic amino acids are brought into the right positions for catalysis to proceed. The activation is reduced due to the positioning and the strain that is put on the substrate molecule. When the products are formed and released, the enzyme returns to its original binding conformation. Chemical reactions depend on the energy of the substrates being sufficient to cause the molecules to move and thus collide. This is usually accomplished with heat. Heat increases the motion of molecules and makes it more likely they will collide. The heat necessary to accomplish this is often inappropriate inside a cell. Enzymes can be used instead of heat to increase the likelihood that reactants will collide, and collide in the correct position. Enzymes bring reactants together and hold them in the correct position with respect to each other.
To increase the concentration of enzyme is to increase the number of enzyme molecules present in solution. More molecules mean more reactions taking place; twice as many molecules mean twice as more reactions. Therefore, if the concentration is doubled, the speed of the reaction also doubles. This means that Rate is proportional to Concentration. The Collision Theory can justify this. If the concentration is increased there are more enzyme molecules, therefore more collisions take place increasing therefore the likelihood of the reactants being brought together increases. The enzymes increase the chances of molecular collisions by drawing reactants together. This is done by the lock and key mechanism. The lock and key hypothesis explains how reactants called substrates and enzymes behave when mixed. Enzyme and substrate molecules combine to form an enzyme- substrate complex before the products of the reaction are released.
Reactions involve making and breaking bonds. Some bonds must be broken at the start of a reaction and this requires energy. No matter how exergonic the overall reaction may be, some energy must be added initially to break the necessary bonds and get the reaction started. This is the energy of activation (Ea). This energy often supplied as heat but this may not be practical in a cell. All organic molecules contain energy that could be released by the breakdown of the molecule. Such a reaction would be strongly exergonic and, in fact, is favoured by the laws of thermodynamics. It does not happen spontaneously because the activation energy must be provided to get the reaction started and this energy is usually not available. There are times, however, when it is advantageous to the cell for such a reaction to occur. To accomplish this it is necessary to provide the activation energy, or else reduce the amount of activation energy needed. Enzymes make reactions more likely by reducing the energy of activation required. Presumably by holding the reactants (substrates) in exactly the right orientation to each other so that contact will result in a reaction each time.
The rate steadily increases when more enzymes are added as there are more active sites of the enzymes to break down the hydrogen peroxide, which results in more reactions so the amount of oxygen released at given time is higher. Once the amount of enzymes (with their active sites) exceeds the amount of substrate molecules available, and then the rate of reaction will no longer go up. This is due to the maximum number of reactions being done at once so any extra enzymes have no substrate molecules to work on, therefore the reaction rate can no longer increase. The time taken for the reaction to take place, or for the set volume of gas to be given off becomes shorter for higher concentrations of enzyme. This is because higher concentrations of enzyme contain more enzyme molecules than the lower concentrations. If there are more molecules, then there are subsequently more collisions taking place over the period of a second (for example.). This means that more
reactions between enzyme and substrate molecules take place in a second, and therefore the product (O2 in this case) is evolved more promptly. So at higher concentrations of enzyme solution, the oxygen is given off more rapidly because there are more enzyme molecules working on substrate molecules in a second.
PRELIMINARY METHOD:
Before the actual experiment was performed, a preliminary experiment was carried out in order to determine certain factors of the experiment, which would need to be researched in order to make sure the main experiment would produce accurate results. These factors included:
- A suitable range of concentrations of yeast solution to test. The concentration levels can neither be too high or too low. If too much water were added to the yeast then solution would be too dilute causing the reaction to be slow due to a lack of collisions taking place and this would waste too much time, however if not enough water were added the yeast may be too concentrated making the reaction rate much quicker, this would result in a lack of time to record the events of the reaction as it would have taken place far too quickly.
- A suitable concentration for the substrate also had to be determined, as it would also contribute to a change in the rate of reaction.
- The controlled variables, for example pH and temperature, will also need 2 be determined, as they may have an effect on the reaction rate, so may therefore give inaccurate results when altering the enzyme concentration levels.
- Another area that will need to be determined is the apparatus that will be used throughout the experiment. This will need to be suitable and enable the experiment to be carried out effectively and correctly in order to produce results, which are both reliable as well as accurate.
Firstly, the following equipment was collected and set up to begin evaluating the effectiveness of the apparatus and the other factors that would need to be considered during this preliminary experiment:
- Hydrogen peroxide (20 volume)
- Yeast solution (10%)
- Hoffman Clamp
- Clamp stand
- Hypodermic needle and bung
- Plastic container
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5cm3 syringe
- Delivery tube
- Measuring cylinder
- Stop clock
Test tube