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An Investigation on the rate of reaction of the enzyme Catalase on the substrate Hydrogen peroxide. Plan Aim: To investigate the rate of the effect of Catalase on hydrogen peroxide. Introduction This investigation will be carried out to investigate the rate of reaction of the enzyme catalase on the substrate hydrogen peroxide. Enzymes are biological catalysts, which speed up the rate of reaction without being used up during the reaction, which take place in living organisms. They do this by lowering the activation energy. The activation energy is the energy needed to start the reaction.
Enzymes are essentially proteins and will only act in an aqueous environment. An enzyme is specific for a certain reaction or type of reaction. The way enzymes work is called the ‘Lock and Key’ mechanism. The enzyme acts as the lock and the substrate acts as the key. The two fit together in the active site of an enzyme, and are said to be complimentary. The substrate and enzyme, when combined together is called an enzyme substrate complex. There are two types of enzymes: – Builder enzymes- These speed up chemical reactions or small chemicals, which have been joint together to make larger ones.
Breaker enzymes: – These speed up chemical reactions breaking down molecules into smaller ones. An example of a breaker enzyme is Catalase. Enzymes have certain conditions in which they work best. They have to be in conditions of a certain temperature and pH or can denature or deactivate. If they are at too high a temperature the enzyme is denatured and destroyed. If kept at too low a temperature the enzyme is deactivated but not destroyed. Catalase is complementary to hydrogen peroxide and breaks it down into water and oxygen.
The enzyme Catalase is found in the body in a number of organs and tissues. This includes the liver where the job of the enzyme is to decompose hydrogen peroxide into oxygen and water. It is necessary for the Catalase to break down the hydrogen peroxide as the hydrogen peroxide is a by product of metabolism and is toxic. catalase Hydrogen Peroxide Water + Oxygen 2H2O2 2H2O + O2 The diagram above illustrates the Lock and Key mechanism. The substrate has a specific shape to fit into the active site of the enzyme.
As both the enzyme and the substrate are moving they only lock into place when the two molecules collide. The diagram above shows the substrate locked in the enzymes active site and then being broken down into the two desired products of water and oxygen. The enzyme remains chemically unchanged and is ready to combine with another substrate molecule. Possible Variables. There are three different types of variables. They are : – ??? Dependant Variables- These are the ones which change automatically due to the experiment. They depend on the experiment and we measure these ourselves.
E. g- Amount of water and oxygen produced. ???Independent variables- These are the variables we alter ourselves. E. g Concentration of the solution. ???Fixed Variables- These Variables remain constant throughout the whole experiment. E. g Time, size, temperature and volume of solution It is important to have fixed variables so we can determine what factors effect the rate of reaction. E. g If we had the same amount of catalase and kept that as the fixed variable and reacted it with different concentrations of the substrate, and very thing else kept constant, we could determine that he factor affecting the rate of reaction would be the concentration of substrate. In our experiment we will only have a single independent variable, the rest shall remain fixed and allow us to measure the dependent variable. There are four possible variables that can enable us to investigate the rate of enzyme activity. They are: – ??? Concentration of hydrogen peroxide (substrate) ???The surface area of the potato(concentration of enzyme catalase) ???pH of the solution ???The temperature of the solution Concentration of Hydrogen peroxide
Different concentrations of the substrate, hydrogen peroxide, can be used to determine how it affects the rate of reaction. This would be a suitable independent variable as it would be easy to change ourselves. If the concentration of the substrate increases the faster the rate of reaction, as more hydrogen peroxide molecules can collide with catalase molecules, so more reactions will take place. As you increase the concentration of substrate this will continue to occur up to a point where all the active sites on the enzyme are being used.
The diagram above shows the theory that the rate of reaction will continue to climb until all the active sites are being used and the rate will level off but does not stop. S. A of the potato Using different sizes of potato could show us whether the concentration of enzyme affect the rate of reaction. However, this would not be a practical independent variable as the S. A to volume ratio would not be proportional and the size of the potato to get significant results would be very hard to change.
It would be very hard to cut the potato tubers to exact measurements and that could lead to the results becoming inaccurate. An option could be to cut the potato tube into small 1 cm bits and pile then up on top of each other in the test tube, but this again would prove to be impractical as then not all of the surface area of the potato would be exposed to the substrate and this would make my results unreliable. It may also prove to be impractical as having the tuber bits piled on top of each other could give the tubers a larger volume than the solution of hydrogen peroxide.
We would expect however, that if the enzyme concentration was to increase so would the rate of reaction, as there would be more active sites available and a greater chance of a substrate molecule to collide with the enzyme in the active site. pH of solution By altering the pH of the solution we would be able to see how the acidity of the solution affects the rate of reaction. However, the variation in the pH may cause the enzyme to slow or denature giving us very limited results beyond the enzymes optimum pH. It would be very difficult to make up the different pHs with the equipment available to us.
We would probably end up with a denatured protein, due to too high an acidity, which cannot combine with a substrate. When an enzyme is in its optimum pH , its rate of reaction would be at its optimum, however, once the pH is over its optimum the enzymes denatures and slows and the rate of reaction decreases. It would be much easier to keep the pH constant and measure it regularly. The temperature of the solution. As temperature plays an important part in all reactions we could see how the temperature affects the rate of reaction. We would not be able to use room temperature for this, as we have no control over the room temperature.
A possible solution for this could be using water baths. However, there would not be enough water baths for everybody to use, and if we had to share that could lead to all the tubes being mixed up. The bath contains hot water and serious accidents could occur. The high temperatures in the baths could also cause the enzyme to denature. However, as the temperature increases to the optimum temperature , the rate of reaction would increase because heat energy causes more collisions between the enzyme and substrate as there is increased kinetic energy of the molecules and therefore greater reactivity.
Factor I shall vary The independent variable will be the concentration of the substrate, hydrogen peroxide. This proved to be the most practical variable and would provide me with a varied set of results, which would enable me to draw a conclusion. The fixed variables will be: – ??? Amount of Catalase ???pH of the solution ???Temperature ???Time The dependent variable could either be water or oxygen, as both are produced when catalase and hydrogen peroxide are reacted. The dependent variable that we will be measuring will be oxygen.
We can measure the amount of oxygen produced easily by using downwards displacement over water. If our dependent variable were to be water we would find it very difficult to measure as the change would be extremely small. Prediction and Explanation I predict that as the concentration of hydrogen peroxide increases the rate of reaction will increase, up until a point where the rate will become constant. I predict this will happen as, while the concentration of substrate is low there will only be a few collisions between the substrate and enzymes and therefore, the rate of reaction will be low.
However, as the concentration of the substrate increases, the number of available enzyme active sites decreases as more substrate molecules are colliding with the enzymes to make enzyme substrate complexes. As the molecules have kinetic energy they collide with each other, if there is a greater concentration of the substrate there is a greater chance of the molecules with kinetic energy colliding with the enzymes. Collision Theory and Activation Energy: The collision theory states that for a chemical reaction to take place, the articles of the substances (whether atoms, ions or molecules) must collide. There must also be enough energy involved in the collision to break the chemical bonds in the reacting molecules. If the energy transfer is not large enough then the particles will simply bounce off one another and no reaction will occur. A collision that does have enough energy to result in a chemical reaction is called an effective collision. The minimum amount of kinetic (movement) energy that two particles need if they are going to react when they collide is called the activation energy
I predict my graph will look similar to the one shown above after I have carried out my experiment. The graph shows us that as the substrate concentration is increasing are using up more and more active sites on the enzymes, the rate of reaction is increasing. V max on the graph is the point where the rate of reaction will become constant as all of the active sites of the enzymes are being used and even with an increase in substrate molecules the rate will not change as there is no enzyme free for a reaction to occur. Preliminary Work
The preliminary work is the work that is carried out before the main experiment. This experiment is carried out in order to determine measurements of certain factors needed for our main experiment and to familiarise ourselves with the equipment. It was decided that we would use a potato as out source of catalase as it is easy to cut a sample of the same size from it, it is easily obtained and it is safe for us to handle. We used our preliminary to determine the different concentrations we wanted to use. The solution we wee given was 100% hydrogen peroxide.
As we can not make it any more concentrated, the only way we would make our different solutions was by diluting it (adding water). We then proceeded in carrying out our practical. Due to the shortage of time we were only able to set up 3 concentrations. Concentration of solution/%Hydrogen peroxide solution/cmDistilled water/cm 100200 501010 0020 I picked a large potato from which all the cylinders could be cut. This is because different potatoes could contain different amounts of catalase, and that could affect my results and make them inaccurate. The length of potato I chose was 4cms and the cork borer size was 5.
This was suitable was the pieces were easy to get in and out of the boiling tube and easy to handle. The concentrations of 100%, 50% and 0% were set up. As we were going to be measuring the amount of oxygen produced by using downwards displacement, we had to familiarise ourselves with a piece of equipment that none of us had used before a Burette. A burette works by filling the long narrow tube with water, ensuring the bottom is closed. You do not fill the entire tube with water, as you need to leave space on the top for the oxygen after it has displaced the water.
You then turn the Burette upside down, placing a finger on the opening to ensure that no water comes out. Then place the Burette in a trough full of water. Then you clamp the Burette so there is enough space underneath to place the rubber tubing, carrying the gas, but the mouth of the Burette is still submerged in water so no water is lost. The temperature and pH was taken of each of the solutions. The first reaction was started, and the amount of oxygen produced was measured by seeing how much the water level had decreased.
The pH and the temperature were taken of the solution again and the same procedure was done with the other two concentrations. My results may have some inaccuracies or anomalies as I found it hard to place the rubber tube under the Burette without the gas escaping. Conc of hydrogen peroxide/%Vol of water at the start/cm? Vol of water at the end/ cm? Vol of oxygen produced/cmpH startpH endTemp at start/? cTemp at end /? c 041410662525 54128. 512. 5662525 100412615772525 From the results of the preliminary we can see that as the concentration of ydrogen peroxide increases so does the amount of oxygen produced.. From the preliminary I have learnt, that in order to make my main experiment as reliable as possible I will :- ???Cut m cylinders from one potato so all the cylinders have a similar amount of catalase present ???Set up at least 7 different concentrations in order to give me more varied and accurate results. ???Repeat the experiment another 2 times. This allows us to have an average result and balances out any anomalies, which may have occurred. ???Leave each concentration for 3 minutes, so it allows the reaction to be completed and not disturbed. Cut the 4cm cylinders in half into 2cm pieces, as the test tube is wide enough to allow both cylinders in without making them sit on top of one another. This will therefore, provide a greater surface area for the substrate to react with. Apparatus and Diagram ???Potato ???Hydrogen peroxide (100%) ???Ruler ???Beaker ???Cutting Tile ???Delivery tube ???Bung ???Thermometer ???Cork borer size 5 ???pH paper ???2 syringes ???Marker pen ???Clamp ???Measuring cylinders ???Burette ???Trough ???Distilled water ???Vernier Calliipers ???Knife ???Boiling tubes ???Test tube rack Method 1. Firstly all safety precautions must be taken.
Goggles and lab coats must be worn. 2. The workbench should be completely cleared, stools should be tucked under the bench and any bags around the area should be cleared to prevent any accidents. 3. All the necessary apparatus should be gathered. 4. 7 boiling tubes should be placed in the rack. 5. Using a marker pen label the tubes 1-7 6. Set up the following concentrations using hydrogen peroxide, distilled water, a measuring cylinder and syringe* Test Tube No. % of conc. Amount of hydrogen peroxide/cmAmount of water/cm 1100200 280164 360128 4501010 540812 620416 70020 When using a syringe, always go a little past the amount needed, and before inserting it into the boiling tube, flick the syringe and press it out to the correct figure. Once that is done then put the solution into the test tube. This allows any air bubbles present in the solution to rise to the surface and be pressed out. 7. The pH will be taken by dipping in a small piece of universal indicator paper. The paper will then be taken out and placed near a pH key so an appropriate pH can be decided. The results will then be recorded. 8. The temperature will be measured by placing a thermometer into solution.
The results will then be recorded. 9. Using a size 5 cork borer, one cylinder will be extracted from the potato 10. This will then be cut down into 4cm using the knife blade and white tile. 11. Using the knife, ruler and cutting tile, cut the 4cm tuber into two pieces each 2cms long 12. Fill the Burette with 41cm? of distilled water, ensuring the tap at the bottom is closed. 13. Place your thumb over the opening in the Burette, and turn it upside down. 14. Place the Burette in a trough full of water. 15. Clamp the Burette so that there is enough space to place the rubber tubing, from the delivery tube, carrying the gas, underneath.
But the mouth of the Burette is still submerged in water. 16. Place the first concentration of 100% in front of you. 17. Insert the two cylinders in the solution and start the stopwatch straight away. 18. Immediately place the bung and delivery tube into the boiling tube. Pinch the rubber tubing at the end to stop any gas escaping. 19. Carefully place the rubber tubing under the water and into the Burette. 20. Leave the reaction for 3 mins 21. While the reaction is taking place, prepare the next cylinder in the same way as the previous one. 22. After 3 mins pinch the end of the rubber tubing so no more gas can escape. 3. Slowly remove the rubber tubing out of the Burette to avoid suck back. 24. Record the new level of water in the Burette. 25. The pH of the solution will be taken and recorded 26. The temperature of the solution will be taken and recorded 27. Fill the Burette with distilled water up to 41cm? for each new concentration and complete all the reactions for the different concentrations in the same way. 28. All apparatus should be cleared and put away. Safety This experiment has few hazards and the following precautions will be taken to ensure a safe working environment. Bags and coats shall be put away to ensure nobody can fall over. ???Benches will be completely cleared so there will be plenty of space for all the equipment so nothing can be knocked over, especially the hydrogen peroxide. ???Goggles will be worn to prevent any damage to the eyes. ???Lab coats will be worn to prevent any damage to clothes. ???The knife will be handled with extreme care as any misuse can lead to serious injury. ???Hair will be tied back properly, to ensure it does not come in the way. ???Paper towels will be readily available should there be any spills. The hydrogen peroxide will be handled with extreme care as it is harmful. ???The Burette will be handle with extreme care as it id fragile and made of glass. Fair Testing This is done to ensure that everything stays the same apart from what I want to change. To ensure this is shall: – ???Cut my cylinders from the same potato. This is t ensure they all have the same amount of catalase present, as different potatoes have different amounts. ???The size of each but of potato will be kept constant at 2cms each. This will be measured using a ruler. ???Each cylinder will be cut with the same cork borer so it has the same thickness. Each concentration shall be carried out 3 times. This makes our results more reliable and enables us to gather an average results. If we only had one set of results, there could be anomalies making my results inaccurate, as I would not have another set of results to compare it with. ???All the concentrations shall be made using measuring cylinders and measuring equipment to ensure exact quantities. ???The time the potato cylinders are left in solution will be the same; this will ensure they all had the same amount of time to react and produce oxygen. A stopwatch will be used for accuracy. The same apparatus will be used throughout the experiment ???The temperature of the solution will be measured at the start and the end of the experiment. This way we can see if the temperature was relatively constant. Obtaining Evidence Modifications The method to the experiment was mostly carried out to plan. There was only one slight change in our original method. After each of the reactions had taken place, we found it very difficult to keep refilling the Burette up to 41cm again. Not only was it difficult to obtain the exact measurements but it wasted a lot of time.
Instead of refilling the Burette after one reaction, we would note down the new measurement the water was at. We would then continue to carry forward the reaction as explained in our method and would then record the new level of water after the oxygen had displaced it. After finding out the difference of the two we were able to figure out how much oxygen was produced. This method was much more practical and allowed us to only have to refill the Burette twice and gave us more time to take care over our experiment and not to hurry it. Analysis Trend
My results make my graph go from the bottom left to the top right. At first my graph line is straight, which shows me that as the concentration of hydrogen peroxide increases the rate of reaction increases proportionally. This means that as the concentration of substrate was increasing it was colliding more often with the enzymes and more active sites of the enzymes were being filled. This caused the rate to increase. After the concentration had reached 80% however the graph levelled off, which means the substrate and enzyme were now reacting at a constant rate as all of the active sites were now being used.
This point on my graph is called Vmax and is the maximum rate of reaction. Conclusion From the results we can see that they agree with the prediction made that when there is a high concentration of hydrogen peroxide the rate of reaction will increase until a certain point at which the rate of reaction will be at a constant speed as all the active sites on the enzymes will be in use. As the concentration of the substrate increased, it was more likely that the substrate molecules would collide with the active site on a catalase molecule to form a enzyme substrate complex.
When the concentrate of the substrate, hydrogen peroxide was lower, molecules collided less often and therefore, it took longer for the reaction to work and longer for the hydrogen peroxide to be decomposed into water and oxygen. That is why when the concentration of the substrate was lower less oxygen was produced. When the concentration of the substrate was higher they bombarded the catalase and increased the breaking down rate. Therefore, more oxygen was produced in the given time. The closer the substrate molecules are with the enzyme molecules the greater the chance of collision.
If the hydrogen peroxide is diluted it means the substrate molecules are more spread out, due to the water molecules and collide less with the enzyme molecules. As the concentration of the substrate increased we can see from the recordings in the results table that the temperature increased slightly. This is due to the enzyme and substrate molecules colliding and producing heat energy, giving other molecules around them more kinetic energy, and increasing the number of collisions. In conclusion I feel my experiment gave me the results I had expected.
Before I had carried out the experiment I predicted that the rate of reaction would increase and the substrate concentration increased up until the point where all the active sites are being used up and the rate becomes constant and it is the same conclusion I have been able to draw after using my graph. My prediction graph and my Results graph are not identical but are similar. This could be due to minor accidents that occurred whilst the experiment was being carried out. Overall my written conclusion and my prediction are the same. Evaluation Accuracy of results I feel my method enabled me to gather accurate and reliable results.
My graph agreed with the one I made as my prediction. My graph shows a smooth line and my results show accuracy as they follow a trend. As the method was followed carefully and precisely I feel my results were very accurate. After doing the preliminary work I knew what mistakes I had made so that they could be resolved so very few errors would occur in the main experiment. My experiment was also carried out three times. This would average out any anomalies, which may have occurred making my results inaccurate. Anomalies An anomaly is one particular result that does not fit in with the rest of your results.
This is why three repeats were done, as if only two were done and one result was an anomaly you would not be able to identify which one it was. Although I had no major anomalies I did have a few minor ones. They occurred at the concentration of 0%. At the concentration of 0% it was expected that no oxygen would be produced. However in one of my repeats it was recorded that 0. 1 cms of oxygen was produced. The reason for such a slight amount of oxygen being produced could be, that a small amount was present in the rubber tubing from the previous concentration where oxygen had been produced and had not escaped.
However on averaging my results for 0% concentration I found that it did not alter my graph. Reasons for inaccuracies ???Not enough readings were taken ???There was not enough time allowed for the displacement of oxygen ???The size of potato (the concentration of catalase) was not sufficient. ???Tubes may have been moved around or shaken. This would cause inaccuracies as it would increase the rate of reaction as it would be giving the molecules more kinetic energy ???It was difficult to place the bung in the boiling tube and to start the stopwatch all at the same time Improvements to method Larger potato pieces, to increase the amount of catalase the substrate had to react with. ???More readings should have been taken and therefore more concentrations should have been made. ???The reaction should be left for longer so displacement can occur fully and not be interrupted. ???Some bungs were not the right size for the boiling tubes. I could therefore check all of my apparatus fully before starting my experiment. Further Work If I were going on to do further work on the rate of reaction of enzymes I would test the other factors, which affect an enzyme.
I would change my independent variable from being the concentration of the substrate to the concentration of the enzyme. I would do this to see if it would have similar effect on the rate of reaction. I would carry out the experiment in a similar way to this one expect I would keep the concentration of the substrate constant and vary the concentration of the enzyme. I would imagine it to have similar effect and as the concentration of the enzyme would increase I would believe the rate of reaction to do so as well. Sources I used to help ???Class notes ???Biological Science ???Google images