Investigating Enzyme Activity

Investigating Enzyme Activity
Aim: To investigate how the concentration of hydrogen peroxide effects the rate of reaction of an enzyme (catalase) Variables: These factors could effect the rate of reaction on an enzyme: ? pH ? Concentration ? Temperature ? Surface Area pH ? Enzymes function at different pH values. In neutral conditions the amount of oxygen gas given of in an enzyme-catalysed reaction will increase. An enzyme is affected by how much acid or alkali is present. Many enzymes work best in neutral conditions but some prefer acids and some prefer alkalis. This graph shows that the enzyme activity reacts best at pH7 (neutral) Concentration ? In concentrated solution there are more collisions between each particle, so the reaction occurs more quickly. This graph shows that increasing the concentration increases the enzyme activity. Temperature ? Reactions go faster as temperature rises
The rate of reaction also increases as the temperature rises, but with enzyme-catalysed reactions the reaction rate starts to decrease when the temperature is above 40 C. This is because enzymes are proteins and their structures start to damage above 40 C. This graph shows that the enzyme activity reacts best at 40?C as the enzyme starts to denature above 40?C Surface Area ? Reactions can react faster when solids are cut into smaller pieces. This is because there is more surface area which is exposed. The more surface area there is, the more collisions that take place between particles so the reaction rate is much quicker. This graph shows that small pieces react better than bigger pieces. Brief Outline I will test the effects of changing the level of concentration. For this variable I will use three different concentrations of hydrogen peroxide with catalase (enzyme). I will change the concentration whilst keeping the time, concentration of catalase and the volume of hydrogen peroxide constant. I will begin all my tests at a constant temperature (room temperature) and I will repeat each test three times so I can obtain an average result. Background Knowledge: Lock and Key Model A catalyst is a substance which alters the rate of reaction without being used up. Enzymes are the catalysts in biological processes. They are large proteins that speed up chemical reactions. The enzyme forms the active site from small numbers of amino acids. The active site is the location on the enzyme where the substrate collides and the reaction takes place. If the shape of and the substrate do not match exactly then they do not bind. This makes sure that the enzyme does not work with the wrong reaction. Enzymes are not affected by the reaction, so when the products have been released, the enzyme is ready to bind with a new substrate. In my experiment the substrate was the hydrogen peroxide, the enzyme that we used was hydrogen peroxide and the product that was formed was oxygen and water. This can be explained by an equation: Enzyme + Substrate ÞProduct In my experiment this is shown as: Catalase + H202 ÞH202 + 02 This equation explains how the catalase in our experiment binded with the H202 to break it down and form oxygen. Induced Fit Theory The induced fit theory states that the binding of a substrate to an enzyme causes a change in the shape of the enzyme. The enzyme and the substrate act on each other to affect the making of the active site to the usual complex between the enzyme and its substrate. As a result, this means the enzyme to catalyze a reaction has changed. This shows that enzymes are specific for specific substrates. I can tell that the catalase in my experiment is a suitable enzyme to break down the H202 as it will form oxygen as a product which is unharmful Denaturing Denaturing is the damage to the protein structure of an enzyme. Most enzymes react faster as the temperature increases. Enzymes also react at low temperatures, but when the temperature rises above 40 C their reaction rate start to decrease. This is because enzymes are proteins and their structures get damaged when the temperature rises above 40 C. When the protein is denatured it becomes less effective as a catalyst and soon the enzyme reaction gets slower and then finally it stops. This is why enzymes in washing powders which clean by breaking down grease and other stains, cannot be used with hot water above 40 C Activation Energy In order for a reaction too occur activation energy must be supplied. The activation energy is the energy required to start a chemical reaction. Some elements and compounds react together to bring themselves into contact. For others it is necessary to supply energy in order to start the reaction. This energy is the activation energy. Enzymes such as catalyst work by lowering the activation energy. The Kinetic Theory of Matter Everything is made of moving particles. The main points of the kinetic theory are: ? All matter is made up of small particles called molecules ? The molecules are always vibrating ? The higher the temperature, the faster the molecules are moving ? As the temperatures rises the particles get hotter. They have more energy and move around faster. Solid Liquid Gas Solid ? In a solid the particles are very close together and have very strong forces between them. Solid particles can only vibrate, this is why they cannot flow. Solids have a fixed shape and a fixed volume Liquid ? In a liquid the particles are a little further apart. The forces are not very strong. Liquids can flow and change shape but they always have a fixed volume. Gas ? In a gas the particles are further apart. There are no forces to hold all the particles together. Thy move about very quickly in the space they find. Gases can flow easily and change their shape and their volume depending on the container. Collision Theory The collision theory explains chemical reactions and the way in which the rate of reaction alters when the conditions alter. For a reaction to occur the reactant particles must collide. Only a fraction of the total collisions cause a chemical change. These are called fruitful collisions. The fruitful collisions have sufficient energy (activation energy) to break the existing bonds and to form new bonds, which then form the products of the reaction. Increasing the concentration of the reactants and raising the temperature make more collisions and therefore more fruitful collisions which increases the rate of reaction. All reactions involve two reactants which need collisions between them for particles to proceed. But not all collisions taking place between particles end up with a reaction. This is because in the middle of a reaction, there is a shape of the particle which is difficult to complete. This is called the transition state. The total kinetic energy of reactant molecules must be at least as high as the activation energy to be able to achieve the transition state, so the reaction can proceed. For a reaction to occur there must be successful collisions in which: 1) Particles must collide 2) Particles must have enough energy for the reaction to take place (activation energy). Which means the reaction must be successful If a collision between particles can produce sufficient energy and the particles collide fast enough in the right direction a reaction will take place. But not all collisions result in a reaction. A reaction is speeded up if the number of successful collisions are increased. The particles in a If the collision has If the collision does not liquid move around enough energy a have enough energy no continually reaction takes place reaction occurs The rate of reaction depends on how many successful collisions there are in a given unit of time. Surface area By breaking solids into smaller pieces the surface area is increased, which gives a greater area for collisions to take place. This causes an increase in the rate of reaction. Temperature When a substance is heated, the particles move faster. When the particles are moving faster they will travel at greater distances which will involve more collisions. This will increase the rate of reaction. Concentration The more concentrated the reactants are the greater the rate of reaction. This is because increasing the rate of reaction increases the number of collisions between particles and therefore increases the rate of reaction. Pressure An increase in pressure can lead to an increase in the rate of reaction. The increase in pressure forces the particles closer together. This causes more collisions and therefore increases the rate of reaction Prediction After looking at the background information I predict that the mass of the oxygen produced will be proportional to the concentration of the hydrogen peroxide which will also be proportional to the rate of reaction. I think that if I increase the concentration of hydrogen peroxide I will increase the number of collisions, which will increase the rate of reaction. Therefore I think that if I double the concentration of hydrogen peroxide I will double the number of collisions which will double the rate of reaction I think this will happen because in general, concentrated solutions react more quickly than dilutes solutions. In a concentrated solution there are more collisions between the reacting particles, so the reaction will proceed more quickly. Dilute solution of Concentrated solution of H202 H202 Conclusion Reaction rates can be speeded up by increasing the concentration of the reacting solution. After observing my results I found that my prediction were correct. Looking back at my experiments and the results I collected I can see that the concentration of hydrogen peroxide increased so did the volume of oxygen produced. This happened because as the concentration of the hydrogen peroxide increased so did the number of particles and this lead to an increase in the number of successful collisions and therefore the reaction rate increased along with the volume of oxygen gas. Dilute solution of Concentrated solution of H202 H202 From looking at the diagram I can see that the dilute solution H202of has only a few particles which means there is less chance of collisions occurring, whereas the concentrated solution of H202 has more particles which means there is a greater chance of collision. However I could not prove the second part of my prediction which was, if I double the concentration of H202 it would double the number of collisions, which would double the rate of reaction. If I pull out some results from my tables I can see that the amount of oxygen produced for each concentration did not double but it did increase. Time (s) Volume of oxygen gas evolved ? 1.5% Volume of oxygen gas evolved ? 3% Volume of oxygen gas evolved ? 6% 10 26.3 37.3 50.0 20 43.5 56.3 76.7 30 57.7 74.5 93.3 Analysing From looking at the calculations of the rate of reaction I can say that all three concentrations of hydrogen peroxide (1.5%, 3%, 6%) had the fastest rate of reaction at the start of the reaction. This can be explained by using the rate of reaction theory. In a reaction, reactants are being used up and products are forming. So the amount of reactants fall and the amount of products rise. In my experiment the hydrogen peroxide decreased and the oxygen rose. Overall the hydrogen peroxide with the concentration of 6% had the fastest reaction because it had more particles for collisions to be successful than the 1.5% and 3% concentration of hydrogen peroxide. From looking at my results I can also see that the 1.5% concentration of H202 had a slower reaction than the 3%. I can explain this by applying the collision theory to this result. There are less particles in the 1.5% concentration than the 3%, this means there would be less chances of collision which would reduce the rate of reaction. This goes the same for the 6% concentration, as the 3% had a slower reaction than the 6%. In my experiment the catalase did not denature at any point. However, if I was to increase the temperature of the catalase the catalase would have denatured above 40oC. Safety ? Safety glasses were used during my experiments harmful chemicals were used ? The measuring cylinder was placed on the table and I bent down to measure the hydrogen peroxide ? I used the syringe slowly and carefully because if I pressed too hard there would be too much pressure and the catalase would spill out ? The catalase was heated in a water bath for safety Fair Test To make my tests fair, I will investigate how the concentration of hydrogen peroxide, effects the rate of reaction by changing the concentration and keeping all other factors constant. Constant Factors: Equipment used ? by using the same equipment throughout all the experiments. Time ? by allowing each experiment to proceed for only 2 minutes (trial experiment ? 1 minute because we will be testing the experiment for any problems) Concentration of ? by using the same concentration of catalase (1%) throughout catalase all the experiments. Volume of catalase ? by using the same volume of catalase throughout all the experiments. Volume of H202 ? by using the same volume of H202 throughout all the experiments. Temperature ? by using the same reaction temperature (room temperature) throughout all the experiments. Variable factor Concentration ? by carrying out the experiment using different concentrations of hydrogen peroxide (1.5%, 3%, 6%). I will use different concentrations of hydrogen peroxide throughout the experiments and carry out three tests for each concentration. This will help me calculate an average result (line of best fit on a graph) and also increase the accuracy of my experiments. Evaluating I can improve my method to give me better results by using a wider range of concentration of hydrogen peroxide. This would then give me more results in which I can use to draw up a better conclusion. I could also improve my experiment by collecting the oxygen gas in a different way. I could use the method of displacement to collect the oxygen gas. This would help me get accurate results of how much oxygen gas evolved. Another improvement I could use in my experiment is by using a bigger gas syringe to collect the oxygen gas. This is because the syringe I used was too small and the oxygen gas filled the syringe causing the end of the syringe to come out. A bigger syringe would help me find out how much oxygen gas was actually being produced. If I was to do this experiment again I would definitely change the time in which we measured the oxygen gas. This is because 5 seconds was a too shorter time to measure the oxygen being evolved. I would rather read the gas syringe every 10 seconds, which will enable me to read the gas syringe more accurately to give me better results. There were two big errors from my results that show up on my graph. The first error (circled) was on the 1.5% concentration. This may have occurred because the conical flask which contained the catalase and the hydrogen peroxide was shaken too much. The second error (circled) was on the 3% concentration. This error may have been a result from an incorrect reading from the gas syringe. Apart from these few improvements I think the equipment and the method I used was an appropriate way of investigating how concentration effects the rate of reaction on an enzyme. A variable that I could try for an extra experiment is temperature. I could try increasing the temperature of the catalase to see if increasing the temperature has the same effect on an enzyme as it does with concentration. This would help me to see which variable, temperature or concentration has a greater effect on the volume of oxygen produced from the reaction of an enzyme. Rate of Reaction I am going to work out the rate of reaction from the average value of each of the concentrations of hydrogen peroxide. I will work out the rate of reaction for every 10 second from the average values by using my graphs. Reaction Rate for the concentration of 1.5% 10s = 20 = 2.2 cm3/s 9 20s = 20 = 2.0 cm3/s 10 30s = 19.5 = 1.4 cm3/s 13.5 40s = 15.5 = 1.1 cm3/s 14 50s = 14 = 0.8 cm3/s 17 60s = 9 = 0.5 cm3/s 17.5 Reaction Rate for the concentration of 3% 10s = 25 = 3.3 cm3/s 7.5 20s = 20.5 = 2.05 cm3/s 10 30s = 17 = 1.2 cm3/s 13.5 40s = 15 = 0.73 cm3/s 20.5 Reaction Rate for the concentration of 6% 10s = 22 = 2.93 cm3/s 7.5 20s = 18 = 2.0 cm3/s 9 30s = 9 = 0.75 cm3/s 12 40s = 5 = 0.29 cm3/s 17.5 From looking at these calculations I can say that for all the concentrations of hydrogen peroxide the reaction rate was fastest at the start of the reaction. During the reaction the rate decreased and eventually the reaction stopped. I can explain this by using the lock and key model. This occurs because the catalase is breaking the particles of the hydrogen peroxide to form into oxygen. This causes the hydrogen peroxide to get used up as the catalyst can alter the rate of reaction without getting used up. This then leaves the catalase to react with no substance. Data Logging Data logging is another experiment I did to extend my experiment. I looked at the enzyme activity experiment in a different way. This time I used the same quantity of catalase and hydrogen peroxide but instead of measuring the volume of oxygen produced, I found out the temperature increase for each of the different concentrations of hydrogen peroxide. Method 1. Collect all equipment and set the experiment up 2. Wear safety glasses as you are using chemicals 3. Pour 25cm3 of each concentration of hydrogen peroxide into a measuring cylinder 4. Pour each concentration of hydrogen peroxide into three different polystyrene cups 5. Measure 1mm of catalase into three different syringes 6. Make sure that the end of the probe is touching the bottom of the cup and that the hydrogen peroxide is completely covering it 7. Inject all three of the catalase at the same time into each polystyrene cup 8. At the same time start the computer as you inject the catalase, to start of the measurement of the temperature, of the hydrogen peroxide 9. Watch how the computer measures the temperature for 2minutes as it transfers all the information into a graph 10. Stop the graph at 2 minutes and print out the results Conclusion From my results I can see that the 6% concentration of hydrogen peroxide has given of the highest temperature. This is because the concentration of 6% has many particles of hydrogen peroxide which will have a greater chance of colliding with the catalase. The concentration of 1.5% and 3% has given of less heat causing a lower temperature. This is because they have less particles of hydrogen peroxide to collide with the enzymes. This result can be explained by the kinetic and collision theory. The increase in heat gave an increase in the kinetic energy. This means that there would be more collision between the hydrogen peroxide particles and the catalase, which would lead to a better chance of collision being successful. This experiment is related to my first experiment as the increase in concentration gave off more oxygen which gave us an increase in temperature. I found out that the 6% concentration gave of the most oxygen which causes a rise in temperature and the 1.5% gave of the least oxygen causing a lower temperature. The 6% also gave off the most oxygen in the shortest time whereas the 1.5% concentration gave off the least oxygen in the longest time. From looking at my graph I can see that the 6% concentration had the steepest slope which if I relate back to my rates of reaction theory. I can say that the steeper the slope the faster the reaction. The 1.5% concentration had the smallest gradient which means it had the slowest rate of reaction. Overall this proves my original prediction, that the 6% concentration of hydrogen peroxide has the fastest rate of reaction and the 1.5% concentration has the slowest. Reaction Rates ====== During a reaction, reactants are being used up and products are forming. The reaction rate tells us how fast the reaction is taking place. You can calculate the reaction rate by measuring how much reactant is used up or how much product forms in a given time. Reaction rate = change in amount of a substance time taken The reaction rate can also be calculated by using a graph. The slope of the graph tells us how quickly the reaction is happening. The steeper the slope, the faster the reaction Results from the Trial Experiment Time (s) Volume of O2 evolved (cm3) ? 1.5% Volume of O2 evolved (cm3) ? 3% Volume of O2 evolved (cm3) ? 6% Average 0 0.0 0.0 0.0 0.0 5 15.0 20.0 25.0 20.0 10 16.0 30.0 45.0 30.3 15 16.0 35.0 62.0 37.6 20 21.0 36.0 64.0 40.3 25 25.0 40.0 73.5 46.2 30 32.0 50.0 77.5 53.3 35 35.0 60.0 80.0 58.3 40 39.0 64.0 100.0 67.6 45 40.0 70.0 100.0 70.0 50 43.0 73.0 100.0 72.0 55 45.0 80.0 100.0 75.0 60 46.0 90.0 100.0 78.0 I did a trial experiment to help me understand the experiment. The trial experiment showed me how quickly each concentration of hydrogen peroxide produced the amount of oxygen gas in one minute. I also learnt that slow pressure is needed with the small gas syringe because too much pressure causes the catalase to spill out. Results from the concentration of 1.5% Time(s) oxygen evolved (cm3) oxygen evolved (cm3) oxygen evolved (cm3) oxygen evolved (cm3) oxygen evolved (cm3) oxygen evolved (cm3) average 0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 5 20.0 17.0 25.0 15.0 20.0 24.0 19.6 10 28.0 24.0 34.0 20.0 25.0 34.0 26.3 15 35.0 26.0 42.0 29.0 37.0 40.5 35.5 20 46.0 29.0 53.0 40.0 41.0 49.5 43.5 25 60.0 33.0 73.0 44.0 46.5 60.0 50.1 30 71.5 35.0 77.0 52.0 52.0 69.0 57.7 35 86.5 39.0 82.0 69.0 60.5 77.0 68.8 40 89.0 44.0 89.0 78.5 68.0 83.0 76.5 45 94.0 49.0 92.0 85.0 75.5 89.0 73.5 50 97.0 53.0 99.0 90.0 85.0 94.0 83.2 55 99.0 59.0 100.0 92.0 87.5 99.0 89.7 60 100.0 65.0 100.0 97.0 94.0 100.0 92.8 65 100.0 72.0 100.0 98.5 97.5 100.0 97.0 70 100.0 79.0 100.0 100.0 99.0 100.0 98.7 75 100.0 84.0 100.0 100.0 100.0 100.0 99.7 80 100.0 88.0 100.0 100.0 100.0 100.0 100.0 85 100.0 94.0 100.0 100.0 100.0 100.0 100.0 90 100.0 100.0 100.0 100.0 100.0 100.0 100.0 95 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100 100.0 100.0 100.0 100.0 100.0 100.0 100.0 105 100.0 100.0 100.0 100.0 100.0 100.0 100.0 110 100.0 100.0 100.0 100.0 100.0 100.0 100.0 115 100.0 100.0 100.0 100.0 100.0 100.0 100.0 120 100.0 100.0 100.0 100.0 100.0 100.0 100.0 I am going to use columns 5,6 and 7 for my average because these columns have the nearest results to each other and the range of results are realistic. Results from the concentration of 3% Time(s) oxygen evolved (cm3) oxygen evolved (cm3) oxygen evolved (cm3) oxygen evolved (cm3) oxygen evolved (cm3) average 0 0.0 0.0 0.0 0.0 0.0 0.0 5 45.0 17.0 25.0 27.0 26.0 26.0 10 51.0 24.0 33.0 41.0 38.0 37.3 15 62.0 30.0 57.5 50.0 40.5 49.3 20 65.0 39.0 60.0 54.0 55.0 56.3 25 72.0 45.0 60.5 61.5 62.5 61.5 30 75.0 54.0 73.0 69.0 81.0 74.5 35 81.0 62.0 80.0 75.0 90.0 81.6 40 88.0 73.0 86.0 80.5 100.0 88.8 45 98.0 86.0 93.5 88.5 100.0 93.8 50 100.0 92.0 100.0 95.5 100.0 98.5 55 100.0 100.0 100.0 100.0 100.0 100.0 60 100.0 100.0 100.0 100.0 100.0 100.0 65 100.0 100.0 100.0 100.0 100.0 100.0 70 100.0 100.0 100.0 100.0 100.0 100.0 75 100.0 100.0 100.0 100.0 100.0 100.0 80 100.0 100.0 100.0 100.0 100.0 100.0 85 100.0 100.0 100.0 100.0 100.0 100.0 90 100.0 100.0 100.0 100.0 100.0 100.0 95 100.0 100.0 100.0 100.0 100.0 100.0 100 100.0 100.0 100.0 100.0 100.0 100.0 105 100.0 100.0 100.0 100.0 100.0 100.0 110 100.0 100.0 100.0 100.0 100.0 100.0 115 100.0 100.0 100.0 100.0 100.0 100.0 120 100.0 100.0 100.0 100.0 100.0 100.0 I will be using columns 4,5 and 6 for my average because these columns have the nearest results to each other and the range of results are realistic. Results from the concentration of 6% Time(s) oxygen evolved (cm3) oxygen evolved (cm3) oxygen evolved (cm3) oxygen evolved (cm3) average 0 0.0 0.0 0.0 0.0 0.0 5 37.0 31.0 35.0 35.0 34.0 10 58.0 45.0 47.0 45.0 50.0 15 66.0 63.0 66.0 50.0 65.0 20 74.0 76.0 80.0 52.0 76.7 25 81.0 87.0 91.0 60.0 86.3 30 86.0 97.0 97.0 95.0 93.3 35 95.0 100.0 100.0 100.0 98.3 40 99.0 100.0 100.0 100.0 99.7 45 100.0 100.0 100.0 100.0 100.0 50 100.0 100.0 100.0 100.0 100.0 55 100.0 100.0 100.0 100.0 100.0 60 100.0 100.0 100.0 100.0 100.0 65 100.0 100.0 100.0 100.0 100.0 70 100.0 100.0 100.0 100.0 100.0 75 100.0 100.0 100.0 100.0 100.0 80 100.0 100.0 100.0 100.0 100.0 85 100.0 100.0 100.0 100.0 100.0 90 100.0 100.0 100.0 100.0 100.0 95 100.0 100.0 100.0 100.0 100.0 100 100.0 100.0 100.0 100.0 100.0 105 100.0 100.0 100.0 100.0 100.0 110 100.0 100.0 100.0 100.0 100.0 115 100.0 100.0 100.0 100.0 100.0 120 100.0 100.0 100.0 100.0 100.0 I will use column 2,3 and 4 for my average because these columns have the nearest results to each other and the range of results are realistic. Time (s) Temperature (oC) 1.5% Temperature (oC) 3% Temperature (oC) 6% 0 20.5 21.0 21.0 2 20.5 21.5 21.5 4 21.0 21.5 21.5 6 21.5 21.5 21.5 8 22.0 22.0 22.0 10 23.0 22.0 22.0 12 24.0 22.5 22.5 14 25.0 23.0 22.5 16 25.5 23.5 23.0 18 26.0 24.0 23.5 20 26.5 24.5 24.0 22 27.0 25.0 24.5 24 27.0 25.5 25.0 26 27.5 26.0 25.5 28 27.5 27.0 26.0 30 27.5 28.0 27.0 32 28.0 29.5 28.0 34 28.0 30.5 29.5 36 28.0 31.5 30.5 38 28.0 33.0 31.5 40 28.0 34.5 33.0 42 28.0 36.0 34.5 44 28.0 37.0

Investigating Enzyme Activity 7.3 of 10 on the basis of 2398 Review.