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AIMEffect of temperature of the action of the Enzyme Catalase.PLANNINGBackground Knowledge
An enzyme is a biological catalyst, it alter the rate of reaction without being changed itself. Enzymes are proteins; they have a very precise three-dimensional shape, which forms a one specific active site on the enzyme. Each enzyme can only convert one kind of substrate molecule in to one kind of product molecule. These are specific.What affects Enzymes?· Temperature- Enzymes stop working if the temperature rises above 40ºC. Increasing the temperature alters the D shape and so the enzyme can no longer fit the substrate.· pH- They work best in neutral conditions neither acidic nor alkaline.What affect does catalase have?Catalase is a very fast reacting enzyme, it is found in many living cells, it breaks down hydrogen peroxide to water and oxygen. In fact one molecule of it can deal with six million molecules of hydrogen peroxide in 1 minute. Hydrogen peroxide is toxic so needs to be changed into harmless substances.CatalaseHydrogen peroxide water + oxygen HO HO + OReferences to practicals referring to enzymes· Biology for You Pg 0 Experiment .1From looking at this I found out that catalase reacts with hydrogen peroxide to give out water and oxygen. Oxygen bubbles produce froth on the surface of the solution. In my forthcoming experiment I will expect to see froth being produced.· Biology- Nelson Science Pg 5 Picture 4From looking at this graph, see below. I have learnt that the affect of temperature does in fact change the rate of reaction. From the graph the reaction reaches 40ºC but then denatures and the rate of the reaction decreases. The rate falls rapidly suggesting denaturing.Taking this information into account I would expect the enzyme catalase to show a similar pattern with respect to the temperature. In order to observe the effect of temperature on catalase we will be maintaining in the amount of oxygen released. The oxygen produces a froth which we will then measure in mm and the volume of oxygen given off which will be measure in cm³Method- measuring the height of froth and volume of oxygen1. Put work shirt on and goggles on. Carry out the rest of safety precautions.. Gather equipment as shown on diagram1.. Using a cork borer make 5 cylinders from the large potato.4. Cut them into all the same length (6cm)5. Using a pestle and mortar mash up each cylinder separately. 6. Measure 5ml of hydrogen peroxide using a measuring cylinder.7. Select the temperature you are going to study0ºC- iced water5ºC-no extra equipment7ºC-water bath required55ºC-water bath required100ºC-beaker of boiling water8. Place on mashed cylinder into a boiling tube add the measured hydrogen peroxide and attach the rubber bung connected to the measuring syringe.. Start stop watch and record volume of gas collected every 0 seconds. At the same time measure the amount of froth produced at 0 seconds intervalsApparatus· 5 beakers· 5 test tubes· Thermometers· Cork borer· Potato· Ruler· Knife· Tile· Measuring syringe · Heat proof mat· Bunsen burner· Tri-pod· Wire gauze· Pestle and mortar· Hydrogen peroxide· Matches· Spills· Ice cubes· Water bath· Goggles · Spatula· Stopwatch· Measuring cylinderFair testIn this investigation I will keep constant the following· The surface area of the potato. I will use the mashed up form as it will be a faster reaction as there is more area to react on, as we have to consider the time span.· The same volume of hydrogen peroxide in each part of the investigation.· The same size equipment e.g. boiling tubes as the readings for the results will be wrong if this is not constant.· Use the same method for each experiment so that there won t be any major differences. Only alter the temperature.· Keep the amount of potato the same amount.· Measure the temperature with a thermometer.AccuracyIn order to make my investigation go to plan I will be as accurate as I can be so I will measure to the correct measuring size.· Measure the volume in cm³ and amount of potato in grams to make sure that they are exactly the same mass before using them in the experiment.· Do the experiment three times to ensure that there isn t an odd result. Three is a good number to use as you can see if there is one odd one where if you just done the experiment twice then you wouldn t know which one odd and which isn t.· Also to average out the results. Safety precautions· Wear goggles· Tuck tie in skirt· Wear work shirt· Handle the hydrogen peroxide with care as it is corrosive and an irritantPredictions and ReasonsFrom my research I think that the enzymes will denature after 40ºC and any other temperature above that. Reason being that enzymes are proteins and their structure is three-dimensional. Increasing the temperature disturbs the intra molecular bonds that hold the D shape. Because of this the shape is altered. Enzymes have an active site. This fits into the substrate molecular (see diagram-lock and key). If the active site is altered the substrate will no longer fit in and so the enzyme doesn t work properly. The rise of reaction rate is also due to the increase in temperature, relating to the kinetic theory. The higher the temperature, the faster they move. This happens but only to an optimum of 40ºC. The curve leading up to the optimum point is gradual but as it is reached it falls dramatically. The reason being that the active site is destroyed therefore no reaction can take place as there is only one specific active site per substrate.OBTAINING EVIDENCEBelow are my table of results which show the height of froth produced in cm and the volume of oxygen in cm³ for each of the three tests at each of the five temperatures studied.TEMPERATURE 10ºCTEST 1 TEST TEST Analysing results and ConclusionFrom my results it appears that catalase works best at 7ºC, and it is virtually denatured at boiling point. Looking at the initial part of the reaction (see graph 1) it is clear that the gradient at the beginning gets steeper when looking at the temperatures between 10ºC-55ºC. At each temperature the line levels off towards the end of five minutes. Looking at graph , there is a steady rise in height of froth up to 7ºC and then a gradual fall up to 100ºC. Looking at my background knowledge and prior experiments using enzymes I can explain my results as follows. Kinetic theory states that particles, which gain heat energy, move more quickly. In our case the reacting particles are the substrate (hydrogen peroxide) and the enzyme catalase. As the temperature is increased the particles of hydrogen peroxide have more energy therefore they collide with the potato more frequently and so increasing the rate at which the product is formed. However at a certain temperature this is no longer the case. This is because enzymes are proteins and proteins can be denatured at high temperatures. This is because proteins have a D shape. In our case the catalase has a certain shape that the substrate fits into. At high temperatures the active site on the enzyme is altered, see diagram below.(Diagram showing active site on the enzyme is altered therefore stopping products being formed)This stops the substrate from 'fittingand so no product is formed. My results do not totally support or undermine my original prediction. The reason being that on graph 1, my results suit my prediction. It shows that the temperature, 7ºC was the fastest and 100ºC is when the enzyme denatures. But in graph , my results undermine my original prediction as at 55ºC the reaction still takes place where as in my prediction I stated that enzymes would denature at 40ºC approximately, I didn t expect this is happen. EvaluationIn my investigation I was pleased with my achievements. In my method, keeping the temperature constant throughout the investigation was hard to maintain, as the temperature of the contents of the tube would change quite quickly and therefore the hydrogen peroxide wouldn t be at the temperature required. To overcome this problem I could keep the test tubes in a hot water bath for all the temperatures making sure that the water bath was the suitable depth. This would ensure constant temperature throughout the whole 5 mins. Also another problem that I encountered was to keep the height of the froth fair. I measured the height of the froth with a 0cm ruler against the test tube rack, with the support of my hand. As I was measuring, my hand would move from time to time and therefore didn t know where I should place my ruler afterwards. To over come this I should attach the ruler onto the test tube rack with cello tape, as it is transparent or maybe use a pointer. With respect to I measured the height of froth in cm, but to be more precise I should have measured it in mm. To over come this I should use a ruler with mm readings. Also another problem that I observed on accuracy was that I didn t allow the temperature to equilibrate to the right temperature. In this case I wasn t using the correct temperature that I wanted, this could have led to some anomalous results. Ideally I should have brought the temperature of the hydrogen peroxide up to the needed temperature before adding to the potato. Looking back at my results I found some anomalous results in my findings. When averaging I used these results, which could of made the average either lower or higher than it should be. To improve this I should have missed these results. Not including some sets of results when making averages may have led to better values. My results are in line with those I predicted. Graphs indicate rise in temperature up a point leads to an increase in oxygen production. This is in line with kinetic theory. However it is very clear that after a certain temperature is reached the enzyme actually virtually stops. This supports my theory of lock and key fit. However optimum activity of enzyme is at about 7ºC this is as we expected. But at 55ºC the enzyme is still not denatured according to my results. This is a higher temperature than I would expect. Possible not allowing solutions to reach temperatures selected has led to an inaccuracy. It may be that in fact that many temperatures of solutions were lower than we stated. Overall, due to reliable repeats and in general predictions being confirmed I feel my results are reliable enough to make a conclusion. The obvious thing I would improve about the measurements I made would be to increase the range of temperatures used. Especially between 55ºC-100ºC. In this way it may be clearer at the temperature which denaturing took place, and would possibly give a graph that resembled the graph in background knowledge. Another way of improving this investigation is to change the method. I measure the volume of oxygen that was produced. In order to get pure oxygen without any other gases that are in the air I would use the same equipment but make sure that the gap between the rubber bung and solution was free from any other gases.AimTo investigate the effect of temperature on the enzyme catalase.PredictionUsing my existing scientific knowledge, I predict that as I raise the temperature to 0, 5, and 40, this is where we will see the greatest reaction. I predict this because enzymes are designed to react best at the body temperatures of the animals to which they belong. For a mammal, this is around 5-6. Catalysts are used to speed up biochemical reactions in the body.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 (microbody 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)Hydrogen Peroxide = HO Hydrogen Peroxide + Catalase = Oxygen + WaterApparatusMeasuring cylinder To hold 100ml of water. This was thoroughly cleaned with tap water beforehand to ensure that the water was not contaminated with anything. This may have led to anomalous results in the long-term.Clay Beehive To provide somewhere to connect the pipe from the mixture to the water. It also acted as a ledge to hold the measuring cylinder as it stood upside-down.Tub To hold water and everything in place.Stopclock To time experiment. I ensured that the experiment was timed as soon as it began because if it hadnt, then the results may have been inaccurate.Water bath To heat chemicals.Conical Flask To hold the yeast and hydrogen peroxide solution together once the experiment had begun. This needed to be cleaned using water to ensure that nothing would contaminate the solution.DiagramMethodI set up the experiment as shown above. I filled the tub full of water. I then placed the beehive in the water. Then I filled a measuring cylinder with exactly 100.0ml of water and placed it on the beehive in the water without letting any of the water in the measuring cylinder escape. I needed it to be exactly 100.0ml so that I could measure it exactly, from a starting point which is relatively easy to remember. After all, 100.0ml is a lot easier to remember than 87.ml! The measuring cylinder was thoroughly cleaned to ensure as little impurities in the water as I could possibly control. Whilst this was being set up, I had already prepared 40.00ml of yeast and 0.00ml of hydrogen peroxide in separate boiling tubes. At this point, it was very important that I kept the two substances apart because if they had been mixed, they would have begun to react The boiling tubes were both cleaned to ensure the chemicals didnt react with anything, and were as pure as possible before the experiment began. When I was certain that it was all prepared, I poured both liquids into a conical flask and fixed on a bung with an attached tube. This operation needed to be practised before the experiment was done for real to ensure it was done as quickly as possible. After all, the hydrogen peroxide and yeast solution will have started reacting as soon as they came into contact. I connected the tube to the clay bee from essaybank.co.uk hive and measuring cylinder, which were both already prepared in the water. From the very beginning, I started the stopclock timing and noted down how much oxygen had been reacted and had travelled down the pipe into the 100ml of water in the measuring cylinder. I noted down the volume of water that was left in the measuring cylinder after five minutes, taking a result every minute. I chose to take down the result for five minutes because any longer than that and there was none, or barely any water remaining in the measuring cylinder. This is because the yeast and the hydrogen peroxide would have finished reacting completely.VariablesVariable The temperatures of the hydrogen peroxide and the yeast.Controlled VariablesVolume of water in the measuring cylinder 100.0mlTimes 0, 1, , , 4, 5 minutesTypes of liquid Water, Hydrogen Peroxide, and Yeast solutionVolumes of substances 40.00ml yeast, 0.00ml hydrogen peroxideRoom temperature 5ºC approximately Temperatures of mixture 0.0?C, 0.0?C, 40.0?C, 50.0?C, and 60.0?C. These must be kept as exactly as possible as yeast is very receptive to changes in temperature.If these variables were altered, it would not be fair test.ResultsGraphs on following pagesConclusionI conclude that my prediction was partly correct. I was correct by saying that the reaction would be quicker as the temperature was 0?, but it was slower at 40? and again quicker at 50?. This may be because the catalyst, in this case catalase worked best at that temperature, allowing for more successful collisions between the yeast and hydrogen peroxide molecules.. I cannot explain these results because I can guarantee that I made sure that it remained a fair test throughout the experiment. I didn t alter any of the other variables. All the other results came out to be what I expected, with the reactions slower at 0? and 60?.EvaluationI was a little surprised at some of the results, and if I were to do this experiment again, I would try to discover what it was that gave these findings. I would also do the experiments for every 5? instead of every 10?. I would also measure other variables, to ensure that there cannot be any more fluke results. I would conduct the experiment more times to get a more accurate average. Although I conducted the experiment as accurately as I could there were many sources of error in the method that I used. Firstly, some help from friends was required to begin the experiment and this lead to a small delay in starting the stopclock. I would have to find a way to be a little more accurate. This would ensure that my results were as accurate and as precise as I could possibly get them.essaybank.coessaybankAim My aim is to find out how temperature affects the breakdown of hydrogen peroxide by the enzyme catalyse. Apparatus & Diagram Delivery tubeMeasuring cylinderBoss & ClampBoiling tube Stand Water basin StopwatchBunsen burnerThermometer Heatproof matWeighing scales TripodBeakerGauzeMeasuring cylinder Safety gogglesScalpel 6 x Boiling tubesPlastic tile PotatoRuler Hydrogen Peroxide Variable to Alter The variable I will alter in my experiment, is the temperature of the catalyse within the potato. This is because; by doing this I will answer my aim and also supply myself with the evidence needed to find out if the temperature of the enzyme (catalyse) does affect the breakdown of hydrogen peroxide. The temperature I measure will be in a regular scale, which starts at 10°c and goes up in tens to 50°c. Variable to keep the same The variables I will keep the same are mainly the amounts of hydrogen peroxide in each of the five boiling tubes, the mass of the potato in each test and that the potato pieces had the same surface area. I have kept these the same, because by doing this I have instantly blocked out any faults in the experiment, which may be due to those variables changing.Safety Used Within the ExperimentThe safety I will have to use is firstly safety goggles through out the experiment and also to have my tie tucked into my shirt, to avoid contact with a naked flame. I will also need a safety tile whilst cutting the potato to ensure that the potato or bench wasn't in any danger of germs. I will also have to consider the fact that other members of the group were to work along side myself, so I will have to be aware of other people's movements. The main term of safety is to be sensible and act quickly if any spillage of Hydrogen Peroxide was to occur. Method To begin with, acquire five boiling tubes and deposit in them ml of Hydrogen Peroxide. Once you placed the liquid in all five boiling tubes, label each one of the boiling tubes starting with 10°c and then ending up with the fifth tube saying 50°c on it. There should still be one empty tube. This boiling tube is used to place the cube of potato in so that it can be heated or cooled to the required temperature, in the water bath. To set up the water bath, you will need a Bunsen burner and the general equipment that is required with it, and a beaker ½ full of water. The water bath for 10°c and 0°c is not filled with water and doesn't require the Bunsen burner; instead, it needs to be ½ full of ice. After you have done this, cut up fifteen pieces of potato 1cm³ and making sure that they have the same properties as each other, for instance the weight, surface area and source of potato must all be the same. Fifteen potato cubes are needed, because the experiment is to be from essaybank.co.uk repeated three times for each temperature, to ensure that I get accurate results and able to draw a average of each temperature. Place one potato piece into the spare boiling tube and according to the label on the tube in which you are going to place the potato in after heating/cooling, put the cube of catalyse (potato) into the water bath for exactly 4 minutes. After the potato has been in the water bath for 4 minutes, carefully take it out of the boiling tube and into the boiling tube in which there is ml of hydrogen peroxide, making sure that the label is correct to the temperature you set in the water bath. The instant you place the potato into the liquid, quickly put the delivery tube that should be placed at one end in a water basin, which leads to a 100 ml measuring cylinder full of water. The water basin should be ½ full of water. The measuring cylinder would collect all the hydrogen being produced. Let the measuring cylinder collect the hydrogen for 4 minutes and the record the amount of water displaced. Repeat this procedure for each temperature three times to insure we have an average amount of hydrogen produced. Prediction By looking at my experiment, I predict that the rate of break down of hydrogen peroxide by the enzyme catalyse will increase as the temperature increases, but I also know that this will only increase to a certain point. This point is the denature stage, which usually occurs around 50°c 60°c. So, I will be expecting to see results that increase and the suddenly decrease. ExplanationI have predicted the above because from my knowledge, I have studied that in general terms enzyme activity increases as the temperature increases. This is because the molecules have more energy and therefore collide more often. This happens to a point where the enzyme becomes denatured and no longer works as usual. This is all because of how enzymes work, which is shown in the diagram below. EnzymeSubstrate This is how the enzyme looks before heating. Notice how the substrate fits exactly in the active site. Enzyme Substrate This is how the enzyme looks after heating of 50°c-60°c. The substrate no longer fits the active site.This sketch graph below shows how the activity of an enzyme increases as the temperature goes up, and it also shows how it suddenly drops as the enzyme is denatured. Preliminary Work I did a brief experiment in order to receive a more knowledgeable view over the final task. The changes that had to be enforced were that the hydrogen peroxide, which I had as ml in the method, had to be increased to 4 ml per boiling tube. This is because the 1cm³ potato didn't fully get coated with the hydrogen peroxide. The usefulness of the preliminary work was that I got the idea of using instruments like a measuring cylinder for example, with more accuracy by measuring below the meniscus. The main advantage of the preliminary experiment, was that in my method I stated that one should use a 100 ml measuring cylinder for the retrieval of oxygen, but this test proved to me that the results were between 0-10 ml. 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