The Effect of Varying CuSO4 Concentration on the Breakdown of Hydrogen Peroxide in the Presence of Catalase

The Effect of Varying CuSO4 Concentration on the Breakdown of Hydrogen Peroxide in the Presence of Catalase
Theoretical Background======An enzyme is a biological catalyst. A catalyst is a molecule whichspeeds up a chemical reaction, but remains unchanged itself.An enzyme is a globular protein and as such has a precise 3D shapewhich incorporates an active site. This is where a specific substratemolecule will bind temporarily by means of Van der Waals forcesbetween the enzymes R-groups and the substrate. A substrate will bindto the active site for a split second; this is the enzyme-substratecomplex. This is when the substrate undergoes a chemical reaction andbecomes the product. The enzyme is then ready to take in anothersubstrate molecule and the process can repeat itself.
Each type of enzyme will usually only act upon one type of substrate molecule. In this case the enzyme catalase works only on hydrogen peroxide (H2O2). An enzyme may catalyse a reaction, which causes the substrate to split, or may join two molecules together. Catalase will split the hydrogen peroxide molecule into water and oxygen and this occurs at a turnover rate of 10^7 molecules per second. Enzymes speed up chemical reactions by lowering the activation energy required for a reaction to take place. This happens because when a substrate binds to the active site of an enzyme, the substrate?s shape is altered slightly. As a result it is easier for the substrate to be broken down into the product. When a volume of substrate is added to an enzyme, the reaction, which turns the substrate into the product, will begin quickly as this is when there are the highest numbers of substrate and enzyme molecules available for collisions to occur. This number of collisions determines how often a substrate will find an enzyme and therefore cause a reaction. As the number of substrate molecules decrease (due to them reacting with the enzymes) there will be less collisions and therefore the rate of reaction will decrease until it stops completely when all of the substrate molecules have become products. There are many factors that affect the reaction rate of an enzyme: 1) Concentration of enzyme: If there is a higher concentration of enzyme than substrate, there will be a large turnover rate at first, then as the substrate is turned into the product by the enzyme the rate will slow down and eventually stop when there is no more substrate left. So the higher the concentration of enzyme, the faster the initial rate of reaction will be. 2) Concentration of substrate: If there is a high concentration of substrate compared to enzyme, the reaction rate will be slower than above but will continue at a steady rate for longer, as there will only be a specific number of enzyme molecules for the substrate to collide with at any one time. 3) Temperature: When a solution of enzyme and substrate is heated up, the reaction rate will increase due to the increased energy provided which causes the molecules to move faster and therefore collide at a higher rate. However when the temperature rises above 40?C the enzyme starts to denature and is no longer able to catalyse a reaction. 4) pH levels: Similarly to temperature pH changes can denature an enzyme, most enzymes work best at a pH of around 7. Some enzymes however work in more acidic or alkaline conditions and any great changes above or below their optimum pH will cause the enzyme to denature. 5) The presence of inhibitors: There are different types of inhibitors which cause varying effects on the rates of reactions: competitive inhibitors bind to the active site of an enzyme temporarily, to briefly stop the substrate being able to bind to the enzyme. This slows the rate of reaction, but does not stop the reaction taking place. non competetive reversible inhibitors bind to a different part of the enzyme, again only temporarily. They cause the active site to change shape, so the substrate will not fit. The active site regains its original shape when the inhibitor leaves the enzyme, this means that the reaction rate slows but does not stop. non competetive irreversible inhibitors bind permanently to the outside of an enzyme causing the enzyme to change shape. The enzyme remains this way so it causes the reaction rate to slow at first, then stop altogether. Hypothesis. In the experiment, I would expect to see a variation in the amount of gas collected, dependant on the molar strength of the inhibitor CuSO4. I would expect to see that the stronger the solution of inhibitor, the lower the volume of gas produced. Prediction. I believe that if I halve the molar strength of inhibitor i.e. from 1m/dm to 0.5m/dm , I would expect to see the amount of gas produced to increase by one quarter. I would expect to see this because if I halve the amount of inhibitor, there will be twice as many enzymes available for the substrate to collide with and as such the reaction rate should increase proportionally and twice as much product produced. The products for this reaction are water and oxygen and therefore if the overall product is twice as much, then each component of the product will show an increase of one quarter compared to the result with twice as much inhibitor present. Variables. There are certain variables that will have an effect on my experiment unless they are made constants. I have listed these variables below and shown how I can keep them constant to allow me to prove which factor is causing the change in results. The mass of liver used: I will use 1.5g of liver in one piece, however I can not measure the amount of enzyme in each piece of liver. The volume and concentration of the substrate hydrogen peroxide: I will use 1cm and 20% concentration. The pH level: I will use 1cm of buffer to keep the pH level constant. The temperature: I will do this experiment at room temperature. The equipment: I will use the same pieces of equipment to measure and record results from. The volume and concentration of inhibitor: I will use 1cm of the inhibitor but I have chosen to vary the concentration. I have chosen to vary the concentration of the inhibitor so that I can show whether it has an effect on the reaction rate of the enzyme catalase. To do this I will dilute the CuSO4 with water in the following quantities. molar value Volume of inhibitor Volume of water (Mol/dm ) (ratio to water) (ratio to inhibitor) 1 1 0 0.75 0.75 0.25 0.5 0.5 0.5 0.25 0.25 0.75 0.1 0.1 0.9 0 0 1 Apparatus. 3, 1cm syringes 3, beakers Side arm test tube 25cm Copper sulphate Gas syringe Test tube rack Bung Delivery tube 25cm Buffer Stopwatch Balance (accuracy of 0.1g) 25cm Hydrogen peroxide Metal clamp 30g liver Method. 1) Set up test tube and gas syringe as shown above making sure that the delivery tube is attached securely and the gas syringe is moving freely. 2) Weigh out 1.5g of liver in one piece, using the balance. 3) Make up 5cm of each of the molar quantities of inhibitor, use the table in variables for the values needed. 4) Place the 1.5g of liver into the bottom of the side arm test tube. 5) Measure out into a beaker 1cm of H2O2, 1cm of inhibitor (from the 5cm made up before) and 1cm of buffer. 6) Add the 3cm of the above solution into the test tube with the liver. 7) Place the bung in the side arm test tube immediately and start the stopwatch. 8) Record the amount of gas produced every 60 seconds. 9) Repeat this experiment at least 3 times per concentration of inhibitor to ensure accurate results. Safety. When handling the raw liver, wear gloves or wash hands thoroughly after contact. The hydrogen peroxide is an irritant to the skin. The inhibitor is a poison and may stain clothing. The reason that my prediction is not accurate is that there are two anomalous results. 1) Looking at the table of results, I notice that for the 1 molar quantity experiment, the first set of results is much higher than the rest. During this test I used 2 small pieces of liver equal to 1.5g, this would have increased the surface area and therefore the amount of enzyme the substrate would have come into contact with. I know that this would cause a higher reaction rate. This would cause the average result to be significantly higher and as such, made it higher than that of the 0.75 molar quantity experiments. 2) During my first experiment with 0.5 molar copper sulphate, I used a piece of liver that was slightly smaller in mass than the rest. When measuring the liver, the balance was flashing between 1.4 and 1.5g. This difference that was due to human error, would have caused the significant difference in the result produced. Evaluation of reliability of results Looking back at my entire experiment I can say that I was reasonably accurate in the way I set up, worked through and retrieved results from my experiment. However there can always be improvements made. When I planned this experiment, I took into account that there would be many variables. I looked at ways in which I could keep everything bat factor constant. I did make two noticeable mistakes: 1) I used two pieces of liver during on of the experiments instead of one. This increased surface area of the liver showed an untrue picture of the rate of reaction with 1m concentration of copper sulphate. 2) In a later test I used a piece of liver that was approximately 0.5g less in mass than all the other pieces used. This inaccuracy was down to my error in reading the balance and also showed an untrue picture of the rate of reaction, this time for copper sulphate of 0.5mol/dm . These two mistakes that I caused, have led to my results not being entirely accurate. With the apparatus I chose there was some degree of inaccuracy: 1) There were limitations using a gas syringe with the accuracy of the measurements that can be taken. The gas syringe only measures to 1cm , eg the measurement taken may have been 13cm but the actual volume of gas could have been between 12 and 13 or 13 and 14 depending on how it was rounded up. When transferring these results to a graph, the inaccuracy of the measurement could show either a higher or lower rate of reaction depending on whether the measurement was rounded up or down and would show an untrue picture of what was happening. 2) Due to the experiment taking place over two days, it was impossible to use the same equipment. This meant that the delivery tube from the side arm test tube may have been a different length, the room temperature may have varied by 1 or 2?C or the gas syringe could have been more or less sticky than the one previously used. Any of these factors could cause a change in the results gained and would also show an untrue picture of reaction rates when they are transferred to a graph. Evaluating the experiment I have completed, I can say there are improvements that can be made to increase the accuracy of the results, were I to repeat this experiment. I would start off by ensuring that all variables were kept constant and I would repeat an experiment if my results were noticeably different from what I had expected to. A better overall result would be obtained by repeating the experiment more times because any errors in one experiment should be compensated for by the other experiments. This would show a truer picture of reaction rates. I would use a wider range of concentrations of the copper sulphate inhibitor to see if I could predict the reaction rates. I would measure the gas produced over a shorter time intervals, possibly every 20 seconds, so that I could get a clearer picture of how the inhibitor was effecting the results over time. The problem of the delay between pouring in the Hydrogen Peroxide, bunging the test tube and starting the stopwatch could have been limited by getting another person to start the stopwatch when the hydrogen peroxide was poured into the tube. I would use a larger volume of substrate so that it does not limit the results. This would enable me to tell if the copper sulphate was a reversible or non-reversible inhibitor. If the inhibitor was reversible then the reaction rate would be slow, but not stop, whereas if it was non-reversible the reaction rate would slow very quickly and stop altogether. I would collect the gas in a measuring cylinder over a beehive as well as in a gas syringe so that I can compare the results of the two separate experiments and see if this caused a change in the result due to the accuracy of the equipment being used and the measurements able to be read. Looking at my table of results I can say that when I halved the molar quantity of the inhibitor, the oxygen produced did in fact increase by one quarter for most of the results that I obtained. The results that did not match up with my prediction were the results where mistakes had been made. This proves that my prediction was correct. Conclusion The experiment detailed above produced results that follow the same general trends as the known facts about the effect of inhibitors on enzyme activity. It is important to note that as a result of completing this experiment I am certain that if someone else were to do the same experiment that they would collate very similar results to my own. This is due to the fact that my results support known facts and as such can be used to prove that inhibitors such as copper sulphate do have an effect the reaction rates of enzyme activity.

The Effect of Varying CuSO4 Concentration on the Breakdown of Hydrogen Peroxide in the Presence of Catalase 8.1 of 10 on the basis of 2238 Review.