Catalase on Hydrogen Peroxide

The aim of this study was to test the rate of reactivity of the enzyme catalase on hydrogen peroxide while subject to different concentrations of an inhibitor. The hypothesis was that hydrogen peroxide will be broken down by catalase into hydrogen and oxygen, where a higher concentration of inhibitor will yield less oxygen, resultant of a lower rate of reaction. Crushed potato samples of equal weight were placed in hydrogen peroxide solutions of various temperatures. The results showed that less gas was produced as the concentration of the inhibitor rose. This Is because more enzymes were inhibited, and so less active sites were available for reaction. Reasearch and rationale Hypothesis: Catalase will break down hydrogen peroxide in water and hydrogen. A higher concentration of inhibitor will yield a slower rate of reaction, thus yielding less oxygen in a given time. Null hypothesis: Catalase will break down hydrogen peroxide in water and hydrogen. The concentration of the inhibitor will have no effect on the rate of this reaction. Supporting knowledge: Enzymes function as biological catalysts, increasing the rate of reactions that take place within organisms. Catalase is an enzyme found in the vast majority of organisms that are exposed to oxygen, making it very common. It catalyses the decomposition of hydrogen peroxide into water and oxygen. Hydrogen peroxide is a molecule that can attack sulphur atoms and metal ions in protein molecules, and with the aid of iron ions is potentionally mutagenic. Catalase is an extremely effective enzyme, and can break down millions of hydrogen peroxide molecules in a single second. As such, it can be seen how important and effective catalase is to oxygen-exposed organisms. As with the majority of enzymes, it has an optimum pH of approximately 7, though the optimum can vary by species. Haliotis discus discus, a type of shellfish, for example, has catalase which works at an optimum pH of 10. 5. There are a number of factors which affect the rate of reaction of catalase. As stated, pH is one. Concentration of both catalase and hydrogen peroxide, concentration of co-enzymes (Fe3+ in human catalase) and temperature are all variables that affect the rate of reaction. These can all be regarded as limiting factors, as they can only affect the rate of reaction to a certain point when alone. That is to say, the reaction will not work as best it can if all factors are not at an optimum standard. The poison cyanide acts as a competitive inhibitor, it strongly binds to the haem of catalase, stopping an enzyme-substrate complex from forming. Heavy metal ions, such as copper ions in copper (II) sulphate, act as non-competitive inhibitors on catalase. Competitive inhibitors act by binding to the actual active site of the enzyme, thus stopping an enzyme substrate complex from forming. Competitive inhibition, this can be reversed by increasing the concentration of the substrate. Non-competitive inhibitors bind to a site other than the active site, known as the allosteric site. As such, it is able to bind even to an enzyme-substrate complex. These inhibitors are usually reversible, but are not affected by concentration of the substrate in the way that competitive inhibitors are. Copper acts as a non-competitive inhibitor of catalase. Copper, as a heavy metal, reacts strongly with sulphydryl groups in proteins. This can cause the protein to precipitate, and thus change the shape of the active site. This type of inhibition is irreversible, as the protein becomes covalently modified. Inhibition affects the rate of enzymatic reactions differently than extremes of pH and temperature, which both affect the protein in a non-specific way. These factors reduce the rate of reaction by a process called irreversible enzyme inactivation. This is where the whole protein structure is destroyed, or denatured. Non-competitive inhibitors work by specifically altering the struc ture of the enzymes active site. Due to the decrease in available active sites, the rate of reaction will decrease. In this sense, it is similar to reducing the concentration of enzymes, as there are less active sites available. Planning I will be studying the rate of catalase activity on hydrogen peroxide while varying the amount of inhibition, which should influence the rate of the reaction, and thus the amount of oxygen, observed in a given time. The concentration of the inhibitor will therefore be the independent variable, while the amount of oxygen will be the dependent variable. There should a smaller volume of oxygen observed as the concentration of the enzyme increases. As I am comparing two variables to each other, it would be wise to calculate the correlation of the two variables. To calculate the correlation, I should use Spearman’s rank correlation coefficient. To find a suitable correlation, I should use at least 8 samples. After plotting a scattergraph, I will proceed to find out the correlation if the correlation looks reasonable enough. Variables Temperature: Temperature is an extremely important variable to consider, as it can affect the rate of reaction in a number of ways. If the temperature of a system is increased, more molecules will reach the activation energy and the rate of reaction will increase. The number of collisions will also increase. As enzymes must collide with substrates, an increase in temperature and thus kinetic energy will result in more collisions occurring in a given time. Increasing the temperature will also increase the heat of the molecules. This will in turn increase the molecules’ internal energy. If the energy increase is too great, the weaker hydrogen bonds that determine the tertiary structure of the protein may break. This may change the structure of the active site, stopping any reaction between enzyme and substrate. Therefore beyond a certain temperature, the rate of reaction will decrease. To control the temperature, each system must be heated to the same temperature. For this experiment, I have chosen to keep each system at 30oC. This is because room temperature can vary in a lab, as some days can be warmer than others, and the room’s ventilation can also affect the temperature of the system. This is also to allow the reactions to happen at an efficient enough rates to collect results. 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 this pH results in denaturation of the enzyme and a slower rate of reaction. The optimum pH for catalase in potatoes is generally 7, but can differ depending on the acidity of the soil it was grown in. In this experiment a pH 7 buffer was used. This is because the optimum pH of most types of catalase is 7, and so that the systems can all be kept constant. Concentration of solutions: the concentration both the enzyme and substrate have to be taken into consideration. The concentration of substrate for example will affect the rate of reaction in a positive way until all the active sites are occupied. The same applies to the concentration of the enzyme. An excess of either will not negatively affect the rate of reaction. Both concentrations will be kept constant for each experiment. I will ensure this by using catalase and hydrogen peroxide from the same batch in each experiment. Potato: Obtaining catalase from potato involves crushing the substance. This can be standardised by first cutting the potato into small cubes, then crushing. This can ensure that there are as little lumps of potato left as possible. If multiple potatoes are used, there is a risk of using catalase of different concentrations. This can be remedied by using a large enough potato. As each sample of potato is 5g, and twelve samples are taken 3 times each, then 180g worth of crushed potato are required. A large potato can easily weigh around 500g, so the problem then comes with different concentrations of catalase distributed within the potato. This can be controlled by evenly mixing the crushed potato. It is then safe to assume the catalase is randomly distributed throughout the potato. Lab air exposure: As the crushed potato would be left exposed to the air during the duration of the experiment, this could affect the enzymes ability to catalyse the reaction, and would yield misleading results. As such, any potato that is not in use should be covered in cling film to prevent exposure as much as possible, although not entirely. Preliminary Method My preliminary experiment involved testing only 5 different concentrations of inhibitor, to see whether the results show any reasonable difference. By this, I mean that the results should show a correlation above the critical level where p = 0. 05, n = 6. By doing a trial experiment, I can make sure my experiment will run safely and successfully, as well as determine any factors of my experiment I should change. Apparatus: -Potato -6% Hydrogen Peroxide solution 5cm -Copper (II) Sulphate solution (2%, 1%, 0. 75%, 0. 25%, 0. 1%) 10cm -Deionised water -Measuring cylinders -Weighing boat -Mass balance -Peeler -Scalpel -Pestle and mortar -Cling film -Test tubes -Test tube rack -Water bath -Thermometer -Bunsen burner Tripod -Gauze -Metal tongs -Delivery tube with bung attached -Gas syringe -Clamp stands, boss and clamp -Stopclock Method Peel and dice the potato in roughly 3x3cm cubes and mash them. Cover the crushed potato when not being used. Set up a gas syringe horizontally supported with stands and clamps, high enough to reach test tubes in a water bath. Attach a delivery tube to the syringe. Prepare a water bath of 30oC. Measure out 5cm3 of Hydrogen Peroxide into 6 test tubes. Add 10cm3 of the 2% Copper (II) Sulphate solution. Heat the solution to 30oC using a water bath. While heating, prepare a weighing boat with 5g of potato. When 30oC, place the potato in the solution, quickly attaching it to the delivery tube and starting the stopclock (a helper is advised here). Allow the catalase to react for 3 minutes, and note the gas collected. Repeat the same process for each concentration. Add 10cm3 of deionised water to the final test tube as a control. Health and Safety Pureed potato may irritate some people’s skin. Wash any potato splashes off skin immediately. Hydrogen peroxide is an irritant, and a powerful oxidising agent and bleach.