Reaction against hydrogen peroxide Essay Example for Free

Reaction against hydrogen peroxide Essay The action of catalase on hydrogen peroxide Aim The aim of this experiment is to discover the relationship between the concentration of the substrate and the rate of the reaction catalysed by an enzyme, by looking at the decomposition of hydrogen peroxide under the action of catalase, and to determine a value for Vmax and the Michaelis constant for catalase. Background Theory An enzyme is a protein biological catalyst. Catalysts speed up or slow down the rate at which chemical reactions occur. They are not used up in the reactions and can be retrieved unchanged afterwards. Biological catalysts control the rate of reactions in living things. Each enzyme is substrate specific it can control only one reaction. For example, the digestion of starch is begun in the mouth by the enzyme amylase. An equation for this reaction can be shown like this: Amylase Starch Simple sugars The enzyme only facilitates the reaction, it is not used up. Each molecule of enzyme can be reused indefinitely, unless it is damaged, or denatured. Enzymes are proteins, so they are denatured if the polypeptide chains, which are precisely coiled and folded to form the active site, become unfolded by the kinetic energy from heat, or the covalent bonds are disrupted. Whilst some heat will increase the rate of reaction because of the increased number of collisions between enzyme and substrate, too much heat will denature the enzyme and render it completely ineffective. Enzymes are also affected by the pH at which they have to work. Charged hydrogen or hydroxide ions in acids or alkalis can cancel out the charges on the active sites of the enzymes, and render them ineffective. Hydrogen peroxide is a toxin produced in every cell of living organisms as a by-product of respiration. It is the same chemical that is used to bleach hair, and so must be broken down before it can damage the cells. It decomposes to give water and oxygen. This reaction will occur naturally, but at a very slow rate. To speed it up an enzyme is used. The enzyme which catalyses the decomposition of hydrogen peroxide is called catalase. Catalase is found in all living cells to decompose the hydrogen peroxide. In this experiment it is being obtained from live yeast in a suspension. The method by which catalase works is called the lock and key method. Catalase works because it has an active site. At this point the enzyme attaches to the hydrogen peroxide molecule, because the opposite charges of enzyme and substrate attract each other, forming an enzyme substrate complex. The enzyme catalyses the reaction, and then the new charges on the product repel the enzyme away to act on a new substrate molecule. (see fig 1) The decomposition of hydrogen peroxide has the following formula: catalase 2H2O2 2H2O + O2 The rate of a reaction is a measure of the change in the amount of reactant or product with time. The rate of decomposition H2O2 can be measured using the volume of oxygen produced, from the formula: Rate of reaction = change in amount of product time The rate of reaction is determined by collision theory:- For a reaction between two substances to occur, the enzyme and substrate particles must collide with each other. If more collisions occur in a reaction, rate will increase. If the reactant particles gain energy and collide faster, then each collision will have more energy, there will be more successful collisions, and rate will also increase. In this case, the more collisions between hydrogen peroxide and catalase molecules, the more hydrogen peroxide will decompose. Anything which increases collisions will increase rate. Increasing the concentration of the substrate (hydrogen peroxide) solution means that there are more substrate molecules in the same volume, causing more collisions, and thus increasing rate. The rate of reaction changes with concentration, but the overall yield of oxygen is independent of factors affecting the rate, so measuring the amount of oxygen produced over the whole reaction is meaningless. Instead, the initial rate of reaction can be estimated by measuring the volume of oxygen produced in the early stages of the reaction. This value can then be compared between the different concentrations of hydrogen peroxide, and used to plot a graph of substrate concentration against rate, from which values for Vmax and the Michaelis constant (Km) can be obtained. The relationship between substrate concentration and rate of reaction is described by the Michaelis-Menton equation: v = Vmax [S] Km + [S]. Vmax is a measure of the maximum rate at which an enzyme can act, and it is the horizontal asymptote of the graph of substrate against time that is when the amount of enzyme is the limiting factor. The Km is defined as the substrate concentration at which the rate of enzyme action is half Vmax. It is measure of the affinity of an enzyme for its substrate molecule the higher Km, the weaker the binding force between the enzyme and substrate. Both Km and Vmax are constants at a specific enzyme concentration and temperature. Pilot Experiment A pilot experiment was carried out in order to see whether the method was practical and could produce good, reliable results, and to choose the concentrations of hydrogen peroxide and the length of time over which the oxygen would be collected so that no more than 50cm3 of gas was given off. Pilot Method 1. 10cm3 of hydrogen peroxide solution was measured into boiling tubes using a syringe, and the apparatus set up as shown below. 2. Using a 1cm3 syringe the yeast suspension was added to the boiling tube and the stopwatch started. 3. Thirty seconds was timed, and then the burette was taken off the end of the delivery tube, but not out of the water, and the volume of gas collected was measured. 4. This was recorded and repeated for each concentration of hydrogen peroxide, made up as shown in the dilution tables below. Pilot Results Concentration of hydrogen peroxide Initial reading on burette (cm3) Final reading on burette (cm3) Volume of gas collected (cm3) Rate of reaction (cm3/s) to 2dOff scale. Analysis of and Modifications to the Pilot These results show a clear increase in the volume of gas collected as the concentration increases, suggesting that with modifications this method will enable clear conclusions to be drawn. Carrying the experiment out over 30s resulted in too much gas being produced at the highest concentration to be recorded with the apparatus available, so for the main experiment the oxygen will be collected over 15s. More readings will be taken to enable a more reliable graph to be drawn at9 and 20%. The experiment will be repeated three times and any anomalous results will be identified and excluded from the average in order to enable more reliable results. Prediction I predict that initially the rate of reaction will increase with the concentration. As the concentration of hydrogen peroxide increases so will the number of collisions between enzyme and substrate molecules, so the hydrogen peroxide will decompose faster into water and oxygen. I predict that this reaction will obey Michaelis-Menton kinetics, and that the graph of rate of reaction against hydrogen peroxide concentration will give a rectangular hyperbola as shown below: The increase in rate of reaction will not continue indefinitely there will be an asymptote when Rate = Vmax, when all the catalase molecules are catalysing the reaction as fast as possible, and so the rate cannot increase without supplying more enzyme. Main Experiment Plan Fair Test A fair test is one from which a reliable conclusion can be drawn. For a fair test only one variable must be changed at a time. In this experiment the concentration of hydrogen peroxide is being changed, and so all others must be controlled. Variables. Independent Variable:- concentration of hydrogen peroxide Dependent Variable:- volume of oxygen gas collected in 15s Controlled Variables:- temperature, volume of hydrogen peroxide, amount of yeast, apparatus, time.   The reaction will be carried out in a water bath at 20? C. Since water is a good thermal buffer it should be fairly easy to keep the temperature constant.   Volume of hydrogen peroxide solution will be controlled quite easily by using two syringes to measure the water and hydrogen peroxide volumes as dictated by the dilution table below.