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Investigation into the effect of substrate concentration on the rate of hydrogen peroxide decomposition by the enzyme catalase.

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Introduction

Investigation into the effect of substrate concentration on the rate of hydrogen peroxide decomposition by the enzyme catalase. Aim An experiment is to be conducted to determine how varying the concentration of the substrate H2O2 used will affect the rate of its catabolic decomposition into oxygen and water due to a fixed amount of the enzyme catalase. The enzyme catalase used is in the form of potato cylinders (since catalase is found in potatoes and that potatoes can be easily cut into cylindrical shapes to give maximum surface area). A chemical equation of the reaction is shown: 2H2O2 (aq) --> 2H2O (l) + O2 (g) It can be seen that the number of oxygen molecules produced will be proportional to the number of H2O2 molecules decomposed. Therefore the amount of O2 produced in a certain time as the product of the reaction can be used to determine the rate of this catabolic reaction. Introduction The above reaction only occurs at a noticeable rate when there is catalase present. Catalase decomposition of hydrogen peroxide has a turn over rate of 5,000,000 molecules (5,000,000 H2O2 molecules are decomposed by one catalase enzyme in one minute). It would require 300 years for the same number of H2O2 molecules to be decomposed without the presence of catalase. (Villee C., Dethier V., 1970, Biological Principles and Processes, W.B. Saunders Co) The enzyme catalase is seen to dramatically increase the decomposition rate of H2O2. This is due to the fact that enzymes (a globular protein) reduce the activation energy of the reaction. This is to say, less energy is required to start the reaction so that more reactions can occur. The energy transfer diagram below illustrates this point: Hypothesis Presently, there are two theories as to how enzyme molecules interact with their substrate molecules to reduce the activation energy: Lock & Key hypothesis: This is the theory that every enzyme has an enzyme active sites of a specific shape corresponding to the substrate that it will attach to, as a key fits a lock. ...read more.

Middle

As oxygen is collected in it, the water is displaced and the receding water level will tell us the volume of oxygen produced. Up turned burette: This will be used in the exact same way as an up turned measuring cylinder. This advantage this will have over the measuring cylinder is that it will measure to �0.1cm3, giving an accuracy ten times that of the measuring cylinder. Bubble counting with up turned boiling tube: An up turned water filled boiling tube could be used for oxygen to be collected in it. Since oxygen is given off in the form of bubbles, the number of bubbles given off could be used to calculate the reaction rate. A boiling tube is preferred to a normal test tube due to its larger volume. Gas syringe: This is a syringe especially adapted so that it self fills as gases (often under low pressures) enters it. It will be able to record the amount of oxygen given off by our reaction to a suitable degree of accuracy. I have decided to use the gas syringe to determine the amount of oxygen produced. This decision was made because: 1. The test tube method will not give us definite quantitative results. Unlike the other methods, the volume of the oxygen produced is unknown. It will also be hard to count the number of bubbles given off if the reaction rate is particularly high. 2. All of the methods of using the displacing of water to measure the amount oxygen produced are sound in theory. In practical, however, they will be far more time consuming to be set up. For example, to reset the syringe to a reading of 0 all that has to be done is for me to push back its plunger. On the other hand, the burette/measuring cylinder will have to be refilled with water and carefully turned upside down under water, this is a far more time consuming technique. ...read more.

Conclusion

This makes sure as little oxygen escape as possible. The stop watch is only started when the rubber bun is placed so that the volume of O2 recorded is still oxygen produced in 60sec. In addition to the above, each test for each concentration of H2O2 will be repeated 3 times so an average will be able to be obtained to further enhance the reliability of the results. Use of results The results for each test will be collected in the following table` Conc. Of H2O2 (%) O2 Produced (cm3) Average O2 produced (cm3)* Reaction rate (cm3 min-1) 1 2 3 20 40 60 80 100 *Any clearly anomalous results will be ignored in the taking of an average. The rate of reaction is measured quantitatively in the form of cm3 of O2 produced per minute. This value can be plotted against the concentration of H2O2 as the variable on the x axis on a graph. The pattern shown by the graph can be characterized with a trend line. From the graph, a relationship between the concentration of the substrate and its reaction rate can be established as well as quantitative analysis for the Michaelis constant (the substrate concentration where the reaction rate is half of Vmax). (M B V Roberts, 1971, Biology a functional approach, Thomas Nelson and Sons Ltd) Risk Assessment Care will have to be taken with laboratory glassware. If any glassware is broken, quickly sweep up and put into broken glass bin. Care should be taken with sharp objects such as the scalpel. Chemical: Chemical Hazards Risks Likelihood Control Measures Hydrogen peroxide solution 20vol. Irritant Irritation to the skin and respiratory system Medium/low Wear Protective clothing and gloves and avoid inhaling. Irritation to the eyes. Low Wear protective goggles May cause nausea, vomiting and internal bleeding if ingested Very low If ingested, wash out mouth thoroughly with water and give plenty of water to drink. OBTAIN MEDICAL ATTENTION. Hazards data obtained from Health and Safety information, BDH. ...read more.

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