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OBJECTIVE: The experiment was carried out to investigate the effects of the increase in the enzyme concentration on the rate of reaction. By using self investigative and experimental skills, the experiment was done in order to determine how the rate of reaction will be altered, whether it will increase, decrease or remain constant when the different concentration of enzymes added. INTRODUCTION: Enzymes are produced naturally in plant, animal, and microbial cell. There are thousands of different enzyme can be found in any cell.
Enzymes can be describe as molecules that are greatly increase the rate of chemical reaction without themselves being changed at the end of the reaction. A chemical reaction involves the conversion of a substrate into a product. This reaction is normally known as an enzyme-catalyzed reaction. In this reaction, the substrate will bind to the active site of the enzyme. Once it is bind, an enzyme-substrate complex is formed. At this form, the substrate is converted into product. Finally, the product will be released.
There are four factors that affect the enzyme activity that is temperature, pH, substrate concentration and enzyme concentration. As for temperature, it will affect the enzyme activity when there is a rise in temperature. When temperature increases, this will automatically increase the kinetic energy. This will then results in the increase of the rate of collision between the enzyme and substrate, but this effect is limited. The rate of reaction will increase up to a certain temperature. At this temperature, the enzyme activity is the greatest.
Changes in pH will results in disruption of hydrogen bond of the protein chain. Hence, this will results in changes in shape of the enzymes’ active site that then will reduce the effectiveness of the enzyme and the rate of reaction. Using different type of substrate concentration will also affect the rate of reaction. Increase in substrate concentration will increase the rate of reaction up to a point where all of the active site of the enzymes is occupied. The fourth factor is the enzyme concentration which is the factor that has been investigated in this experiment.
When using different concentration of enzyme, the rate of reaction will also change. Any increase in the concentration of enzyme will also increase the rate of reaction up to a point where all substrate are occupied. Substrate concentration will become the limiting factor of the experiment. In this experiment, the enzyme used is catalase. Catalase is an enzyme normally found in many plant and animal tissues. Generally, catalase break down toxic substances and specifically, it catalyze the decomposition of hydrogen peroxide to oxygen and water as stated clearer in the equation below: H2O2 ? 2 H2O + O2 Catalase is the fastest known enzyme. It has one of the highest turnover numbers of all enzymes. A molecule of enzyme can convert millions of molecules of hydrogen peroxide to water and oxygen per second where a normal enzyme would catalyze thousands of molecules per minutes rather than millions. Catalase consists of four polypeptide chains with each over 500 amino acids long. It also contains four porphyrin heme (iron) which act as the enzyme cofactor that will allow the enzyme to react with the hydrogen peroxide.
Approximately, the optimum pH value for enzyme catalase is 7, while its optimum temperature depends on the sources of the enzyme but it works at an optimum temperature of 37??C, which is approximately the temperature of human body. Besides, catalase also can break down other toxic substances such as alcohol, formic acid and phenols. Catalase has its inhibitor that is copper (II) sulphate, a heavy metal ion and cyanide poison. Therefore, such enzymes’ inhibitor is avoided while conducting the experiment. Hydrogen peroxide is the substrate in this experiment.
This substance is the one who will react with enzyme catalase to produce the product or enzyme-substrate complex. In this experiment, hydrogen peroxide occurs as a colorless liquid in its diluted solution. Genuinely, it is a very pale blue liquid. It is slightly more viscous than water. Hydrogen peroxide is a weak acid but it is a strong oxidizing agent that acts as a bleaching agent, disinfectant, oxidizer, antiseptic and the like. It is produced as a byproduct of oxygen metabolism and by all organisms that possessed enzyme peroxidase.
Hydrogen peroxide always decomposes exothermically into water and oxygen gas spontaneously. The rate of decomposition is dependent on the temperature and concentration of the peroxide, as well as the pH and the presence of impurities and stabilizers. Hydrogen peroxide is incompatible with many substances that catalyse its decomposition, including most of the transition metals and their compounds. Common catalysts include manganese dioxide and silver apart from enzyme catalase as has been used in this experiment.
A common concentration for hydrogen peroxide is “20 volumes”, which means that when 1 volume of hydrogen peroxide is decomposed, it produces 20 volumes of oxygen. A 20 “volume” concentration of hydrogen peroxide is equivalent to 1. 67 mol/dm3 (Molar solution) or about 6%. Hydrogen peroxide available at drug stores is three percent solution. In such small concentrations, it is less stable, and decomposes faster. It is usually stabilized with acetanilide, a substance which has toxic side effects in significant amounts.
Buffer solution is used in this experiment as to keep the pH at a constant value while the chemical reaction occurs. As for this experiment, a citric acid sodium phosphate buffer solution of pH 6. 5 is used. It is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. Buffer solution is needed to keep the correct pH for enzymes in many organisms to work effectively. This is because many enzymes work only under the correct end precise condition.
Hence, if the pH is too far out of margin, the enzymes will slow up or stop working and denatured, thus, will cause the enzyme activity become disabled. Buffer solutions are commonly used in industrial sector such as in fermentation process and in setting the correct conditions for dye used in coloring fabrics. Some of them are also used as calibration mark for pH meters. HYPOTHESIS: There is a clear positive correlation between the concentration of enzyme and the rate of reaction. Increase in enzyme concentration results in increase in the rate of reaction up to a point where all substrate has occupied with the enzyme.
VARIABLES: Manipulated variable: Concentration of enzyme Responding variable: Volume of oxygen produced Fixed variables: pH, temperature, concentration of substrate APPARATUS: ??? 250 ml beaker ??? 50 ml measuring cylinder ??? 5 ml measuring cylinder ??? Dropper ??? Bung ??? Conical flask ??? Test tube ??? Spatula MATERIALS: ??? Blended potato ??? pH 6. 5 buffer solution ??? Hydrogen peroxide ??? Tap water PROCEDURE: 1. All the apparatus was rinsed with tap water to remove any foreign substances. 2. A test tube was weighed and the weight is recorded. 3.
A spatula of blended potato was added into a test tube. 4. The blended potato altogether with the test tube was weighed. The weight is recorded and the difference between the weight of the test tube and the test tube with blended potato is recorded as the weight of the blended potato. 5. The test tube then was added with 5 cm 3 of 6. 5 buffer solution by using 5cm 3 measuring cylinder. 6. The test tube was then swirled in order to mix the blended potato and buffer solution well. 7. A beaker and an upside down 50 ml measuring cylinder inside the beaker was set up.
Both beaker and measuring cylinder are filled with water. It is sure that there is no air bubble inside the measuring cylinder. 8. The delivery tube of the bung was connected into the upside down measuring cylinder. 9. 2. 5 cm 3 of hydrogen peroxide, H2O2 was added into the test tube and immediately, the stopper of the bung is placed into the test tube. 10. The volume of oxygen, O2 produced was recorded after 30 seconds. 11. Step 2 to 10 was repeated for 2 and 3 spatula of blended potato, but the test tube is replaced by a conical flask. 12.
The result was all recorded in Table 1 and Graph 1 was plotted to show a clear data of the results. RESULTS: |Number of spatula of |Mass of blended potato |Initial reading of |Final reading of |Concentration of enzyme (g/|Volume of oxygen produced | |blended potato |(g) |measuring cylinder (ml) |measuring cylinder |cm 3) |(ml) | | | | |(ml) | | | |1 |1. |50 |43 |1. 6 ? (5 + 2. 5) = 0. 213 |50 ??? 43 = 7 | |2 |3. 2 |50 |34 | 3. 2 ? (5 + 2. 5) |50 ??? 34 = 16 | | | | | |= 0. 427 | | |3 |4. 8 |50 |33 | 4. 8 ? (5 + 2. ) |50 ??? 33 = 17 | | | | | |= 0. 640 | | Table 1 DISCUSSION: 1. Table 1 shows all the data that has been colleted in the experiment and Graph 1 shows clear relationship between all the data that has been collected. 2. The concentration of catalase enzyme in this experiment was obtained by dividing the weight (mass) of blended potato with the total volume of buffer solution and hydrogen peroxide as stated clearer in the formula below :
Weight (mass) of blended potato (g) Volume of buffer solution (5 cm 3 ) + hydrogen peroxide (2. 5 cm 3 ) 3. The volume of oxygen produced was obtained by subtracting the initial volume of water in the measuring cylinder that is 50ml with the final reading of the measuring cylinder after the reaction has occur as stated in clearer below: Initial volume of water in measuring cylinder (50ml) ??? Final volume of water in measuring cylinder 4. Based on Table 1 and Graph 1, when a spatula of blended potato with mass of 1. g is used, the volume of oxygen produced 7ml with the concentration of 0. 213 g/ cm 3 . When 2 spatula of blended potato with mass of 3. 2g is used, the volume of oxygen produced y the reaction is 16ml with the concentration of 0. 427g/ cm 3 . When 3 spatula of blended potato with mass of 4. 8g is used, the volume of oxygen produced y the reaction is 16ml with the concentration of 0. 640g/ cm 3 . 5. From the overall observation of Graph 1, it can be seen that the volume of oxygen increase as the concentration of enzyme increase.
There is a large jump for the volume of oxygen produced when 2 spatula of blended potato and 1 spatula of blended potato used that is from 7ml to 16ml compared to from 2 spatula to 3 spatula of blended potato used which is just results in slight increase. 6. There is a large jump from the used 1 spatula to 2 spatula of blended potato because the mass of blended potato used has doubled. In other words, the increase in concentration of enzyme catalase results in increase of volume of oxygen produced.
The increase in catalase enzyme has increased the rate of collision between the enzyme which is the catalase, and the substrate, which is the hydrogen peroxide. There are more collision between the active site of the catalase and hydrogen peroxide, results in decomposition of hydrogen peroxide, hence producing more oxygen. 7. There is just a slight increase between the used of 3 spatula and 2 spatula of blended potato because the substrate that is the hydrogen peroxide is limited to the catalase enzyme. The substrate, hydrogen peroxide has become the limiting factor in the experiment.
This is because all of them had become occupied with the enzyme. Hence, it can be concluded that at 0. 640 g/ cm 3 of catalase enzyme used, there will be no more enzyme activity occur. As for example, if 4 spatula or 6. 4g and 0. 853 g/ cm 3 of blended potato used, the volume of oxygen released will also be 17ml since it is the limiting point in the activity. 8. Besides that, there are also other possibilities that why there is just a slight increase between the used of 3 spatula and 2 spatula of blended potato. It is maybe because of some errors occurring while conducting the experiment.
It is possibly that that while the experiment was conducted, there is oxygen released to the surrounding. It is maybe because the stopper did not so immediately put into the mouth of the conical flask or the stopper did not fit the mouth of conical flask nicely. There might be some empty spaces between them without us realizing it. 9. By referring to the graph, any concentration of enzyme used can be obtained. For example, if the concentration of 0. 30 g/ cm 3 of enzyme, there will be 11. 6ml of volume of oxygen produced as previously shown in Graph 1. 10. The blended potato might have the possibility to be oxidized.
Therefore, the blended potato should not be prepared too early before conducting the experiment. In addition, there might be some suspension of blended potato which will not react with the hydrogen peroxide. This can decrease the volume of the oxygen gas liberated which then can affect the accuracy of the result of the experiment. 11. The mixture of the solutions has to be shaken so the mixture is well reacted. If the mixture is not shaken, the oxygen gas will still be released but the exact amount of the oxygen gas produced at that is will not be obtained.
Thus, it will affect the result of the experiment. Care must also be taken for not letting the solution of the reacting mixture to go into the delivery tube because it will affect the volume of the gas released during the reaction. 12. The calibrated tube was not big enough that another calibrated tube was added to collect the gas. Therefore, it had taken some time to transfer the delivery tube from the first calibrated tube to the second calibrated tube. During the transfer, some oxygen gas may be lost. In order to overcome this, bigger calibrated tube or measuring cylinder can be used to replace the mall calibrated tube. 13. The reaction between catalase and the hydrogen peroxide is very quick, that the test tube must be connected to the delivery tube as soon as the hydrogen peroxide is poured into the test tube containing blended potato. This is to minimize the loss of oxygen gas to the surrounding because the reaction is very fast. 14. Hydrogen peroxide is a strong oxidizing agent, hence safety measures should be taken to avoid any hazard occur while conducting the experiment since it is a toxic that can bleach hair and corrosive towards our skin.
While using the substances, it is important to not make contact with it and avoid direct exposure towards eyes or mouth. 15. To make sure that this experiment can achieve its objective that is to investigate the effect of enzyme concentration on the enzyme activity, the other three factors that also affect the rate of reaction that are pH, temperature and substrate concentration should remain constant in the experiment. This has been done by using buffer solution in order to keep the pH at constant level, conducting the experiment at room temperature only and maintaining the amount of substrate used.
CONCLUSION: It is proven that, by the experiment that has been conducted, the rate of reaction increase when the concentration of enzyme increases up to a point where the substrate concentration become limited o the enzymes. In the experiment, it can be concluded that the maximum of enzyme concentration that can be used is 0. 640 g/ cm 3 ,that is the mixture of 4. 8g of blended potato with 2. 5 cm 3 of hydrogen peroxide and 5 cm 3 of buffer solution produced maximum volume of oxygen that is 17ml.