Potato Enzyme Lab Report
I. Aim
The aim of the three experiments is actually to investigate the effects of different factors such as temperature, pH, and concentration of substrate, on the activity of the enzymes. By conducting these three separate experiments also, three graphs are able to be obtained where the trend of each factor affecting on the enzyme activity is shown and described clearly.
II. Hypothesis
Experiment 1 (Effect of Temperature):
As the temperature increases, the height of the bubble will increase too, indicating a faster rate of reaction.
Experiment 2 (Effect of pH):
Enzymes are affected by changes in pH. Extremely high or low pH values generally result in complete loss of activity for most enzymes.
Experiment 3 (Effect of Substrate Concentration): …show more content…
The more substrate added to the reaction the faster the rate of the reaction will be. However, at a saturation point, the enzyme will start to reach a maximum velocity slowing down the enzyme activity compared to the increased rate before this saturation point.
III. Introduction
Enzymes
Enzymes are defined as biological catalysts, present in living systems in which it increases the rate of a chemical reaction without being changed itself. It performs its function by lowering down the activation energy of the substrate. In these three experiments, the enzyme that is being used is potatoes. After doing some research, potatoes are proven to contain several enzymes such as catalase (the most well known and common), oxidase, and peroxidase.
Catalase is a common enzyme found in nearly all living organisms that are exposed to oxygen, where it functions to catalyze the decomposition of hydrogen peroxide to water and oxygen. Catalase has one of the highest turnover numbers of all enzymes; one molecule of catalase can convert 40 million molecules of hydrogen peroxide to water and oxygen each second. Catalase is a tetramer of four polypeptide chains, each over 500 amino acids long. It contains four porphyrin heme (iron) groups that allow the enzyme to react with the hydrogen peroxide. 1
Oxidases in potato includes tyrosinase, cathecol oxidase and cytochrome oxidase. Peroxidase have the same function as with oxidase but they need a co-factor in order to complete its required action. The co-factor phenol oxidase is found abundant in potatoes. Hence, it will have a positive reaction once a substrate will be added indicating the presence of peroxidase in potatoes. 2
Substrate
The substrate, which is the reactant in an enzyme-catalyzed reaction, being used in the three experiments is hydrogen peroxide (H2O2). Hydrogen peroxide is a toxic chemical that is produced in many organisms during metabolism. Organisms must get rid of this toxin to survive. One reaction turns the hydrogen peroxide into water and oxygen. The enzyme that helps to perform this reaction is the catalase enzyme, which exists in potatoes. 3 Moreover it is also a colourless liquid that resembles water in many respects. Its physical properties are very similar to those of water, except that it is 40% denser.
The main difference between hydrogen peroxide and water, however, is in its chemical behaviour. The single bond between the two oxygen atoms is weak, so that H2O2 readily fragments into either H and HO2 or two OHs. Either way, the resulting species are free radicals, which means they are very reactive, and this makes H2O2 a very powerful oxidising agent. Its oxidising properties are effective in inhibiting growth and killing microbes. What makes hydrogen peroxide unique is the foaming action that takes place whenever it is placed on a cut.4
Hydrogen Peroxide is not considered explosive, but explosive vapors can be formed when peroxide is mixed with certain organic materials. In addition, Hydrogen Peroxide is not flammable, but does generate large amounts of oxygen during decomposition that supports combustion.5
Reaction
Hydrogen peroxide bubbles when it comes into contact with an enzyme called catalase. Most cells in the body contain catalase, so when the tissue is damaged the enzyme is released and becomes available to react with the peroxide. Catalase allows hydrogen peroxide (H2O2 to be broken down into water (H2O) and oxygen (O2). The bubbles appearing when the peroxide is poured to the enzyme are bubbles of oxygen gas. Here is the word equation of the reaction: 3
2H2O2 2H2O + O2
The reaction between catalase and hydrogen peroxide is a very speedy and dangerous reaction and may cause harm to those surrounding it. This is because oxygen is formed and it decreases with time due to enzyme damage. This reaction is important to cells because hydrogen peroxide (H2O2) is produced as a byproduct of many normal cellular reactions. If the cells did not break down the hydrogen peroxide, they would be poisoned and die.6
Factors affecting enzyme activity
The factors include temperature, pH, enzyme concentration, substrates concentration and also inhibitors and activators molecules. At optimal conditions of temperature, pH, substrate concentration, enzyme concentration, absence of inhibitors etc, this reaction may be at its highest rate.7
IV. Materials
Experiment 1:
1. Electric stove
2. Potato
3. Ice cube
4. Water
5. Hydrogen peroxide solution
6. 150 ml beaker
7. Potato borer
8. 250 ml beaker
9. Pipette with rubber dispenser
10. Thermometer
11. Testube
12. Test tube holder
13. Stopwatch
14. Ruler
Experiment 2:
1. hydrogen peroxide (H2O2)
2. sodium Hydroxide (NAOH)
3. hydrochloric acid (HCl)
4. fresh potato
5. universal indicator paper
6. pipettes (10 mL) with rubber dispenser
7. dropper
8. test-tubes (5)
9. test-tube rack
10. marking pens
11. labels
12. potato borer
13. ruler to measure in cm and mm
14. stop watch
15. 150 mL beaker
Experiment 3:
1. Hydrogen peroxide (H2O2)
2. Distilled water
3. 5 test tubes
4. 1 of 150 mL beaker
5. Test Tube rack
6. 2 of 10 mL pipettes with rubber dispenser ( 1 for distilled water & 1 for H2O2 )
7. Potato borer and cutter (if necessary)
8. Fresh Potato
9. Labels
10. Pen
11. Ruler
12. Stopwatch
V. Procedures
Experiment 1:
1. The hydrogen peroxide solution is being measured on its temperature by using a thermometer
2. The temperature data is recorded, marking that this is the control (room temperature).
3. 4 mL of hydrogen peroxide is being sucked by the pipette with the rubber dispenser.
4. It is then being poured out into one of the 5 test tubes.
5. The potato is being bored to get 5 cylinder-like shape potatoes with a length of 3 cm and diameter of 0.5 cm.
6. This cylinder potato is then put into the test tube as the stopwatch is started.
7. After 2 minutes, the oxygen level is measured by using the ruler and is recorded into the table.
8. Distilled water is poured into the 150 mL beaker and 250 mL beaker (each half full).
9. The water in 250 mL beaker is being heated by the electric stove until it reaches a specific temperature required, by placing a thermometer inside the beaker.
10. The water in 150 mL beaker is added with some ice cubes to lower down the temperature until it reaches a specific temperature, by also placing another thermometer inside the beaker.
11. After the named temperature is reached, hydrogen peroxide is being sucked by the pipette with rubber dispenser for 4 mL and poured to a test tube.
12. This test tube is held by using the test tube holder and placed inside the heated or chilled water beaker.
13. After it is placed for 1 minute, the cylinder potato is added into the test tube as the stopwatch is started.
14. After 2 minutes, the oxygen level is measured on its height by using a ruler
15. The height is then recorded in the table
16. Procedure 11-16 are repeated for measuring the reaction in a temperature either above or below the room temperature
Independent variable: temperature
Dependent Variable: height of the oxygen level
Constant Variable: volume, concentration and pH of the hydrogen peroxide, size of the potato, duration of reaction
Control: test tube with the room temperature
Safety Precaution:
Use glove and lab coat to avoid any irritation due to the contact of Hydrogen peroxide solution towards human skin
Use pipettes to avoid hand direct touch to the solution
Be careful in starting and heating the water using electric stove
Experiment 2:
1. 5 test-tubes labelled 1 to 5 are setted up in the rack.
2. The potato is bored to create 5 cylinder-like potato pieces with the size of 0.5 cm in diameter and 3 cm in length.
3. The hydrogen peroxide is poured into one fifth of the beaker and is measured on its pH by submerging half of the pH paper in the solution.
4. After a couple of second, the pH paper is observed upon its color to know the pH number by referring to the list of patterns for each pH number. (This pH is the control for it is the natural pH of the H2O2 )
5. Then hydrogen peroxide is being sucked for 4 mL by using the pipette with rubber dispenser and poured into test tube 1.
6. One cylinder potato is then placed into the test tube as the stopwatch is started.
7. After 2 minutes, the height of the bubbles of oxygen that formed over the contents of the tubes was measured by a regular ruler.
8. To increase the pH value (more basic) of the hydrogen peroxide, 3-4 drops of sodium chloride (NaOH) are added into the hydrogen peroxide solution in the beaker.
9. Then the two liquid are mixed together by shaking the beaker steadily.
10. Half of the pH paper is then dipped into the solution and Procedure 4 is carried out.
11. After the pH value of the solution matches with the desired pH, procedures 5-7 are repeated.
12. Procedure 8-11 are repeated if a more basic solution is required.
13. To decrease the pH value (more acidic) of hydrogen peroxide, 3-4 drops of hydrochloric acid (HCl) are added into the hydrogen peroxide solution inside the beaker.
14. Procedure 9-11 are then repeated.
15. Procedure 13-14 are repeated if a more acidic solution is required.
16. The pH values of acid and basic solutions were setted up as follow:
Test tube 1: pH 1
Test tube 2 : pH 3 (control)
Test tube 3 : pH 5
Test tube 4 : pH 7
Test tube 5 : pH 8
17. Trial 2 was carried out for more accurate results.
Independent variable: pH value of the hydrogen peroxide
Dependent variable: height of the oxygen level (rate of reaction)
Control: original pH of the hydrogen peroxide
Constant: volume and concentration of hydrogen peroxide in each test tube, temperature of all the solution, size of the potato, duration of the reaction
Safety precaution:
Hydrogen peroxide is a reactive substance to our skin. Therefore, wear a pair of safety gloves and a lab coat to prevent skin damage whenever an accident spill occurs.
Experiment 3:
1. 5 test tubes are being set up and labeled from 1 to 5.
2. Distilled water is being poured into the 150 mL beaker just enough
3. A volume of 10 mL of distilled water is being pipette by using one of the 10 mL pipettes with rubber dispenser
4. That volume of water is then poured into test tube 1.
5. While this process is being conducted, the potato is being bored into 5 cylinder-like shapes with same size of 3 cm in length and approximately 0.5 cm in diameter. (Cutter may be used to keep the length of the potato uniform by cutting it)
6. A cylinder of potato is then dropped into test tube 1, as the stopwatch is being started.
7. Test tube 1 is then placed back in the test tube rack
8. After 2 minutes, the height of the oxygen bubble in test tube 1 is measured by using a ruler
9. The height is then recorded in the table in cm
10. The above procedures are then repeated for test tube 2,3,4,and 5, according to the volumes of hydrogen peroxide and water being mixed together as shown in the table below:
Test tube
Duration
Volume of H2O2 (mL)
Volume of water (mL)
1
2 minutes
0
10
2
2.5
7.5
3
5
5
4
7.5
2.5
5
10
0
Independent variable: concentration of hydrogen peroxide
Dependent variable: the height of the oxygen bubble (the higher the height the faster the reaction is)
Control: Test tube 5 (pure hydrogen peroxide)
Constant: temperature, pH, enzyme (the size of the cylinder-like potato), amount of the substrate (the volume of the solution before reaction, which is 10 mL), duration of reaction
Safety precaution:
Wear gloves and lab coat during the experiment, because the hydrogen peroxide may cause a skin irritation when it makes contact with your skin.
Avoid direct contact with hydrogen peroxide
Do not play around with the potato borer and cutter because it is sharp
VI. Result
Experiment 1:
Trial 1
Water Temperature (°C)
Time
Height of the oxygen level
8
2 min.
0.9 cm
15
1.4 cm
25
2 cm
35
1.9 cm
46
1.3 cm
Trial 2
Water Temperature (°C)
Time
Height of the oxygen level
8
2 min.
1.2 cm
15
1.3 cm
25
1.5 cm
35
1.8 cm
46
1 cm
Average between trial 1 and 2
Water Temperature (°C)
Time
Height of the oxygen level
8
2 min.
1.05 cm
15
1.35 cm
25
1.75 cm
35
1.85 cm
46
1.15 cm
Experiment 2:
Trial
Test-tube no. pH Measure
Time
Oxygen Height (cm)
1
1
2 min.
1
2
3
3
3
5
3.3
4
7
2.9
5
8
0.9
Trial
Test-tube no. pH Measure
Time
Oxygen Height (cm)
1
1
2 min.
0.9
2
3
2.6
3
5
2.9
4
7
2.5
5
8
1.5
Average between trial 1 and 2:
Test-tube no. pH Measure
Time
Oxygen Height (cm)
1
1
2 min.
0.95
2
3
2.8
3
5
3.1
4
7
2.7
5
8
1.2
Experiment 3:
Trial 1
Test tube
Duration
Volume of H2O2 (mL)
Volume of water (mL)
Height of the bubble (cm)
1
2 minutes
0
10
0
2
2.5
7.5
0.7
3
5
5
0.8
4
7.5
2.5
0.8
5
10
0
0.9
Trial 2
Test tube
Duration
Volume of H2O2 (mL)
Volume of water (mL)
Height of the bubble (cm)
1
2 minutes
0
10
0.2
2
2.5
7.5
2.4
3
5
5
2.2
4
7.5
2.5
1.6
5
10
0
2.3
Average between trial 1 and trial 2
Test tube
Duration
Volume of H2O2 (mL)
Volume of water (mL)
Height of the bubble (cm)
1
2 minutes
0
10
0.1
2
2.5
7.5
1.5
3
5
5
1.6
4
7.5
2.5
1.3
5
10
0
1.6
VI. Discussion and Analysis
Experiment 1: The data result based on the experiment shows that as the temperature increases the height of the oxygen will increase, which means that the rate of enzyme activity is rapidly speeding up until it reaches a certain temperature where the height begins to decrease, meaning the rate of enzyme activity is slower. The hypothesis is incorrect, since both trials show that the height of the oxygen level does not always increase whenever the temperature increases. Seeing from the average graph, starting from 8 degree Celsius, the height of the oxygen bubble keeps on increasing constantly and steadily as the temperature increases. Then comes to the point of approximately 32 degree Celsius where the activity reaches its optimum temperature and the height of the oxygen bubble begins to drop down when the temperature is above 32 degree Celsius, indicating that the enzyme activity begins to fail. The decreasing rate of the activity is much more rapid and severe compared to the increasing rate of the activity when it is below 32 degree Celsius. This is proven by looking at the average graph. The curve before 32 degree Celsius is not as steep as the curve after 32 degree Celsius, indicating that the dropping of the enzyme activity happens much more rapidly and severely and more sensitive to the difference in temperature. This trend happens because the enzyme catalase that contain in the potato is not suitable for higher temperature, specifically above 32 degree Celsius. If compared to the secondary information, the result shows the same. Catalase enzyme is not suitable for higher temperature, in this case is more than 32 degree Celsius. The information wrote the catalyst enzyme became thermally denatured, meaning the heat energy has been able to break the bonds that cause the protein to fold, so destroying the active site. The substrate no longer has an active site with which to bind so there is little or no activity. Denaturation by heat is also irreversible and the active site is not restorable. The accuracy of this experiment remains quite high because we use ruler in measuring the height of oxygen bubble and thermometer in measuring the temperature of the environment. We also measure as accurate as possible for the hydrogen peroxide to be 4 mL in each of the test tube, before the reaction began. By starting the stopwatch the second when the potato is submerged in the hydrogen peroxide also keeps the accuracy in this experiment. However, the accuracy may be defected by some factors such as the ruler being used is only to the nearest cm, the temperature within the test tube may not be equal to the temperature within the water inside the beaker when we start the reaction, and a different eye level or perspective when measuring the height of the oxygen bubble for each reaction. The reliability of this experiment however, is high because there are two trials being performed to get closer and much more accurate result, by taking the average of both trials as the result data. But one thing that defects the reliability is the inconsistency of the data being received. For example in the room temperature of 25 degree Celsius, trial 1 shows a result of 2 cm (higher than the next result) while trial 2 results in 1.5 cm (lower than the next result). The validity of this experiment is high because there is only one independent variable being added in this experiment which is the temperature variations. Moreover, the result of this experiment corresponds with the aim of the experiment in the first place, showing how the temperature affects enzyme activities. However, there are still some factors that prevent this experiment to be highly valid. These factors are related to the factors that defect the accuracy stated above, during the experiments. Since some of the variables are not accurately measured and done, there may be some additional independent variables, causing a lowly valid experiment. So, to improve this experiment, there are some several actions needed to be done. Firstly, by using more advance measurement tools such as rulers that can measure to the nearest mm and modern test tube that can indicate the temperature within and so on, the accuracy level of the experiment may be increased. The reliability of the experiment may also increase if more trials are done and making sure that everything stay the same among each trials to keep the consistency in the experiment. The validity too can be improved if the accuracy level is improved.
Experiment 2: As you can see from the average result, the enzyme activity increases as the pH value approaches pH of 5 then it slowly decreases when it approaches 7, and finally drops severely when it approaches 8. The graph also shows that the curve remains quite high at the pH ranging from 3.5 to 6.5, and there is little change happening within this range. On the other hand, the curve drops or increases heavily when it is below 3.5 or above 6.5. This analysis proves that the enzyme catalase allows little range of pH within which it is active. From the graph also it can be obtained the optimum pH of the enzyme is approximately 4.5, in which the active site is maintained most efficiently. Therefore this result has proven that the hypothesis is correct: Enzymes are affected by changes in pH. Extremely high or low pH values generally result in complete loss of activity for most enzymes. When being compared to the second hand information, it shows that there is a difference in the result of optimum value of pH in which the enzyme catalase can best react. The internet shows it should have been a pH of 7 8, while our experiment shows a pH of 5 as the optimum pH. But, some sources also state that the result of this experiment will not always be the same which is the pH of 7 being the optimum pH, due to some several factors of difference in temperature, solution density, inhibitors, and so on. That is why lots of trials are needed to be performed to achieve the most accurate result.9 The accuracy of this second experiment is similar with the first experiment which is quite high, because we use the similar materials for conducting the experiment. One factor from this experiment that adds on the accuracy level is the use of different pipettes for different solution. One pipette for NaOH and one pipette for HCl, which prevents any contamination and distortion of the pH level. The reliability however is not as high because as the hydrogen peroxide being sucked up for test tube 1,2,3, and 4, the remaining will become less than the initial. Then more NaOH or HCl is being added. Since the concentration of hydrogen peroxide is much lesser than the first time its being mixed, there may be a distortion in the pH value of the solution, creating a less reliable experiment results. Secondly, because the experiment result of optimum pH value doesn’t match with the research, this experiment is considered to be highly unreliable, especially knowing that the difference is by 2 pH value (which is a lot). The validity of this experiment is quite high, similar to the first experiment because the result corresponds with the aim of the experiment at the very first place. The experiment also only consists of one independent variable which is the pH value of the substrate, creating a valid experiment. Similar to that of the accuracy level, if the accuracy level is not high, so does the validity because it will create more than one independent variable. Things that need to be improved in this experiment include the need of more repeated trials, especially knowing that the result is very different than what it is supposed to be according to the second-hand information. This unreliable result has become the evidence that the experiment must have been taken much more carefully and precisely. More advance tools are also needed to increase the accuracy and validity level of this experiment.
Experiment 3: Based on the final graph of the average rate of enzyme activity depending upon the concentration of substrate, it is clearly seen that as the concentration of enzyme increases the rate of reaction increases too. From 0-25%, the enzyme activity increases steadily and constantly. Then between the concentration of 25% and 50% the enzyme activity remains very constant showing how there are no increases or decreases in the rate of the activity. However, after it reaches 50% of concentration, the activity starts to slow down and increase again when it is 100% concentration. This overall creates a fluctuating curve, although not that volatile. This third experiment has produced a result that does not match with the theory of how the different concentration of substrate may affect enzyme activities, because the result should not fluctuate in a way where the enzyme activity drops in 75% of concentration. This happens in this third experiment because of a predicted reason, which is the use of different type of potatoes. In the first trial as you can see, the data of the height of oxygen bubble does not flow the way the second trial’s data does. It never drops down; it either increases or remains the same. During the second trial, another potato has to be taken because the first potato being used in the first trial has already been wholly bored, no more spaces. But there are no more potatoes of the same age as the one being used in the first trial, this time all of them looks much younger. So there is no choice but to take one of them. However, the first and fourth test tube in the second trial have the same potatoes as the first trial, leaving the second, third and fifth test tube of trial 2 with the younger potatoes. Therefore, this explains why the 0% and 75% concentration to be so low. And because of this also, we may conclude that younger potatoes can perform much faster reaction than the old ones. Due to this major problem affecting the result of the third experiment, it still cannot be said whether the hypothesis of “the more substrate added to the reaction the faster the rate of the reaction will be, but it will not be as increasing as rapid as before after reaching maximum velocity” correct or not. In this experiment, however, it is indicated that the maximum velocity will be when the concentration is around 50%. The accuracy of this experiment has the same level as the two experiments before because the instruments being used are similar. The reliability and validity of this experiment however is quite low, because of the problem being stated before, creating an inconsistent result that distorts the final result of the experiment severely. The validity has been defected also due to the existences of two independent variables, in this case, are the concentration of substrate and the type of the enzymes (potato). But a part of the experiment that is still being valid is that it once again relates to the first aim of the experiment which is to know the effect of concentration of substrate on the enzyme activity. The most important thing to be improved in this experiment is the awareness of the independent variable, meaning that the independent variable must be kept single all the time. It should not be more than two. The potatoes being used for both trials should have been the same type, or at least in one trial there should only be one type of potato being used, instead of mixing the different types of potatoes in one trial. By doing more and more trial also, this experiment can be improved furthermore.
VII. Conclusions
Experiment 1:
Enzyme activity will increase steadily as the temperature increases, but after it peaks at its optimum temperature, the enzyme activity will drop down significantly.
Enzyme catalase has an optimum temperature of 32 degree Celsius.
Enzyme catalase does not suit with temperature above 32 degree Celsius because it is when the enzyme thermally denatures and active sites being destroyed
Denaturation by heat is irreversible and active enzyme cannot be restored
Experiment 2:
Enzyme catalase is affected by changes in pH. Extremely high or low pH values generally result in complete loss of activity for most enzymes.
It allows little range of pH for the enzyme to be highly active and within this range the optimum pH is located.
Enzyme catalase has an optimum pH of 5 according to this experiment, but it is supposedly 7.
Experimet 3:
Enzyme activity will increase as the concentration of the substrate increases but then after it reaches its saturation point or maximum velocity; it will remain constant or have only a slight increase in activity.
Enzyme catalase should have reached its maximum velocity during 50% concentration of substrate.
General Conclusions:
The reaction between enzyme catalase and hydrogen peroxide will produce two harmless products of water and oxygen gas.
Enzymes are highly affected on temperature, pH, substrate concentration, enzyme concentration, inhibitors and activators
It is very important that these enzymes work in its optimum level of each factor to be able to function in each living organisms.
References
Bennett, T. P., and Frieden, E.: Modern Topics in Biochemistry, pg. 43-45, Macmillan, London (1969).
Catalase. (n.d.). Retrieved October 29, 2013, from http://www.princeton.edu/~achaney/tmve/wiki100k/docs/Catalase.html
Enzyme Catalase. (n.d.). Retrieved October 29, 2013, from http://www.csun.edu/~aef21890/coursework/695/science_kit/enzyme-catalase.htm
Harrow, B., and Mazur, A.: Textbook of Biochemistry, 109, Saunders, Philadelphia (1958).
Holum, J.: Elements of General and Biological Chemistry, 2nd ed., 377, Wiley, NY (1968).
Hydrogen Peroxide. (n.d.). Retrieved October 29, 2013, from http://www.ch.ic.ac.uk/rzepa/mim/environmental/html/h2o2_text.htm
Martinek, R.: Practical Clinical Enzymology: J. Am. Med. Tech., 31, 162 (1969)
Pfeiffer, J.: Enzymes, the Physics and Chemistry of Life, pg 171-173, Simon and Schuster, NY (1954)
Potatoe Enzyme lab by Hadley McCollester on Prezi.
(n.d.). Retrieved October 29, 2013, from http://prezi.com/wii0fgw48euc/potatoe-enzyme-lab/
What enzyme is present in liver and potatoes. (n.d.). Retrieved October 29, 2013, from http://wiki.answers.com/Q/What_enzyme_is_present_in_liver_and_potatoes#slide2
What Is the Optimum PH for Catalase - Ask.com. (n.d.). Retrieved October 29, 2013, from http://www.ask.com/question/what-is-the-optimum-ph-for-catalase
What is the optimum PH for potato catalase and why? - Yahoo! Answers. (n.d.). Retrieved October 29, 2013, from http://answers.yahoo.com/question/index?qid=20071201155345AADgypg
Retrieved October 29, 2013, from http://www.fmcchemicals.com/Portals/chem/content/docs/engineering/Properties%20of%20Hydrogen%20Peroxide.pdf
Retrieved October 29, 2013, from lab-biology-blue-2-gray-2a.sbsd.sbhs.schoolfusion.us%2Fmodules%2Flocker%2Ffiles%2Fget_group_file.phtml%3Fgid%3D1176791%26fid%3D13813691%26sessionid%3Dc93577&ei=SU9vUv-xI8eNrQfq0YCwCg&usg=AFQjCNE-Np-noyT-M_c8DrdYeo8bZ38zzQ&sig2=j-0hFL2Dtuq3hAhtXAsw9w&bvm=bv.55123115,d.bmk
"Science Fair Projects - How Temperature Affects the Peroxidase Enzyme." Science Fair Projects - How Temperature Affects the Peroxidase Enzyme. N.p., n.d. Web. 28 Oct. 2013.
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Retrieved October 29, 2013, from www.preservearticles.com/2011112217708/what-are-the-factors-that-affect-the-enzymatic-activity.htmlH
The aim of the three experiments is actually to investigate the effects of different factors such as temperature, pH, and concentration of substrate, on the activity of the enzymes. By conducting these three separate experiments also, three graphs are able to be obtained where the trend of each factor affecting on the enzyme activity is shown and described clearly.
II. Hypothesis
Experiment 1 (Effect of Temperature):
As the temperature increases, the height of the bubble will increase too, indicating a faster rate of reaction.
Experiment 2 (Effect of pH):
Enzymes are affected by changes in pH. Extremely high or low pH values generally result in complete loss of activity for most enzymes.
Experiment 3 (Effect of Substrate Concentration): …show more content…
The more substrate added to the reaction the faster the rate of the reaction will be. However, at a saturation point, the enzyme will start to reach a maximum velocity slowing down the enzyme activity compared to the increased rate before this saturation point.
III. Introduction
Enzymes
Enzymes are defined as biological catalysts, present in living systems in which it increases the rate of a chemical reaction without being changed itself. It performs its function by lowering down the activation energy of the substrate. In these three experiments, the enzyme that is being used is potatoes. After doing some research, potatoes are proven to contain several enzymes such as catalase (the most well known and common), oxidase, and peroxidase.
Catalase is a common enzyme found in nearly all living organisms that are exposed to oxygen, where it functions to catalyze the decomposition of hydrogen peroxide to water and oxygen. Catalase has one of the highest turnover numbers of all enzymes; one molecule of catalase can convert 40 million molecules of hydrogen peroxide to water and oxygen each second. Catalase is a tetramer of four polypeptide chains, each over 500 amino acids long. It contains four porphyrin heme (iron) groups that allow the enzyme to react with the hydrogen peroxide. 1
Oxidases in potato includes tyrosinase, cathecol oxidase and cytochrome oxidase. Peroxidase have the same function as with oxidase but they need a co-factor in order to complete its required action. The co-factor phenol oxidase is found abundant in potatoes. Hence, it will have a positive reaction once a substrate will be added indicating the presence of peroxidase in potatoes. 2
Substrate
The substrate, which is the reactant in an enzyme-catalyzed reaction, being used in the three experiments is hydrogen peroxide (H2O2). Hydrogen peroxide is a toxic chemical that is produced in many organisms during metabolism. Organisms must get rid of this toxin to survive. One reaction turns the hydrogen peroxide into water and oxygen. The enzyme that helps to perform this reaction is the catalase enzyme, which exists in potatoes. 3 Moreover it is also a colourless liquid that resembles water in many respects. Its physical properties are very similar to those of water, except that it is 40% denser.
The main difference between hydrogen peroxide and water, however, is in its chemical behaviour. The single bond between the two oxygen atoms is weak, so that H2O2 readily fragments into either H and HO2 or two OHs. Either way, the resulting species are free radicals, which means they are very reactive, and this makes H2O2 a very powerful oxidising agent. Its oxidising properties are effective in inhibiting growth and killing microbes. What makes hydrogen peroxide unique is the foaming action that takes place whenever it is placed on a cut.4
Hydrogen Peroxide is not considered explosive, but explosive vapors can be formed when peroxide is mixed with certain organic materials. In addition, Hydrogen Peroxide is not flammable, but does generate large amounts of oxygen during decomposition that supports combustion.5
Reaction
Hydrogen peroxide bubbles when it comes into contact with an enzyme called catalase. Most cells in the body contain catalase, so when the tissue is damaged the enzyme is released and becomes available to react with the peroxide. Catalase allows hydrogen peroxide (H2O2 to be broken down into water (H2O) and oxygen (O2). The bubbles appearing when the peroxide is poured to the enzyme are bubbles of oxygen gas. Here is the word equation of the reaction: 3
2H2O2 2H2O + O2
The reaction between catalase and hydrogen peroxide is a very speedy and dangerous reaction and may cause harm to those surrounding it. This is because oxygen is formed and it decreases with time due to enzyme damage. This reaction is important to cells because hydrogen peroxide (H2O2) is produced as a byproduct of many normal cellular reactions. If the cells did not break down the hydrogen peroxide, they would be poisoned and die.6
Factors affecting enzyme activity
The factors include temperature, pH, enzyme concentration, substrates concentration and also inhibitors and activators molecules. At optimal conditions of temperature, pH, substrate concentration, enzyme concentration, absence of inhibitors etc, this reaction may be at its highest rate.7
IV. Materials
Experiment 1:
1. Electric stove
2. Potato
3. Ice cube
4. Water
5. Hydrogen peroxide solution
6. 150 ml beaker
7. Potato borer
8. 250 ml beaker
9. Pipette with rubber dispenser
10. Thermometer
11. Testube
12. Test tube holder
13. Stopwatch
14. Ruler
Experiment 2:
1. hydrogen peroxide (H2O2)
2. sodium Hydroxide (NAOH)
3. hydrochloric acid (HCl)
4. fresh potato
5. universal indicator paper
6. pipettes (10 mL) with rubber dispenser
7. dropper
8. test-tubes (5)
9. test-tube rack
10. marking pens
11. labels
12. potato borer
13. ruler to measure in cm and mm
14. stop watch
15. 150 mL beaker
Experiment 3:
1. Hydrogen peroxide (H2O2)
2. Distilled water
3. 5 test tubes
4. 1 of 150 mL beaker
5. Test Tube rack
6. 2 of 10 mL pipettes with rubber dispenser ( 1 for distilled water & 1 for H2O2 )
7. Potato borer and cutter (if necessary)
8. Fresh Potato
9. Labels
10. Pen
11. Ruler
12. Stopwatch
V. Procedures
Experiment 1:
1. The hydrogen peroxide solution is being measured on its temperature by using a thermometer
2. The temperature data is recorded, marking that this is the control (room temperature).
3. 4 mL of hydrogen peroxide is being sucked by the pipette with the rubber dispenser.
4. It is then being poured out into one of the 5 test tubes.
5. The potato is being bored to get 5 cylinder-like shape potatoes with a length of 3 cm and diameter of 0.5 cm.
6. This cylinder potato is then put into the test tube as the stopwatch is started.
7. After 2 minutes, the oxygen level is measured by using the ruler and is recorded into the table.
8. Distilled water is poured into the 150 mL beaker and 250 mL beaker (each half full).
9. The water in 250 mL beaker is being heated by the electric stove until it reaches a specific temperature required, by placing a thermometer inside the beaker.
10. The water in 150 mL beaker is added with some ice cubes to lower down the temperature until it reaches a specific temperature, by also placing another thermometer inside the beaker.
11. After the named temperature is reached, hydrogen peroxide is being sucked by the pipette with rubber dispenser for 4 mL and poured to a test tube.
12. This test tube is held by using the test tube holder and placed inside the heated or chilled water beaker.
13. After it is placed for 1 minute, the cylinder potato is added into the test tube as the stopwatch is started.
14. After 2 minutes, the oxygen level is measured on its height by using a ruler
15. The height is then recorded in the table
16. Procedure 11-16 are repeated for measuring the reaction in a temperature either above or below the room temperature
Independent variable: temperature
Dependent Variable: height of the oxygen level
Constant Variable: volume, concentration and pH of the hydrogen peroxide, size of the potato, duration of reaction
Control: test tube with the room temperature
Safety Precaution:
Use glove and lab coat to avoid any irritation due to the contact of Hydrogen peroxide solution towards human skin
Use pipettes to avoid hand direct touch to the solution
Be careful in starting and heating the water using electric stove
Experiment 2:
1. 5 test-tubes labelled 1 to 5 are setted up in the rack.
2. The potato is bored to create 5 cylinder-like potato pieces with the size of 0.5 cm in diameter and 3 cm in length.
3. The hydrogen peroxide is poured into one fifth of the beaker and is measured on its pH by submerging half of the pH paper in the solution.
4. After a couple of second, the pH paper is observed upon its color to know the pH number by referring to the list of patterns for each pH number. (This pH is the control for it is the natural pH of the H2O2 )
5. Then hydrogen peroxide is being sucked for 4 mL by using the pipette with rubber dispenser and poured into test tube 1.
6. One cylinder potato is then placed into the test tube as the stopwatch is started.
7. After 2 minutes, the height of the bubbles of oxygen that formed over the contents of the tubes was measured by a regular ruler.
8. To increase the pH value (more basic) of the hydrogen peroxide, 3-4 drops of sodium chloride (NaOH) are added into the hydrogen peroxide solution in the beaker.
9. Then the two liquid are mixed together by shaking the beaker steadily.
10. Half of the pH paper is then dipped into the solution and Procedure 4 is carried out.
11. After the pH value of the solution matches with the desired pH, procedures 5-7 are repeated.
12. Procedure 8-11 are repeated if a more basic solution is required.
13. To decrease the pH value (more acidic) of hydrogen peroxide, 3-4 drops of hydrochloric acid (HCl) are added into the hydrogen peroxide solution inside the beaker.
14. Procedure 9-11 are then repeated.
15. Procedure 13-14 are repeated if a more acidic solution is required.
16. The pH values of acid and basic solutions were setted up as follow:
Test tube 1: pH 1
Test tube 2 : pH 3 (control)
Test tube 3 : pH 5
Test tube 4 : pH 7
Test tube 5 : pH 8
17. Trial 2 was carried out for more accurate results.
Independent variable: pH value of the hydrogen peroxide
Dependent variable: height of the oxygen level (rate of reaction)
Control: original pH of the hydrogen peroxide
Constant: volume and concentration of hydrogen peroxide in each test tube, temperature of all the solution, size of the potato, duration of the reaction
Safety precaution:
Hydrogen peroxide is a reactive substance to our skin. Therefore, wear a pair of safety gloves and a lab coat to prevent skin damage whenever an accident spill occurs.
Experiment 3:
1. 5 test tubes are being set up and labeled from 1 to 5.
2. Distilled water is being poured into the 150 mL beaker just enough
3. A volume of 10 mL of distilled water is being pipette by using one of the 10 mL pipettes with rubber dispenser
4. That volume of water is then poured into test tube 1.
5. While this process is being conducted, the potato is being bored into 5 cylinder-like shapes with same size of 3 cm in length and approximately 0.5 cm in diameter. (Cutter may be used to keep the length of the potato uniform by cutting it)
6. A cylinder of potato is then dropped into test tube 1, as the stopwatch is being started.
7. Test tube 1 is then placed back in the test tube rack
8. After 2 minutes, the height of the oxygen bubble in test tube 1 is measured by using a ruler
9. The height is then recorded in the table in cm
10. The above procedures are then repeated for test tube 2,3,4,and 5, according to the volumes of hydrogen peroxide and water being mixed together as shown in the table below:
Test tube
Duration
Volume of H2O2 (mL)
Volume of water (mL)
1
2 minutes
0
10
2
2.5
7.5
3
5
5
4
7.5
2.5
5
10
0
Independent variable: concentration of hydrogen peroxide
Dependent variable: the height of the oxygen bubble (the higher the height the faster the reaction is)
Control: Test tube 5 (pure hydrogen peroxide)
Constant: temperature, pH, enzyme (the size of the cylinder-like potato), amount of the substrate (the volume of the solution before reaction, which is 10 mL), duration of reaction
Safety precaution:
Wear gloves and lab coat during the experiment, because the hydrogen peroxide may cause a skin irritation when it makes contact with your skin.
Avoid direct contact with hydrogen peroxide
Do not play around with the potato borer and cutter because it is sharp
VI. Result
Experiment 1:
Trial 1
Water Temperature (°C)
Time
Height of the oxygen level
8
2 min.
0.9 cm
15
1.4 cm
25
2 cm
35
1.9 cm
46
1.3 cm
Trial 2
Water Temperature (°C)
Time
Height of the oxygen level
8
2 min.
1.2 cm
15
1.3 cm
25
1.5 cm
35
1.8 cm
46
1 cm
Average between trial 1 and 2
Water Temperature (°C)
Time
Height of the oxygen level
8
2 min.
1.05 cm
15
1.35 cm
25
1.75 cm
35
1.85 cm
46
1.15 cm
Experiment 2:
Trial
Test-tube no. pH Measure
Time
Oxygen Height (cm)
1
1
2 min.
1
2
3
3
3
5
3.3
4
7
2.9
5
8
0.9
Trial
Test-tube no. pH Measure
Time
Oxygen Height (cm)
1
1
2 min.
0.9
2
3
2.6
3
5
2.9
4
7
2.5
5
8
1.5
Average between trial 1 and 2:
Test-tube no. pH Measure
Time
Oxygen Height (cm)
1
1
2 min.
0.95
2
3
2.8
3
5
3.1
4
7
2.7
5
8
1.2
Experiment 3:
Trial 1
Test tube
Duration
Volume of H2O2 (mL)
Volume of water (mL)
Height of the bubble (cm)
1
2 minutes
0
10
0
2
2.5
7.5
0.7
3
5
5
0.8
4
7.5
2.5
0.8
5
10
0
0.9
Trial 2
Test tube
Duration
Volume of H2O2 (mL)
Volume of water (mL)
Height of the bubble (cm)
1
2 minutes
0
10
0.2
2
2.5
7.5
2.4
3
5
5
2.2
4
7.5
2.5
1.6
5
10
0
2.3
Average between trial 1 and trial 2
Test tube
Duration
Volume of H2O2 (mL)
Volume of water (mL)
Height of the bubble (cm)
1
2 minutes
0
10
0.1
2
2.5
7.5
1.5
3
5
5
1.6
4
7.5
2.5
1.3
5
10
0
1.6
VI. Discussion and Analysis
Experiment 1: The data result based on the experiment shows that as the temperature increases the height of the oxygen will increase, which means that the rate of enzyme activity is rapidly speeding up until it reaches a certain temperature where the height begins to decrease, meaning the rate of enzyme activity is slower. The hypothesis is incorrect, since both trials show that the height of the oxygen level does not always increase whenever the temperature increases. Seeing from the average graph, starting from 8 degree Celsius, the height of the oxygen bubble keeps on increasing constantly and steadily as the temperature increases. Then comes to the point of approximately 32 degree Celsius where the activity reaches its optimum temperature and the height of the oxygen bubble begins to drop down when the temperature is above 32 degree Celsius, indicating that the enzyme activity begins to fail. The decreasing rate of the activity is much more rapid and severe compared to the increasing rate of the activity when it is below 32 degree Celsius. This is proven by looking at the average graph. The curve before 32 degree Celsius is not as steep as the curve after 32 degree Celsius, indicating that the dropping of the enzyme activity happens much more rapidly and severely and more sensitive to the difference in temperature. This trend happens because the enzyme catalase that contain in the potato is not suitable for higher temperature, specifically above 32 degree Celsius. If compared to the secondary information, the result shows the same. Catalase enzyme is not suitable for higher temperature, in this case is more than 32 degree Celsius. The information wrote the catalyst enzyme became thermally denatured, meaning the heat energy has been able to break the bonds that cause the protein to fold, so destroying the active site. The substrate no longer has an active site with which to bind so there is little or no activity. Denaturation by heat is also irreversible and the active site is not restorable. The accuracy of this experiment remains quite high because we use ruler in measuring the height of oxygen bubble and thermometer in measuring the temperature of the environment. We also measure as accurate as possible for the hydrogen peroxide to be 4 mL in each of the test tube, before the reaction began. By starting the stopwatch the second when the potato is submerged in the hydrogen peroxide also keeps the accuracy in this experiment. However, the accuracy may be defected by some factors such as the ruler being used is only to the nearest cm, the temperature within the test tube may not be equal to the temperature within the water inside the beaker when we start the reaction, and a different eye level or perspective when measuring the height of the oxygen bubble for each reaction. The reliability of this experiment however, is high because there are two trials being performed to get closer and much more accurate result, by taking the average of both trials as the result data. But one thing that defects the reliability is the inconsistency of the data being received. For example in the room temperature of 25 degree Celsius, trial 1 shows a result of 2 cm (higher than the next result) while trial 2 results in 1.5 cm (lower than the next result). The validity of this experiment is high because there is only one independent variable being added in this experiment which is the temperature variations. Moreover, the result of this experiment corresponds with the aim of the experiment in the first place, showing how the temperature affects enzyme activities. However, there are still some factors that prevent this experiment to be highly valid. These factors are related to the factors that defect the accuracy stated above, during the experiments. Since some of the variables are not accurately measured and done, there may be some additional independent variables, causing a lowly valid experiment. So, to improve this experiment, there are some several actions needed to be done. Firstly, by using more advance measurement tools such as rulers that can measure to the nearest mm and modern test tube that can indicate the temperature within and so on, the accuracy level of the experiment may be increased. The reliability of the experiment may also increase if more trials are done and making sure that everything stay the same among each trials to keep the consistency in the experiment. The validity too can be improved if the accuracy level is improved.
Experiment 2: As you can see from the average result, the enzyme activity increases as the pH value approaches pH of 5 then it slowly decreases when it approaches 7, and finally drops severely when it approaches 8. The graph also shows that the curve remains quite high at the pH ranging from 3.5 to 6.5, and there is little change happening within this range. On the other hand, the curve drops or increases heavily when it is below 3.5 or above 6.5. This analysis proves that the enzyme catalase allows little range of pH within which it is active. From the graph also it can be obtained the optimum pH of the enzyme is approximately 4.5, in which the active site is maintained most efficiently. Therefore this result has proven that the hypothesis is correct: Enzymes are affected by changes in pH. Extremely high or low pH values generally result in complete loss of activity for most enzymes. When being compared to the second hand information, it shows that there is a difference in the result of optimum value of pH in which the enzyme catalase can best react. The internet shows it should have been a pH of 7 8, while our experiment shows a pH of 5 as the optimum pH. But, some sources also state that the result of this experiment will not always be the same which is the pH of 7 being the optimum pH, due to some several factors of difference in temperature, solution density, inhibitors, and so on. That is why lots of trials are needed to be performed to achieve the most accurate result.9 The accuracy of this second experiment is similar with the first experiment which is quite high, because we use the similar materials for conducting the experiment. One factor from this experiment that adds on the accuracy level is the use of different pipettes for different solution. One pipette for NaOH and one pipette for HCl, which prevents any contamination and distortion of the pH level. The reliability however is not as high because as the hydrogen peroxide being sucked up for test tube 1,2,3, and 4, the remaining will become less than the initial. Then more NaOH or HCl is being added. Since the concentration of hydrogen peroxide is much lesser than the first time its being mixed, there may be a distortion in the pH value of the solution, creating a less reliable experiment results. Secondly, because the experiment result of optimum pH value doesn’t match with the research, this experiment is considered to be highly unreliable, especially knowing that the difference is by 2 pH value (which is a lot). The validity of this experiment is quite high, similar to the first experiment because the result corresponds with the aim of the experiment at the very first place. The experiment also only consists of one independent variable which is the pH value of the substrate, creating a valid experiment. Similar to that of the accuracy level, if the accuracy level is not high, so does the validity because it will create more than one independent variable. Things that need to be improved in this experiment include the need of more repeated trials, especially knowing that the result is very different than what it is supposed to be according to the second-hand information. This unreliable result has become the evidence that the experiment must have been taken much more carefully and precisely. More advance tools are also needed to increase the accuracy and validity level of this experiment.
Experiment 3: Based on the final graph of the average rate of enzyme activity depending upon the concentration of substrate, it is clearly seen that as the concentration of enzyme increases the rate of reaction increases too. From 0-25%, the enzyme activity increases steadily and constantly. Then between the concentration of 25% and 50% the enzyme activity remains very constant showing how there are no increases or decreases in the rate of the activity. However, after it reaches 50% of concentration, the activity starts to slow down and increase again when it is 100% concentration. This overall creates a fluctuating curve, although not that volatile. This third experiment has produced a result that does not match with the theory of how the different concentration of substrate may affect enzyme activities, because the result should not fluctuate in a way where the enzyme activity drops in 75% of concentration. This happens in this third experiment because of a predicted reason, which is the use of different type of potatoes. In the first trial as you can see, the data of the height of oxygen bubble does not flow the way the second trial’s data does. It never drops down; it either increases or remains the same. During the second trial, another potato has to be taken because the first potato being used in the first trial has already been wholly bored, no more spaces. But there are no more potatoes of the same age as the one being used in the first trial, this time all of them looks much younger. So there is no choice but to take one of them. However, the first and fourth test tube in the second trial have the same potatoes as the first trial, leaving the second, third and fifth test tube of trial 2 with the younger potatoes. Therefore, this explains why the 0% and 75% concentration to be so low. And because of this also, we may conclude that younger potatoes can perform much faster reaction than the old ones. Due to this major problem affecting the result of the third experiment, it still cannot be said whether the hypothesis of “the more substrate added to the reaction the faster the rate of the reaction will be, but it will not be as increasing as rapid as before after reaching maximum velocity” correct or not. In this experiment, however, it is indicated that the maximum velocity will be when the concentration is around 50%. The accuracy of this experiment has the same level as the two experiments before because the instruments being used are similar. The reliability and validity of this experiment however is quite low, because of the problem being stated before, creating an inconsistent result that distorts the final result of the experiment severely. The validity has been defected also due to the existences of two independent variables, in this case, are the concentration of substrate and the type of the enzymes (potato). But a part of the experiment that is still being valid is that it once again relates to the first aim of the experiment which is to know the effect of concentration of substrate on the enzyme activity. The most important thing to be improved in this experiment is the awareness of the independent variable, meaning that the independent variable must be kept single all the time. It should not be more than two. The potatoes being used for both trials should have been the same type, or at least in one trial there should only be one type of potato being used, instead of mixing the different types of potatoes in one trial. By doing more and more trial also, this experiment can be improved furthermore.
VII. Conclusions
Experiment 1:
Enzyme activity will increase steadily as the temperature increases, but after it peaks at its optimum temperature, the enzyme activity will drop down significantly.
Enzyme catalase has an optimum temperature of 32 degree Celsius.
Enzyme catalase does not suit with temperature above 32 degree Celsius because it is when the enzyme thermally denatures and active sites being destroyed
Denaturation by heat is irreversible and active enzyme cannot be restored
Experiment 2:
Enzyme catalase is affected by changes in pH. Extremely high or low pH values generally result in complete loss of activity for most enzymes.
It allows little range of pH for the enzyme to be highly active and within this range the optimum pH is located.
Enzyme catalase has an optimum pH of 5 according to this experiment, but it is supposedly 7.
Experimet 3:
Enzyme activity will increase as the concentration of the substrate increases but then after it reaches its saturation point or maximum velocity; it will remain constant or have only a slight increase in activity.
Enzyme catalase should have reached its maximum velocity during 50% concentration of substrate.
General Conclusions:
The reaction between enzyme catalase and hydrogen peroxide will produce two harmless products of water and oxygen gas.
Enzymes are highly affected on temperature, pH, substrate concentration, enzyme concentration, inhibitors and activators
It is very important that these enzymes work in its optimum level of each factor to be able to function in each living organisms.
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Retrieved October 29, 2013, from www.preservearticles.com/2011112217708/what-are-the-factors-that-affect-the-enzymatic-activity.htmlH