TITLE: EFFECT OF ENZYME CONCENTRATION ON THE RATE OF REACTION OBJECTIVE To study and correlate what is the effect of enzyme

e on the rate of reaction. This experiment used enzyme (smashed potato as catalyze) on rate of decomposition of Hydrogen Peroxide produce water and oxygen by measuring the initial rate of reaction. To develop experimental and investigate skills. PROBLEM STATEMENT What is the effect of enzyme concentration on the rate of reaction?

HYPOTHESIS The higher the enzyme concentration used (smashed potato) the higher the rate of reaction until it reaches a point where the entire enzyme is saturated. Beyond this point, the rate of reaction remains the same, provided that the amount of substrate (Hydrogen Peroxide) used is constant. NULL HYPOTHESIS Enzyme concentration of catalase makes the reaction of decomposition of Hydrogen Peroxide slower. VARIABLES MANIPULATED : Enzyme concentration ( no of spatula full of blended potato used ) RESPONDING : Initial rate of reaction ( the gradient of the rate of reaction graph ) CONSTANT : Volume of hydrogen peroxide, H2O2 used Temperature in which the experiment was conducted. APPARATUS Beaker, graduated tube, bung stopper, conical flask, dropper, spatula, measuring cylinder, delivery tube, stopwatch. MATERIALS Distilled water, buffer solution of pH 6.5, hydrogen peroxide, H2O2 solution, blended potato, pair of gloves

TIME,s 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300

Volume of oxygen gas produced,cm3 0 1.8 3.8 6.0 8.0 9.5 11.8 13.6 15.6 18.6 21.6 23.6 25.0 26.6 28.4 28.6 29.2 29.6 30.0 30.6 31.2 31.4 31.6 32.0 32.2 32.8 32.8 32.8 32.8 32.8 32.8

Table 1.2: shows the results after 2.5cm3 of hydrogen peroxide is placed into the conical Flask containing 2 spatula of blended potato mixed with 5cm3 buffer solutions.

VOLUME OF OXYGEN GAS COLLECTED AGAINST TIME

VOLUME OF OXYGEN GAS COLLECTED, cm3
35 30 25 20 15 10 5 0 0 50 100 150 TIMES,s 200 250 300 350

Graph 1.2 shows the volume of oxygen gas produced when 2.5cm3 of hydrogen peroxide is placed into the conical flask containing 2 spatula of blended potato mixed with 5cm3 buffer solutions.

Initial rate of reaction = gradient of tangent = volume of oxygen gas collected ( cm3) Time (s) = 15.5 - 0 80 - 0 = 0.19 cm3s-1

VOLUME OF OXYGEN GAS COLLECTED, AGAINST TIME

40 VOLUME OF OXYGEN GAS COLLECTED,,cm3 35 30 25 20 15 10 5 0 0 50 100 150 TIME,s 200 250 300 350

Graph 1.3 shows the volume of oxygen gas produced when 2.5cm3 of hydrogen peroxide is placed into the conical flask containing 3 spatula of blended potato mixed with 5cm3 buffer solutions.

Initial rate of reaction = gradient of tangent = volume of oxygen gas collected ( cm3) Time (s) = 4.5 - 0 20 - 0 = 0.23cm3s-1

Times,s 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300

Volume of oxygen collected,(cm3) 0 2.9 4.9 6.8 8.7 11.2 16.2 19.2 20.6 23.4 25.6 27.6 29.6 30.6 31.6 32.8 33.4 34.6 35.2 36.3 36.3 36.3 36.3 36.3 36.3 36.3 36.3 36.3 36.3 36.3 36.3

Table 1.3: shows the results after 2.5cm3 of hydrogen peroxide is placed into the conical Flask containing 3 spatula of blended potato mixed with 5cm3 buffer solutions.

Result

Times(s) 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300

Volume of oxygen gas,O2 collected(cm3) 0 1.4 2.6 4.4 6.0 7.6 9.4 10.2 11.4 12.4 13.8 14.8 16.0 17.0 17.6 18.2 19.2 19.6 20.0 20.8 21.6 22.2 22.6 23.2 23.8 23.8 23.8 23.8 23.8 23.8 23.8

Table 1.1: shows the results after 2.5cm3 of hydrogen peroxide is placed into the conical Flask containing 1 spatula of blended potato mixed with 5cm3 buffer solutions.

Volume of oxygen content against time

30

25

Volume of Oxygen gas,O2 collected(cm3)

20

15

10

0 0 50 100 150 200 250 300 350 Time(s)

Graph 1.1 shows the volume of oxygen gas produced when 2.5cm3 of hydrogen peroxide is placed into the conical flask containing 1 spatula of blended potato mixed with 5cm3 buffer solution.

Initial rate of reaction = gradient of tangent = volume of oxygen gas collected ( cm3) Time (s) = 7.5 - 0 50- 0 = 0.15 cm3s-1

concentration of enzyme catalase/ number of spatula 1 2 3

Initial rate of reaction(cm3s-1) 0.15 0.19 0.23

Table 1.4 shows the increases in number of spatula of blended potato show the increase in number of enzyme concentration lead to increase in initial rate of reaction.

0.25 INITIAL RATE OF REACTION,cm3s-1 0.2 0.15 0.1 0.05 0 0

INITIAL RATE OF CONCENTRATION AGAINST CONCENTRATION OF ENZYME CATALASE

0.5

1.5

2.5

3.5

CONCENTRATION OF ENZYME CATALSE/NUMBER OF SPATULA

Graph 1.4 shows the increases in number of spatula of blended potato show the increase in number of enzyme concentration lead to increase in initial rate of reaction.

DISCUSSION This experiment investigates about the effect of concentration of enzyme catalase (smashed potato) reaction decomposition of Hydrogen Peroxide. As we know before the main function of the enzyme is acts as catalyst which speed up the rate of reaction. In this experiment character of enzyme catalase take over by smashed potato which breakdown the decomposition of the Hydrogen Peroxide which converts to the water and Hydrogen gas. The catalase acts as lowering activation energy to make reaction faster as usual and give effect to Hydrogen Peroxide to gain more energy to break the bond in order to overcome the high energy in beginning of reaction. This vice versa without reaction which most probably reaction occurs slowly in the decomposition of the Hydrogen Peroxide. So in the experiment to achieve main objective we manipulating the enzyme concentration by using different number spatula of enzyme catalase( smashed potato) to get initial rate of reaction. Then initial rate of reaction is calculated based on the tangent of volume of oxygen gas collected against time. Based on the table 1.1 and graph 1.1 we can see that there is positive linkage between the data in this experiment. We can observe from the graph plotted, the gradient of the graph is increasing and steeper at 0 s until 60 seconds and regularly decreasing as the time increased. This is because when one spatula of the smashed potato is used and there is abundant of Hydrogen Peroxide which is not all reacted. As we look back, the catalase react to speed up the reaction of decomposition of Hydrogen Peroxide, the amount of Hydrogen Peroxide will decrease as time taken increased. As a consequences, the when the molecules of Hydrogen Peroxide decreased so, the volume of the oxygen gas collected also decreased. This reason why the gradient of the graph decreasing together with increasing time. Other than that we can see the initial rate of reaction recorded was 0.15 cm3s-1.Then we can see that the final volume of the oxygen gas collected is 23.8 is lower compared to the other spatula of the experiment. This because less Oxygen produce due to the low concentration of enzyme catalase. Based on table 1.2 and graph 1.2, also show positive correlation of the data between the oxygen gas collected and the time. The gradient of the graph at the first 80s is the highest when compared to the next seconds. This shows, the rate of the reaction is the highest at the first 80s compared to first spatula but different in time for first experiment which is time taken is 60s. The gradient of the curve start to decrease after the 80s. In the time from 1s to 80s, the catalase is decomposing the hydrogen peroxide molecules. The number of hydrogen peroxide molecule is decreasing lead to decrease in oxygen gas production. Thus, lead to decrease in rate of reaction. Then the final oxygen molecule produced is the higher compared to the first experiment which is 32.8 cm3 and this shows more molecules of oxygen gas can be produced from the decomposition as the concentration of catalase increased. Based on table 1.3 and graph 1.3, the graph also shows positive correlation between the volumes of oxygen gas collected by time. The first point that located on the straight line (tangent of the graph) indicates the initial rate of reaction. Based on the graph, we can see as

the time increase, the point is getting further away from the straight line. This shows the gradient of the curve is decrease as the time increase. The initial reaction is steeper at point 0 to 20 s is more steeper compared to the experiment using 1 spatula and 2 spatula. The Final volume of oxygen collected is the highest in the experiment because the concentration of the catalase (smashed potato) used is the highest and more oxygen molecule produced as product of decomposition. Based on the three graphs, there are many similarities we can see obviously while conducting the experiment. Firstly, the initial rates of reactions for the graphs are always higher. In this experiment, concentration of substrates is kept constant by using the same volume of hydrogen peroxide which is 2.5 cm3.Besides that, the rate of reaction also decrease in all three graphs. As potato acts as catalyst and Hydrogen Peroxide acts as substrate, the catalase will decompose one molecule of Hydrogen Peroxide to produce one molecule of water and one molecule of oxygen gas. As the unique function of catalase can be used up again in the next reaction of decomposition to produce bundle of product again.So we can see that the concentration of catalase in experiment remains constant whereas the concentration kept decreasing through time running. If we can see softly, we can see some confusing look about the data in this experiment. We use the same volume and concentration of substrate in each experiment and we suppose to get same final volume of oxygen collected in each experiment. Unfortunately in each experiment reading recorded is varies and It can be cause by the error made by student. When the student gets constant value or there is no air bubbles after ten second, they conclude that the reaction had finished. Other than that, another characteristic we can see varies in this experiment are the initial rate of reaction in each experiment. As in table 1.4 and graph 1.4 shows that the higher concentration of catalase (3 spatula of smashed potato) has highest initial rate of reaction (0.15cm3s-1) compared with 1 spatula of smashed potato has lowest initial rate of reaction (0.23cm3 s-1).whereas 2 spatula of smashed potato indicate reading of 0.19cm3s-1).This suited and explained very well how precise the hypothesis as when concentration of the catalase increased, the initial rate of reaction also increased. This theory can be explained by restating on increasing the enzyme concentration increase rate of collision between the molecules, in same time frequency of effective collision also increase, thus rate of reaction also increased. But the increasing rate of reaction is temporary as when the concentration of substrate becomes limiting factor, the rate of reaction will be decreased and finally become constant.

EVALUATION There are few possible errors that I can detect while I am conducting throughout this experiment. Firstly, there are some air bubbles in the inverted graduated tube. This is due to error when inverting the tube into the beaker containing water. The bubbles cause the results to be less accurate. Other than that, the amount of blended potato in a spatula full is not accurate. The weakness of using Spatula is not an appropriate tool to measure substance as it has no scale. Hence, the amount of blended potato in a spatula full is not in fixed volume, so the enzyme concentration is varied from original. As consequences this result in inaccuracy of the results and affects its reliability. Next, there is some oxygen bubbles formed under the meniscus level when the reading was taken. The bubbles affect the meniscus shape, making it hard for me to take the reading. Making it worst, There is the rapid increase in rate of reaction and short time interval( 10 second each) causes hurry to take reading and impossible for me to take the reading properly and accurately. This causes the results to be less accurate and reliable. LIMITING FACTOR Certain limiting factors that can be observed from this experiment are the oxidation of potato and the insufficient calibration of graduated tube. After the potato was blended, it was left in an opened beaker as another procedure was carried out. When left in open air, the blended potato become oxidised, causing the enzyme concentration become affected in a way. This is afraid to be the cause of inaccuracy of the results obtained. Besides, the rapid reaction of the experiment causes the oxygen gas formed to flow really fast. The water in the graduated tube decreases rapidly in short time. This causes the time taken for experiment 3 spatula full of blended potato to differ from the other two experiments because the water in the tube already finished when the time reached 80 second instead of 250 seconds in the other experiments. The graph plotted cannot be compared directly as the scale on each graph differs. This makes it hard for showing a comparison of the initial rate of reaction of the graphs.

SAFETY PRECAUTION There are some precautions that should be taken notice in this experiment which involves usage of harmful chemicals. First of all, wear a lab coat to prevent any stain on the clothes. Next, wear a pair of rubber gloves and closed shoes to protect the hand and foot from corrosives chemical which is the hydrogen peroxide solution. If the skin is in contact with the solution, quickly wash the part in contact with plenty of water. Other than that, handle the apparatus with care to prevent any breakage. Report any accidents to the lecturer or the lab assistant so the administration is aware of the students welfare.

IMPROVEMENT From the experiment, it can be seen there are a few weaknesses that can affect the results. Hence, we need to improve on how to conduct the experiment so that accurate results can be obtained. The first cause of the inaccurate results is the oxidation of blended potato in the open air. We can avoid this by covering the beaker containing blended potato with a flat object such as books and even filter paper can be used. Next, the cause of inaccuracy results is insufficient calibration on the graduation tube. This can be improved by using bigger scale graduation tube or as simply as using a big measuring cylinder. However, please remember, if you use measuring cylinder, calculation would be needed because the scale is inverted in this experiment. Other than that, random error such as parallax error is common in experiments which require taking of readings. Thus, to solve this problem, ensure that the eye position is perpendicular with the calibration when taking the reading. Dont forget that the reading is at the lower part of the meniscus. Another problem is the calibration cannot be read properly due to lighting. This problem can be overcome by placing a plain coloured paper behind the graduated tube so that the calibration can be seen clearly. The short time interval of 10 seconds makes it hard to take the reading. To have more accurate results, use longer time interval such as 30 seconds. The use of spatula to determine the enzyme concentration also leads to inaccuracy of the results. This is because the spatula is not a measuring tool and it cannot gives a fixed amount of the substance it holds. Thus, to overcome this problem, use syringe or a weighing scale to measure out the amount of blended potato used. The presence of air bubbles in the graduation tube also affects the results of the experiment. This can be overcome by carefully inverting the graduated tube into the beaker containing water. When the oxygen gas come out from the delivery in form of bubbles, it goes into the bottom of the inverted graduation tube. However, some of the bubble do not pop out and remain intact with the meniscus. This makes it hard to take the reading. To overcome this problem, shake the graduation a bit so that the bubble can pop out and do not affect the reading. Make sure that when shaking the tube, it does not affect the conical flask or more oxygen gas would come out of the delivery tube rapidly and will cause another inaccuracy in the results of this experiment.

FURTHER STUDY Enzyme is a protein which acts as a biological catalyst. It speed up the rate of reaction why lowering the activation energy. Enzyme are highly specific which mean only one substrate can bind with specific enzyme. It have active site which the part of substrate binding. Enzyme work optimumly at certain pH and temperature. Substrate concentration also affects the rate of reaction.

The pH also has a great effect on enzyme activity. Certain enzymes need certain pH to work optimum. Pepsin work optimum at acidic condition where the pH is about 3.5. trypsin work optimum at alkaline conditions where the pH is about 8. Different enzymes work at different pH because of the particular arrangement of weak bonds which holds their shape together. But too acidic or too alkaline solution will disrupt the formation of the hydrogen bond and sulphur bridges that hold together the three-dimensional structure of the protein. Thus, the rate of reaction will be decrease. Temperature effect the activity of enzyme. Enzyme work optimum at body temperature, 37`C. Any changes of the temperature will cause the changes in enzyme activity. If the temperature decreases from the body temperature, the rate of reaction is also decrease. This is due to the decrease in heat energy that cause decrease in kinetic energy between the enzyme and substrates. So, the frequency of collision between the enzyme and the substrate is also decrease. Less substrate is bind to the active site of the enzyme. So, the product form is also decrease, the rate of reaction decrease. As the temperature increase beyond the body temperature, the bonds such as hydrogen bond, ionic bond are disrupted resulting in change of the enzyme structure. The active site will also change which prevent it from catalyzing a substrate. Any increase in temperature at 60`C, the enzyme is denatured. This is because almost the entire bond that holds the 3D structure of the enzyme is destroyed. And the reaction stop completely as the enzyme loses its ability to catalyzed reaction. Substrate concentration also affects the enzyme activity. When the concentration of substrate increase, the rate of reaction is also increase. This is due to increase in frequency of collisions between the substrates molecules and the enzymes increase. But the rate of reaction will remains constant at some point, as all enzyme active site are full with substrate molecule. This means, enzyme concentration becomes a limiting factor, which means any increase in substrate concentration does not increase the rate of reaction. Another substance that can affect the activity of enzyme is inhibitor such as sarin. Present of inhibitor decrease the rate of reaction. There are two types of inhibitors, competitive and non-competitive inhibitor. For competitive inhibitors, the inhibitors bind at the active site, competing with the substrate for the active site. This reduces the productivity of enzymes by blocking substrates from entering active site. As for noncompetitive inhibitor, it does not bind to the active site, but bind to another part of the enzyme. It alters the shape of the enzymes. Even though the substrate still can bind, but the active site function less effectively.

Conclusion
As the concentration of catalase increase, the initial rate of reaction is also increase. Thus, as the concentration of enzyme increase, the initial rate of reaction increases. The hypothesis is accepted. REFERENCE http://www.h2o2-4u.com 23 September 2011 http://www.newworldencyclopedia.org/entry/Hydrogen_peroxide - 22 September 2011 http://h2o2.com/products-and-services/us-peroxide-technologies- 24 September 2011 http://www.nlm.nih.gov/medlineplus/ency/article/002353.htm - 25 September 2011 http://www.worthington-biochem.com/introbiochem/enzymeconc.html - 25 September 2011 http://encyclopedia2.thefreedictionary.com/ENZYME+STRUCTURE+AND+FUNCTION - 25 September 2011 http://www.anyvitamins.com/enzymes-info.htm - 25 September 2011 24 September 2011

http://www.chemguide.co.uk/physical/acidbaseeqia/buffers.html - 24 September 2011 http://www.ebi.ac.uk/interpro/potm/2004_9/Page2.htm - 24 September 2011

NAME GROUP STUDENT ID LECTURERS NAME

: ABDUL RAHMAN BIN MOHAMED : 12M13 : 2011894958 : MR.MOHD HAFIZ BIN MOHD ROTHI

DATE OF SUBMISSION: 27 September 2011

INTRODUCTION What is enzyme?

Enzyme is well known for its function in our metabolism. Enzyme is one of the biological catalysts. The term enzyme origin from zymosis, the Greek word meaning fermentation process performed by yeast cells and well known to the brewing industry. Enzyme also complex protein that will make all the part in our body experienced specific change and facilitating most of the body's metabolic processes We can take example from our daily life activity which from our food that we consume every day. The food is one complex molecule and needed to broke down into simpler molecule before can be absorbed, so the enzyme function is breakdown the food that we take. Enzyme also functional on other things such as for supplying energy, free our body from waste product, in our digestive system and purifying blood also done by enzyme. They also balance the triglyceride and cholesterol levels and strengthen the endocrine system by supplying the required hormones. At no time and in no way can natural enzymes harm the body. Enzymes are found in body such as in saliva (mouth), gastric juice (stomach) and other part of the body Enzyme is made up of protein. Protein enzyme is made up from one or more amino acid chains called polypeptides chain. The amino acids sequences determine the folding pattern of the protein structure which essential to enzyme specification. Enzyme is made up from the tertiary structure of protein. With enzyme as globular protein, so many chemical reactions are under the enzyme. Enzyme function as useful catalyst which trigger the process of chemical reaction without altering and destroyed at the end of the reaction, so it can be used up again in the next reaction. For example we take decomposition of hydrogen peroxide is definitely slowly without catalyze but with catalyse rate of decomposition of Hydrogen Peroxide ,increased rapidly 40 million per molecule. That`s fact show how superb the enzyme effect on rate of reaction. Another one more fact that more important about enzyme is about the unique 3D shape which make the enzyme more specific which is only certain substances can work with enzyme. This can be explained more in the `lock and key which definitely tells that the substrate must be fit to the enzyme. Enzyme working mechanism by bind itself to the substrates molecule called as enzyme-substrate complex. Then enzyme substrate complex molecules will breakdown releasing its product and the enzyme molecules will used back again to produce new enzyme substrate molecules. Enzyme and substrate that fail to bind if their
shape is not suit each other. This ensures that the enzyme does not participate in the wrong reaction.

Enzyme also categorized into two large groups which they are from cofactor and non protein component called prosthetic group Cofactors always related to enzyme. Cofactors are small

molecules which necessary to complete the catalytically structure of enzyme. Enzyme without cofactor called Apoenzyme while Holoenzyme is Apoenzyme cofactor complex. In other hand coenzyme (other name of prosthetic group) is organic molecule usually found as vitamin deravitative or in metal ion. The function of coenzyme is carrier of group transferred from one substrate to another. It also interacts directly in catalytic reaction. Some coenzyme tightly binds to the protein and in some cases coenzyme dissociate readily. As fact, coenzyme and apoenzyme can`t separated which can affect effectiveness of the Holoenzyme.By mixing them together the fully active holoenzyme can be arranged. The same coenzyme gives different catalytic reaction which in every reaction it depends on nature of the apoenzyme in order to determine the specificity of the reaction As molecule react they become unstable, high energy intermediates, but only momentarily, while in transition state. Effectively, the products are formed immediately. The minimum amount of energy needed to raise substance molecules to their transition state is called the activation energy.This is the energy barrier that has to be overcome before the reaction can happen. Enzymes work by lowering the amount of energy required to activate the reacting molecules. The products have a lower energy level than the substrate molecules.
Hydrogen Peroxide Hydrogen Peroxide is water like liquid that has a lot of application. Hydrogen Peroxide is made up of two Hydrogen atoms and two oxygen atoms. With extra oxygen in the Hydrogen Peroxide compared to water molecules, make it most powerful oxidiser. Hydrogen Peroxide good in kill bacteria, some viruses and fungi on surfaces. This experiment use enzyme known as catalase, it is a commonly founded enzyme in almost all of living organisms that are exposed to oxygen . It is a redox enzyme which contains heme groups. It can be found in peroxisomes (organelles in eukaryotic cells) . Let us see how the enzyme works. As we all know, enzymes have specific active sites that work only for certain reactions. For catalase, it works in the decomposition of hydrogen peroxides, H2O2 to form water and oxygen .

2H2O2

2H2O + O2

Catalase decomposes the H2O2 rapidly in two steps . First, a H2O2 molecule binds at the active site of catalase and broken aparts. From the breaking of H2O2 molecule, one oxygen atom is

extracted, then attached at a porphyrin heme (iron) and a molecule of water is released from the reaction. H2O2 H2O + O

The second step is the same with step one. The only difference is the oxygen atom in the second H2O2 molecule binds with the iron-bound oxygen atom, forming an oxygen molecule, O2 or we call as oxygen gas. The reaction is very fast because of the structure of catalase molecule which consists of four heme groups that is required in the decomposition of the H2O2 molecules. Catalase in human body works at optimum pH between 6.8 and 7.5. Other catalases work optimally in different pH and temperature according to the variation of species. Other than that, the structure of catalase is very rigid and stable; make it resistant to unfolding, thus, more resistant to pH, thermal denaturation compared to other enzymes. Now, let us go through about the hydrogen peroxide, H2O2 solution. The solution is an acidic solution which usually has pH 6.2. However, it can go lower until 4.5 when diluted at 60%. H2O2 is the simplest peroxide (a compound with one oxygen-oxygen single bond) and a powerful oxidizing agent. Buffer solution is an aqueous solution which consist a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. Buffer solution is used to keep the pH at nearly constant value in a wide variety of chemical application. Most of living organisms have relatively small pH range. One example of buffer solution is blood. Buffer solution is very necessary to keep the correct pH value for enzyme to work at optimum. In this experiment, we used buffer solution with pH value of 6.5 which is the optimum temperature for the catalase to catalyze the decomposition of hydrogen peroxide to water and oxygen.