Title

The effect of enzyme concentration on rate of reaction.

Objective

To investigate the effect of enzyme concentration on the rate of reaction by measuring the initial rate of reaction.

Introduction Enzyme is a globular protein that is very important to living things especially human and animals. It acts as biological catalyst which speeds up the rate of reactions. Without enzymes, the substrate might take a long time for the reaction to happen. In fact, it may end up with no reaction. Enzymes help to overcome this problem. The lock and key theory suggests that the substrate slots into the active site of the enzyme, and a reaction takes place which turns the substrate into product. As the enzyme is catalysts, it remains the same unchanged and used back for reactions. As we can see, the enzyme is very important to the body because, without it reaction will not take place. Every reaction shown in the metabolism section has a corresponding enzyme which ensures that the reaction happens. Metabolism is the combination of build-up new chemicals process known as anabolic reaction and the break substance down process known as catabolic reaction. Even though when in equilibrium, the catalyst will speed up the reaction in both directions, sometimes one enzyme is more appropriate for one direction than the other. In the body, if a reaction needs to go both forwards and backwards, then one enzyme may be used for the forward reaction and another for the backwards reaction.

Diagram 1 : the lock and key theory. Picture adapted from, emptyemptyme93.blogspot.com

An enzyme has a three dimensional shape which is very precise and detailed. The polypeptide chains of the enzyme are folded to form a cleft called active site. The active site of an enzyme has a distinctive shape and charges. So, the shape of the substrate must be a match with the active site precisely for the reaction to happen. This is the reason why enzymes are highly specific. The lock and key theory explains the way the substrate binds to the enzyme to allow reaction to take place. The substrate molecule binds to the active site to form an enzyme-

substrate complex. After reactions take place, the substrate which becomes the product will be released from the active site of the enzyme and the enzyme is free to bind to other substrates. There are few factors that affect the enzymes activities such as the pH value of the surrounding, the temperature of the surrounding, the enzyme concentration and the substrate concentration. The first factor is the pH value of the surrounding. Because of the alteration of pH, the enzyme or the active site might not able to bind to the substrate at all. This is because, when the pH increases, it means that the hydrogen ions decreased in the solution. This can lead to reactions taking place that alter the functional groups of the amino acids, which in turn leads to the enzyme changing shape. Similarly if the pH decreases, there are more hydrogen ions in solution, leading to the possibility of hydrogen ions causing other group changes. If the functional group changed, the active site will be different as the shape also changed. This may cause the substrate unable to fit the active site. When the pH changes sufficiently, the enzyme will be completely altered due to this effect, and it is said to be denatured. Extreme temperature also may affect the enzyme reaction. The way the temperature affects the enzyme reaction is the relation of the movements of particles. When the temperature increase, generally, the particles of the enzyme molecules and substrate molecules will move faster and collide with each other many times. The more they collide with each other, the more likely the substrate will slot into the active site of the enzyme leading to the product being formed. But, when the temperature gets too high, the enzyme does not respond really well as it is a biological molecule. When the temperature gets extremely high, the enzyme denatured. As we know, enzyme made up of proteins. When the temperature increases, the bonds between the functional groups of amino acid break down and change the shape of protein. When the shape changes, the active site of the enzyme changed as well causing it to be useless for enzyme reaction as substrate cannot bind to it. So, at certain temperature, the enzymes will stop working and further increase of temperature will not help the enzyme reaction. Other than that, the substrate concentration is also one of the factors. At low substrate concentrations, few substrate molecules are present. As such, there are many active sites which are available. An increase in substrate concentration means more substrate molecules are available. This means there are more chances of collision between the substrate molecules and enzyme molecules for a catalytic reaction to take place. As more substrate molecules fill the active sites, more products are formed per unit time. The increase in substrate concentration will only lead to an increase in the rate of reaction if there are enough enzyme molecules which are available to catalyse the additional substrate. But, there are limit to the rate of the reaction to happen. At a certain substrate concentration, the rate of reaction will not increase further and become constant. The reaction is at maximum rate. All active sites are filled with substrate and there are still some left, waiting to be reacted with the enzyme. The enzyme molecules are said to be saturated. So, the enzyme concentration is the limiting factor when the substrate concentration increase results the rate of reaction becomes constant.

Diagram 2 : the enzyme concentration provides a lot of extra active sites. Picture adapted from, blobs.org

The last factor is the enzyme concentration. This goes the same as the substrate concentration factor. When the enzymes concentration increases, it means that the enzyme molecules increase producing a lot of active sites. The rate of reaction will keep increasing if there are no limiting factors such as the limited substrate concentration. When the enzyme concentration increased, there are more enzyme molecules colliding about in a given volume. So, there are more enzyme molecule to perform the reaction and more free active sites. This is the factor that we are going to test in this experiment.

REPORT OF PRACTICAL
Problem Statement : How does concentration of enzyme affect the initial rate of reaction?

Hypothesis

The higher the enzyme concentration, the higher the rate of reaction, providing the substrate concentration is not a limiting factor in the reaction. Variables :

Manipulated variable Responding variable

: the enzyme concentration : the rate of oxygen gas released to determine the rate of reaction.

Fixed variable

: volume of Hydrogen Peroxide used (substrate concentration), the temperature and the buffer.

Apparatus

Blended potato (catalase ), 2.5 cm3 of Hydrogen peroxide solution (H2O2), 5 cm3 of buffer solution with pH 6.5

Material

Conical flask, dropper, spatula, delivery tube with bung, measuring cylinder, beakers and stopwatch.

Procedure

1. One spatula of blended potato is placed inside a conical flask and then 5 cm3 of buffer solution with pH 6.5 is added.

2. The mixture is swirled.

3. The measuring cylinder is filled with water and inverted carefully into a half-filled beaker. The end of the delivery tube is inserted into the inverted measuring cylinder in the beaker.

4. 2.5 cm3 of H2O2 is measured into the syringe and added into the flask.The bung is replaced in the flask immediately.

5. The stopwatch is started and volume of Oxygen collected every 30s interval is measured.

6. The experiment is repeated using 2,3 and 4 spatulas of blended potato.

7. The results are recorded in a suitable table,showing the time and volume of Oxygen collected.

8. The graph is plotted to find the initial reaction for each of the experiment.

Results

Volume of Oxygen (cm3) Time (s)

1 30 60 90 120 150 180 210 240 270 300 5.0 9.0

Enzyme concentration 1 spatula

2 6.0 11.0 Average 5.5 10.0 14.0 17.0 19.5 21.5 23.0 24.5 25.5 25.5 1 6.0

2 spatulas
2 9.0 Average 7.5 15.0 20.0 24.0 27.0 29.5 31.5 33.0 34.5 34.5 1

3 spatulas
2 Average 16.0 26.5 35.0 40.0 44.0 45.5 46.5 46.5 46.5 46.5 1 17.0 15.0 27.0 26.0 34.0 36.0 39.0 41.0 43.0 45.0 45.0 46.0 46.0 47.0 46.0 47.0 46.0 47.0 46.0 47.0

4 spatulas
2 Average 20.5 31.0 38.5 44.0 46.5 48.5 49.5 50.0 50.0 50.0 21.0 20.0 31.0 31.0 39.0 38.0 43.0 45.0 46.0 47.0 48.0 49.0 49.0 50.0 49.0 51.0 49.0 51.0 49.0 51.0

14.0 16.0 20.0 20.0 24.0 24.0 27.0 27.0 29.0 30.0 31.0 32.0 33.0 33.0 35.0 34.0 35.0 34.0

13.0 15.0 17.0 17.0 19.0 20.0 21.0 22.0 22.0 24.0 24.0 25.0 25.0 26.0 25.0 26.0

30

25

Volume of Oxygen produced ( cm3)

20

15

10

0 0 50 100 150 200 250 300 350

Time (s) Initial rate of reaction using 1 spatula of blended potato : Volume (cm3) = (18-5)cm3_ Time (100-30) s = 0.186 cm3 s-1

40

35

30

Volume of Oxygen produced (cm3)

25

20

15

10

0 0 50 100 150 200 250 300 350

Time (s)
Initial rate of reaction using 2 spatulas of blended potato : Volume (cm3) = (25-5)cm3_ Time (100-20) s = 0.250 cm3 s-1

50

45

40

Volume of Oxygen produced ( cm3)

35

30

25

20

15

10

0 0 50 100 150 200 250 300 350

Time (s) Initial rate of reaction using 3 spatulas of blended potato : Volume (cm3) = (40-5)cm3_ Time (70-10) s = 0.583 cm3 s-1

60

50

Volume of Oxygen produced ( cm3)

40

30

20

10

0 0 50 100 150 200 250 300 350

Time (s) Initial rate of reaction using 4 spatulas of blended potato : Volume (cm3) = (36-0)cm3_ Time (50-0) s = 0.720 cm3 s-1

Graph 5: The rate of reaction ( cm3/s ) against the concentration of enzymes

0.8

0.7

0.6

Rate of reaction (cm3 s-1)

0.5

0.4

0.3

0.2

0.1

0 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 Enzyme Concentration

Graph 6: Volume of oxygen gas produced ( cm3 ) against time for 1,2,3 and 4 spatulas

60

50

4 spatula 3 spatula

Volume of Oxygen produced (cm3)

40

2 spatula
30

1 spatula
20

10

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

Discussion According to the graph, the oxygen released is gradually increasing for every graph of enzyme concentration providing there are no limiting factors. The reading is taken at every 30 seconds intervals for five minutes (300 seconds). As the graph keep on increasing with the increasing amount of blended potato (the enzyme), there are more active sites available for the substrate to react becoming the products. This means, at one time, more substrate can be reacted by the enzyme. The rate of reaction of this experiment can be found by using the graph. The gradient of the graph describes the rate of reaction. Generally, the higher the enzyme concentration, the steep the gradient will be as more oxygen gas is released per seconds. In this experiment, the enzyme reaction is potrayed by the blended potato and hydrogen peroxide which act as the enzyme and the substrate respectively. The blended potato which acts as the enzyme is manipulated in this experiment. The different masses used symbolize the different concentration of enzyme. The other factors such as the temperature, the time taken (intervals), the pH (pH6.5) and the volume and concentration of hydrogen peroxide (substrate) are all kept constant as this experiment is about how the enzyme concentration will affect the enzyme activity or reaction. When the other conditions are kept constant and unchanged, theoretically, the rate of reaction will increase as the concentration of enzyme (blended potato) increased because there are more active sites available for binding. The substrate, hydrogen peroxide is a used to be reacted with the enzyme to release oxygen gas. Since the concentration of hydrogen peroxide has to be kept constant throughout the experiment, the bottle has to be always capped so that the hydrogen peroxide will not react with the air and decomposed. If this happen, the concentration of the hydrogen peroxide will change, thus affecting the result. Other than that, the condition of the buffer is important as well. The buffer must be in a suitable pH so that the reaction can take place. The volume of the buffer also must be the same throughout the experiment. Besides that, rather than using normal plain cubic potato, the potato is blended so that the concentration is uniform and standardized. It is easier for us to determine or to differentiate the concentration of enzyme. It also provides a large total surface area allowing the reaction to take place faster so, less time is needed for us to determine the reaction or responding variable. Based on the result produced, we can see that the highest rate of reaction is produced when 4 spatulas are used as the hypothesis stated since 4 spatulas of blended potato provide more active sites at once to bind with the substrate. The lowest is when 1 spatula of blended potato is used. As the concentration of enzyme is increased, the volume of oxygen produced in every 30 seconds also increase. Hence, it can prove that the higher the concentration of enzyme used in a reaction, the faster the rate of reaction.

Limitation There are some limitations in this experiment. One of it comes from the blended potato. As we know, blended potato is easily oxidized when in contact with air. Since the experiment took quite a long time because of the repetition, the blended potato may be already oxidized when we are doing the next step for different number of spatula. When the potato is oxidized there are some oxygen gas released from the oxidization. This really affects the result. The oxidized blended potato can be seen by the changes in the color of the potato. It changed from pale brown to dark brown. Next, the problem always comes when we need to replace the bung at almost the same time as we pour the hydrogen peroxide into the mixture of buffer and blended potato. As we know, it is almost impossible to replace the bung at almost the same time. So, some of the earlier reaction producing oxygen may be released out into the surrounding causing the result is not really reliable. Even though there are repetitions, the result will still be the same as we keep on making the same mistake. Another limitation is parallax error. Although we tried very best in making sure the reading is as accurate as it can be, parallax error still may occur because it is hard to read the reading or the meniscus on the measuring cylinder as the light is slightly refracted by the water making the numbers or the scale on the measuring cylinder hard to be read.

Source of error and ways to overcome Errors also happened in this experiment. By the time we put in the blended potato, some of the residue might be stuck on the conical flasks wall. This will affect the reading or the reaction as not all enzymes are used at the same time. But then, the reading will suddenly rises when the residue left slowly move down to the bottom of the conical flask producing more active sites at once than at the beginning of the experiment. By using the buffer to wash away the residue left on the wall of the conical flask, all the blended potato will be present when the reaction occurs giving a more accurate and reliable results. Other than that, the beaker used to occupy the measuring cylinder is quite small. So, the measuring cylinder was slanted and has to be held all the time. This result in difficulty when taking the reading of the volume of oxygen released. By using a bigger beaker, it will be easier for us to take the reading and also to invert the measuring cylinder into the beaker containing water. Because of the reaction sometimes are so fast and ongoing, the reading taken at the specific time might be slightly affected as there are some extra time taken to read the volume. This may cause unreliable and inaccurate result. Since the reading is hard to be taken due to confusion while taking the reading, using a dyed water solution will help. Besides that, sometimes there is air bubbles trapped inside the measuring cylinder during inverting it into the beaker with water. So, zero error had occurred. To avoid this from happening, we have to be extra careful when inverting the measuring cylinder.

Safety precaution The laboratory coat must always be wear during the experiment. This is because to avoid the blended potato form staining the clothes. Other than that, we should be extra careful when handling the glassware such as the measuring cylinder, beakers and the delivery tube. This is because they are fragile and may cause injuries when broken. Besides, the hydrogen peroxide is a corrosive substance if concentrated. So, we must be very careful when using it. Use eye goggle and wear protections such as the proper shoes, and laboratory coat. Do not play or do any attempt to swallow it. We also must wipe and clean the respective area if any spills happened. In addition, the blended potato cannot be consumed in the laboratory as it may be contaminated by the chemicals in the lab.

Further studies Since this experiment is about what affect the rate of reaction of the enzyme, there are a lot of things to be investigated. For example, the pH value of the solution, the temperature of the solution and the substrate concentration. It will be very interesting to investigate on how the pH value affects the enzyme activities. Since we learnt that, theoretically the enzyme will changes shape when exposed to unsuitable pH value, doing experiment on this factor will helps to explain more about the reasons.

Conclusion When the concentration of enzyme is increasing, the rate of reaction will keep increasing if there are no limiting factors such as the limited substrate concentration. The hypothesis is accepted.

References Edexcel Biology For AS, Hodder Education, C.J Clegg, 2008. Edexcel AS Biology, Pearson Education Limited, 2008.
http://www.blobs.org

http://en.wikipedia.org/wiki/Enzyme_kinetics