Enzyme Concentration

8/3/2019 Enzyme Concentration

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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 andanimals. 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 thesubstrate 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 itreaction 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 ascatabolic 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 ofan enzyme has a distinctive shape and charges. So, the shape of the substrate must be a matchwith 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-
http://www.blobs.org/science/article.php?article=24#3http://www.blobs.org/science/article.php?article=35#1http://www.blobs.org/science/article.php?article=35#1http://www.blobs.org/science/article.php?article=24#3


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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 thesurrounding, 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 leadto 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 thefunctional 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 movefaster 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 beingformed. But, when the temperature gets too high, the enzyme does not respond really well as it isa 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 shapechanges, 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 substrateconcentrations, few substrate molecules are present. As such, there are many active sites whichare 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 theactive 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 tohappen. 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 concentrationincrease results the rate of reaction becomes constant.


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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 ifthere 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.


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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 : the enzyme concentration

Responding variable : 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.


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Procedure

1. One spatula of blended potato is placed inside a conical flask and then 5 cm3 of buffersolution 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-filledbeaker. 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 isreplaced 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 Oxygencollected.

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


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Results

Time

(s)

Volume of Oxygen (cm3)

Enzyme concentration

1 spatula 2 spatulas 3 spatulas 4 spatulas

1 2 Average 1 2 Average 1 2 Average 1 2 Average

30 5.0 6.0 5.5 6.0 9.0 7.5 17.0 15.0 16.0 21.0 20.0 20.5

60 9.0 11.0 10.0 14.0 16.0 15.0 27.0 26.0 26.5 31.0 31.0 31.0

90 13.0 15.0 14.0 20.0 20.0 20.0 34.0 36.0 35.0 39.0 38.0 38.5

120 17.0 17.0 17.0 24.0 24.0 24.0 39.0 41.0 40.0 43.0 45.0 44.0

150 19.0 20.0 19.5 27.0 27.0 27.0 43.0 45.0 44.0 46.0 47.0 46.5

180 21.0 22.0 21.5 29.0 30.0 29.5 45.0 46.0 45.5 48.0 49.0 48.5

210 22.0 24.0 23.0 31.0 32.0 31.5 46.0 47.0 46.5 49.0 50.0 49.5

240 24.0 25.0 24.5 33.0 33.0 33.0 46.0 47.0 46.5 49.0 51.0 50.0

270 25.0 26.0 25.5 35.0 34.0 34.5 46.0 47.0 46.5 49.0 51.0 50.0

300 25.0 26.0 25.5 35.0 34.0 34.5 46.0 47.0 46.5 49.0 51.0 50.0


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Initial rate of reaction using 1 spatula of blended potato :

Volume (cm3) = (18-5)cm3_

Time (100-30) s

= 0.186 cm3

s-1

0

5

10

15

20

25

30

0 50 100 150 200 250 300 350

VolumeofOxygen

produced(cm3)

Time (s)


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Initial rate of reaction using 2 spatulas of blended potato :

Volume (cm3

) = (25-5)cm3

_Time (100-20) s

= 0.250 cm3 s-1

0

5

10

15

20

25

30

35

40

0 50 100 150 200 250 300 350

VolumeofOxyge

nproduced(cm3)

Time (s)


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Volume (cm3) = (40-5)cm

3_

Time (70-10) s

= 0.583 cm3

s-1

0

5

10

15

20

25

30

35

40

45

50

0 50 100 150 200 250 300 350

VolumeofOxygen

produced(cm3)

Time (s)


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Volume (cm3) = (36-0)cm

3_

Time (50-0) s

= 0.720 cm3

s-1

0

10

20

30

40

50

60

0 50 100 150 200 250 300 350

VolumeofOxygen

produced(cm3)

Time (s)


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

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Rateofreactio

n(cm3s

-1)

Enzyme Concentration


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Graph 6: Volume of oxygen gas produced ( cm3

) against time for 1,2,3 and 4 spatulas

0

10

20

30

40

50

60

0 50 100 150 200 250 300 350

VolumeofOxygenproduce

d(cm3)

Time (s)

4 spatula

3 spatula

1 spatula

2 spatula


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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 30seconds 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 reactedby 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 thedifferent 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 theexperiment, 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. Thebuffer 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 takeplace 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 atonce 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.


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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 experimenttook 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 frompale 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 occurbecause 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 thereaction 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 easierfor 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 themeasuring 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.


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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 whenhandling 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 anyattempt 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 thesolution 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 increasingif there are no limiting factors such as the limited substrate concentration. The hypothesis is

accepted.


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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
http://www.blobs.org/http://www.blobs.org/http://www.blobs.org/

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Enzyme Concentration

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