Concentration Of Substrate On Rate Of Enzymic Reaction Biology Essay

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Enzymes are very efficient natural biological catalysts; they speed up chemical reactions in living organisms. They are large protein molecules which are made up of long chains of amino acids. The shape of the enzyme is determined by the shape of the amino acid chain - the number of amino acids. The shape of an enzyme is the key feature to how it works. Depending on the enzyme, some may break down large molecules to smaller ones and some enzymes join two molecules together. How enzymes work can easily be portrayed as a key fitting into a lock. Enzymes are specific in their purpose just like each key has its own lock. Molecules will only fit into the active site of an enzyme if they are the right shape and the right chemical composition. A key will only work in a lock if it is the right shape.

Without enzymes, our digestive system wouldn't be able to digest our food, our muscles, nerves and bones would not work properly. Enzymes speed up reactions without the body temperature being raised. One way of speeding a reaction up is to increase the temperature, this causes particles and molecules to move faster due to the increase in energy. Collisions will happen more often and far quicker. The problem with raising the temperature of the body is that body cells will die. This is why enzymes are the best option and why most living things cannot function without enzymes. We rely on enzymes for our survival. The shape of an enzymes active site can be changed by pH and temperature. Each enzyme has an optimum pH at which it works most effectively. The enzymes in the stomach work best at high level of acidity. They would denature in a highly alkaline environment.

A catalyst is any substance which causes a chemical reaction to occur faster by lowering the activation energy, without its physical and chemical properties being changed. A catalyst can be used over and over again in a chemical reaction: it does not get used up. Enzymes are very much the same except that they are easily denatured by a high level of heat. This is why are body temperature is 37°c because the enzymes work best at that temperature without denaturing.

This graph shows how the activation energy of a reaction is decreased with a catalyst. The products will react more readily and quicker. Catalysts are used in industry because of this property they can reduce to heat at which a reaction takes places saving money and eliminating safety worries.

In my investigation, I am going to look at how the concentration of the substrate affects the rate of reaction. I will look at the reaction rate at different percentages of substrate (H2O2) from 100% down to 10%. I will keep the same quantity of enzyme (homogenised celery) to ensure that reliable results are gained. I have chosen to investigate the concentration of the substrate because I discovered that it is the most controllable experiment with regard to the variables involved and to see how big the change in reaction rate is. I found that investigating how temperature affects reaction rates was hard to carry out due to controlling the temperature and the fact that it was very time consuming. I also found that there were similar problems while trying to investigate the change in pH on reaction rates. It was hard to monitor the readings accurately and the temperature needed to be controlled. After this preliminary work, I decided that I would experiment with the concentration of the substrate because of the reasons I gave above.

I can't include preliminary results from the first attempt of my experiment due to fact that I had too large a quantity of enzyme and a measuring cylinder that was far too small. My second preliminary experiment results are shown below. This second preliminary experiment was after I had made adjustments from the mistakes in my first experiment. In this experiment I was using 2.5ml of the enzyme which was homogenised celery instead of the 5ml I was using before. I also increased the time interval between recordings from 15 seconds to 20 seconds as I found that it was very hard to record the data with such a short time interval. The measuring cylinder I was using in the first experiment was not big enough to hold all the gas being produced; it went off the scale so I used a bigger measuring cylinder.

The only drawback from using a bigger measuring cylinder was that my results would not be as precise as they had been before. I carried out the experiment three times to eliminate the effect of any outliers and to increase reliability of results.

This was at 20°c just as all the other experiments were.

The equipment I used:

1 conical flask

1 large measuring cylinder

1 delivery tube

1 water bath

1 stopwatch

3 syringes - pH 7 buffer, Hydrogen peroxide, 2.5mm³ enzyme (extract of celery juice)

1 clamp stand

1 clamp

Method of Experiment

I set up the experiment just as the diagram below shows. I filled a water bath with water and made sure that the water was at 20°C. I set up a clamp stand next to the water bath to hold the measuring cylinder in place for the experiment. I filled the measuring cylinder with water ensuring that no air bubbles were prevalent. I then got my conical flask, not the test tube that is shown in the diagram and made sure that it was not contaminted by washing and drying it. I then collected my homogenised celery, the pH 7 buffer and the hydrogen peroxide that I would be using as my substrate in the reaction. To add the both the enzyme and the substrate I used syringes. It was an accurate way to measure out the correct volume and easy to use quickly so that no time was wasted when starting the stopwatch. First I added the 2.5ml of enzyme to the conical flask, I then added the correct amount of pH 7 buffer depending on the concentration of the substrate I was investigating. Finally this left the substrate, I added the substrate last because otherwise the reaction would have started before I had added the pH buffer.

After I had added the substrate to the conical flask, I quickly put the bung on the conical flask which was connected to the delivery tube. The gas produced in the conical flask

Preliminary Experiment

Conical flask containing hydrogen peroxide, homogenised celery and pH7 buffer

Investigation Experiment

2H2O2 - 2H2O + O2

the pH of the experiment will be controlled by the volume of pH buffer 7, it will keep the pH of the experiment at pH7 as well as changing the concentration of the substrate. I will monitor the temperature ensuring it stays at 20°c.

The enzyme I have decided on using is homogenised celery. I tried using yeast and I found it hard to accurately weigh out the right amount. Some of the yeast was suspended on the bubbles it produced so not all of it was being used up. The reason I chose homogenised celery was because it was easy to measure out the correct amount I needed with the syringes and that fact that it had been homogenised meant that the cells and enzymes where exposed and mixed up in the mixture. I had increased the surface area of the celery cells. If I had used chopped potato, not only would it have been difficult to get the exact same size potato but the surface area with enzymes showing and ready to react would have been much smaller. It is not really possible to homogenise potato without adding water which I was not prepared to do as it would dilute the potato.

I tried to make sure that I pressed the start button on the stopwatch at exactly the same time I poured the substrate in and attached the delivery tube. There is an obvious limitation with this method as it is slow and could affect results by not pressing the start at the right time or failing to attach the delivery tube quickly.

This is the table shows the results from my experiment. As you can see after making more adjustments from my preliminary I ended up using this table. I decided that it was not necessary to carry out the experiment over the time period of two minutes as it was very time consuming and the graph to show the volume of gas being produced, levelled off. Not much gas was being produced after 1 minute and 20 seconds as all the substrate had reacted with the enzyme. There was a large spread of data if my experiment which is shown by the error bars on my graph. These fluctuations may have been caused by the table being knocked which did happen on one occasion. I had to start a test again because of this. Other factors may have been that I was slightly inaccurate in the measuring out of my enzyme or substrate.

To get the average reaction rate I added all of the readings up from after 60 seconds and divided by three to give a mean average. I did this for each concentration to show the volume of gas produced per minute, in other words this is the reaction rate.

On the graph the reaction rate shows a relatively steady and straight, upward sloping curve on the graph. After a period of time the graph will level off as the substrate is reacted with the enzyme. There is a rapid rise at the beginning and this is the activation of the reaction just like on the graph showing a catalyst reduces activation energy.

The reactions I have investigated all have shown similar rises at the beginning and levelled off at the end.

There is 27cm3 of gas produced at 100% substrate concentration per minute more than there is at 10% and this is because collisions of the enzyme and substrate occur less often meaning that the reaction takes place slower. At 100% concentration the graph levels off sooner than it does at 10%.

Test

% substrate

20 secs

40 secs

60 secs

Average Rate of reaction

1

100

12

22

29

2

100

15

23

33

32 cm3/min

3

100

10

21

33

1

90

7

17

23

2

90

13

21

31

28 cm3/min

3

90

12

22

31

1

80

5

12

21

2

80

9

18

24

23 cm3/min

3

80

8

19

25

1

70

5

12

20

2

70

7

14

20

20 cm3/min

3

70

7

15

21

1

60

3

10

19

2

60

5

10

18

19 cm3/min

3

60

7

13

19

1

50

4

9

16

2

50

4

10

15

16 cm3/min

3

50

6

11

18

1

40

3

7

10

2

40

4

8

13

12 cm3/min

3

40

3

10

15

1

30

2

5

8

2

30

2

7

9

9 cm3/min

3

30

2

6

11

1

20

0

2

8

2

20

1

3

5

7 cm3/min

3

20

3

7

10

1

10

0

2

4

2

10

0

2

4

5 cm3/min

3

10

0

3

6

Improvements that I could have made to equipment would be that the stopwatch was of greater precision so that recording could be taken at more precise times. This would have enabled me to accurately find out at a precise time where the graph levels of and gas is produced at a far slower rate.

A more precise measuring cylinder would have allowed me to get readings of the volume of gas to more decimal places. This would aid the validity of results.

The problem with conducting this experiment on your own is that at the beginning of the experiment where you need to add the substrate, attach the delivery tube and start the stopwatch in quick succession, means that gas maybe lost or the conical flask is knocked meaning the solution is shaken creating a lot of gas to be produced at once due to the knock. This is probably the reason for the fluctuations in my results. If I had had someone at hand to help then the method of conducting this experiment would have run more efficiently and would have given more reliable results.

If I had not carried out the experiment three times then the outliers would have been very prominent and would have given unreliable data. The fact that my graph has a near straight upwards curve shows that after working out an average my results are accurate and reliable. The graph looks what I expected it to look like.

I think that the experiment went well and that the results I have recorded are accurate and reliable enough to be happy with my work. As always improvements can be made and I did my best to keep variables and take account of others. I am confident that my results have concluded that there is a large change in reaction rate when substrate concentration is changed.

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