In order to carry out a fair, accurate and reliable investigation to find the effect of copper sulphate on pepsin activity I have decided to keep all of the variables constant and only alter the concentration of copper sulphate solution. I have described why I intent to keep the following variables constant below:
Volume of copper sulphate: If I increase volume of copper sulphate solution more copper sulphate molecules will inhibit more pepsin particles therefore slowing down the rate of reaction making the test unfair and altering my results.
Concentration of pepsin: If I increase the concentration of pepsin more pepsin molecules will collide with more casein molecules and as a result a faster rate of reaction would occur. This is turn would affect my results of only finding the affect of copper sulphate concentration.
Volume of pepsin: If I increase volume of pepsin more pepsin particles will catalyse the hydrolysis of casein therefore resulting in a faster rate of reaction. Thus affecting my results of only finding the affect of copper sulphate concentration.
Concentration of casein: If I alter the casein concentration by increasing the concentration for example, my results would be also affected, as more casein molecules would collide with pepsin molecules and result in successful reactions therefore increase the rate of reaction and biased in my results.
Volume of casein: If the volume of casein was altered say by decreasing the amount, less successful collision would occur between the casein and pepsin thus decreasing the rate of reaction. This would create biased amongst my results.
pH of casein, copper sulphate and pepsin solution: The pH of the solution could affect the efficiency of my enzyme, pepsin as small changes in pH affect the hydrogen bonds used to make the structure causing a change in active site. This in turn would change the rate of reaction and cause my experiments to be unfair.
Temperature of the casein, copper sulphate and pepsin solution: For example if the temperature of the solution is increased, the particles tend to gain more kinetic energy therefore travel faster and collide more often and produce more successful reactions. Thus the rate of reaction increases causing biased in my experiment.
I will control the:
Volume of copper sulphate: By using the same amount of copper sulphate solution through out my investigation.
This will be 10 cm3
Concentration of pepsin: By using the same concentration of pepsin throughout my investigation.
This will be 0.5% concentration
Volume of pepsin: By using the same amount of pepsin throughout my investigation.
This will be 10cm3
Concentration of casein: By using the same concentration of pepsin throughout my investigation.
This will be 2% concentration
Volume of casein: By using the same amount of casein throughout my investigation.
This will be 10cm3
pH of casein, copper sulphate and pepsin solution: By using same casein, and pepsin solution through the experiments to avoid differences in pH .
Temperature of the casein, copper sulphate and pepsin solution: By using the same temperature throughout my investigation using a water bath.
Which will be body temperature 37oC.
Apparatus list and materials
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Copper Sulphate stock solution at 0.5% concentration – This is my inhibitor
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Casein at 2% concentration – This is my substrate
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Pepsin at 0.5% concentration – This is my enzyme
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1x Safety goggles – This is a safety measure used for protection
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1x Stopwatch – To measure the amount of time casein take to go clear
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3x Thermometers – To measure the temperature of casein, copper sulphate and pepsin
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3x Boling tubes – To hold the copper sulphate, casein and pepsin before they are mixed together
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1x Testube rack – To hold the boiling tubes.
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1x Glass rod – To mix solutions
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1x Graduated pipette – To accurately measure out solutions
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1x Pipette filler – Used with the graduated pipette to measure out solutions accurately
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1x Colorimeter & Cuvetts – To accurately test the change in colour of casein and to produce quantitative results.
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Distilled Water – To make different concentrations of copper sulphate solution from the stock solution.
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Electronic Water bath – To heat the casein, pepsin and copper sulphate to 37oC
Diagram
Pilot Study Results
In order to iron out any problems before I start my main practical I decided to do a series of pilot study to achieve the best and most suitable set of results.
The first test was to acquire a suitable end point for my experiment, i.e. an absorbency reading. In order to obtain this end point I added my enzyme, pepsin to my substrate, casein and waited till the solution had become clear.
Table 1:
From the above results it is evident that my experiment was unsuccessful as after even 300 seconds the absorbency reading did not change. In order to overcome this problem I decided to change my enzyme from pepsin to trypsin.
I then repeated the same experiment once again to find my end absorbency reading with trypsin and casein.
Table 2:
Table 2 shows that not only did the trypsin work, but that the end absorbency reading is 74%
My second series of pilot studys was to see whether my chosen concentrations of copper sulphate were suitable for my investigation. I did this by testing my highest and lowest concentrations:
Table 3: Summary of full pilot study results
From table 3 it is evident that the 0.5% concentration is far too strong an inhibitor as it took over 1200 second (20 minutes) for the casein to go clear. However the 0.01% concentration worked well.
Modifications
The following modification were made to my plan after my pilot study:
- I changed my enzyme from pepsin to trypsin.
- I changed the concentrations of copper sulphate from 0.5, 0.25, 0.1, 0.05, 0.025 & 0.01 to 0.25, 0.1, 0.05, 0.025, 0.01 & 0.005.
- I have also decided to change the time of my reading from every 30 seconds to every 60 second as I did not get enough time to make accurate reading every 30 seconds
New Method With Modifications
I first prepared the following concentrations of copper sulphate solution by mixing the 0.5% stock solution with distilled water in the following parts as shown by the table below:
Table 4:
Once the six different concentrations of copper sulphate had been made up I put 10cm3 of the first concentration of copper sulphate, 0.25%, into a boiling tube using the graduated pipette and pipette filler. Then, as with the copper sulphate I put 10cm3 of casein, 2%, into another boiling tube using the graduate pipette and pipettes filler. I also put 10cm3 of trypsin, 0.5%, into another boiling tube using the graduated pipette and pipette filler. This meant that I had three separate boiling tubes, one with 10cm3 of copper sulphate, my inhibitor, one with 10cm3 of casein, my substrate and one with 10cm3 of trypsin, my enzyme.
I placed all three boiling tubes into a testube rack whilst I set up the electronic water bath, to 37oC. After I had set up my water bath I placed the testube rack along with the three solutions into the water bath. In order to maintain accuracy when considering temperature I placed one thermometer into each of the different solutions, the copper sulphate, casein and trypsin.
When all three solutions were at 37oC, I poured the trypsin solution into the copper sulphate solution. I then allowed the two solutions to incubate together in the electronic water bath for 2 minutes, which I timed using the stopwatch. After the incubation period I immediately mixed the casein solution with the copper sulphate and trypsin solution and placed the copper sulphate, trypsin and casein solution back into the water bath. Whist doing so, I reset the stopwatch and began timing again as well as taking the first absorbency reading.
To take an absorbency reading I used the colorimeter along with the cuvettes. I would extract a sample of 3cm3 of the solution from the boiling tube using the graduated pipette and pipette filler. I would then put this sample in a cuvette. I would place the cuvette within the colorimeter and take a reading of the percentage absorbency of the solution. (The percent of light that is able to pass through the solution). The higher the percentage reading the clearer the solutions more light is able to pass through the solution easily.
After I had taken an absorbency reading I would place the sample back into the boiling tube, along with the rest of the solution and give the whole solution one stir with the glass rod. I would then take an absorbency reading every 60 seconds until the solution had gone clear (i.e. the end point had been reached).
I would repeat the same method for all 6 copper sulphate solution. I would repeat the test three times for every different concentration of copper sulphate for a reliable set of results.
Risk Assessment
Copper sulphate
Harmful if swallowed. Solutions equal or greater the 1M should be labelled HARMFUL. It may also be irritating to the eyes, skin and has been known to sensitise the skin. It is very toxic to the aquatic environment and may cause long-term adverse effects.
If swallowed: wash out mouth and give a glass or two of water
If contacted with eyes: Flood eyes gently with running tap water for 10 minutes
If split on to skin: Wash off with plenty of water
Pepsin (powder form) – Used in pilot study
Irritating to the eyes, respiratory system and skin. May cause sensation by inhaling. WEAR EYE PROTECTION.
If swallowed: Have victim drink 1-3 glasses of water to dilute stomach content
If contacted with eyes: Immediately flood the eye wit plenty of water for at least 10 minute.
If split on to skin: Wash hand or skin with large quantities of water after spillages.
Trypsin (powder form) – Used in main Experiments
Irritating to the eyes, respiratory system and skin. May cause sensation by inhaling
If swallowed: Immediately call doctor.
If inhaled: Supply fresh air and be sure to call for a doctor.
If contacted with eyes: Immediately flood the eye wit plenty of water for at least 10 minute.
If split on to skin: Wash hand or skin with soap and water after spillages.
Casein
Low risk. No adverse health effects expected from inhalation. Not expected to be a health hazard via ingestion. Not expected to be a health hazard from skin exposure.
If swallowed: If large amounts were swallowed, give water to drink and get medical advice
If contacted with eyes: It is not expected to require first aid measures. Wash thoroughly with running water. Get medical advice if irritation develops.
If split on to skin: Not expected to require first aid measures. Wash exposed area with soap and water. Get medical advice if irritation develops
Disposal
Copper sulphate: Very toxic to environment, dissolve salts in 10 litres of water and wash down drains.
Pepsin: Wash down sink with plenty of water to dilute solution.
Trypsin: Wash down sink with plenty of water to dilute solution.
Casein: Wash down sink with water, is not toxic to environment so no extreme measures are needed to be taken.
Results
Table 5: Summary of results with rate of reaction
Table 6: Showing range of error within data for each concentration
Conclusion
Trends and Patterns
From my results you are able to see that the concentration of copper sulphate significantly changes the average time taken for the casein to be hydrolysed by the trypsin. From graph 1it is quite evident that the main trend seems to be that as the concentration of copper sulphate increases so does the time taken for the casein to be hydrolysed. As a result, the rate of reaction decreases as the concentration of copper sulphate increases which is demonstrated in graph 2. Due to this it is evident my hypothesis that as the concentration of copper sulphate increases the time taken for trypsin to catalyse the hydrolysis of casein will increase.
Graph 1 shows that the relationship between the concentration of copper sulphate and the time taken for casein to hydrolyse is not proportional as otherwise the line of best fit would be a straight line. Instead the relationship is comparable to that of a curve, which starts of steeper and then begins to level off towards the end. This means the gradient between 0.005% & 0.01% should be far greater then 0.1% & 0.25%. This is illustrate below:
Gradient between 0.005% & 0.01%:
Gradient between 0.1% & 0.25%:
This means that as the concentration increases the rate at which casein is being hydrolysed by the trypsin is slower. For example at 0.005% the rate of reaction was 55.56, which is fairly fast, where as at 0.25% the rate of reaction is at a mere 9.8, which is more than 5 times slower.
My raw data in table 7 shows that throughout my experiment the results were reasonably consistent as the end point of my replicates for the majority of concentrations all ended around the same sort of time. This is reinforced by table 6, which shows the range of the end point for the different concentrations. However, I did have some anomalies within my results. My first anomaly as shown on my table 7 was the 2nd reading on the 0.005% concentration. Unlike the 1st and 3rd reading, the 2nd reading finished at 540 second which is 3 times slower the other two which finished at 180 seconds. Due to this I decide not to include this reading in my average. My second anomaly was with the 3rd reading of the 0.25% concentration. Whilst the other two reading ended at 1020 seconds the 3rd reading ended far early at 900 seconds. In spite of this I still included this repeat in my results
Explanation of trends and patterns
The active site of an enzyme is a relatively small area that binds temporally to a substrate molecule such as casein. The way an active site of an enzyme works is explained by the ‘lock and key’ theory, which means each enzymes active site is specific to the shape of a substrate. If the shape is changed of either the active site on the enzyme or substrate, the enzyme will no longer be able to hydrolyse the substrate.
Copper sulphate is an effective non-competitive inhibitor of enzymes such as trypsin. When it is added to the trypsin during the incubation period of the experiment, a copper sulphate molecule will bind itself permanently to a trypsin enzyme causing the active site of the globular protein to change. As a result the substrate, casein, is unable to bind with the enzyme, trypsin, and will no longer be hydrolysed. This is how the copper sulphate inhabits the trypsin, causing the time taken for the trypsin to catalyse the casein to increase.
In spite of this, as the copper sulphate is a non-competitive inhibitor, when it is added to the trypsin only a certain number of trypsin molecule are inhibited permanently. This means that the rest of the casein molecules are able to bind with trypsin enzyme and be hydrolysed.
For example, if 50 Copper sulphate molecules are added it 100 trypsin enzymes, the copper sulphate molecules will bind to exactly 50 of the 100 trypsin enzymes, and as a result inhibiting them. The other 50 trypsin enzymes that are not inhibited are not affected and can carry on hydrolysing the casein.
When the concentration of copper sulphate is increased more trypsin enzymes are made inactive, leaving less active enzymes. Due to this more time is needed for fewer enzymes to hydrolyse the same amount of substrate. This is why more time is taken for the casein to got clear when the concentration of copper sulphate is increased.
Evaluation
The overall variability of my investigation was reasonably consistent throughout my test with the exception of the 2nd reading on the 0.005% concentration, which appears to be a major anomaly in my collection of data. By considering the range of my data, it is evident that my results are reliable as my largest spread was 120 seconds from the 0.25% concentration and my smallest was 0 seconds from 0.05%, 0.025% and 0.01%.
The accuracy and precision of my experiment is demonstrated by my results and the lack of common anomalies. I believe that 0.005% concentration anomaly can be justified by human error rather the error due to accuracy of data collection or equipment. If this anomaly is not considered it is quite clear that my results reinforce my hypothesis.
Considering my whole investigation, it is important to state that I had only done a single investigation with a limited set of resources and that it is entirely possible for my results to be coincidental, however I personally do not believe this to be true as I carried out my experimentation with up most accuracy and consistency. In spite of this to overcome this possibility, further investigation will be needed.
Limitations
The largest limitation of my experiment was recording accurate results every 60 seconds. Even though I used a colorimeter to obtain my result that made my reading quantitative, it is still was not a very accurate method of acquiring results as it was a discrete method of acquiring results rather then continuous. A way of overcoming this problem would be by using a logIT, which digitally records the change in absorbency as time goes on, rather then every 60 or 30 seconds.
When carrying out my investigation I was unable to control the pH of the solutions using a buffer solution, as it would have made the experiment overly complicated. However even thought the pH was not controlled it is unlikely this would effect my overall results as this inconsistency was present throughout my experiment which makes my result still reliable enough to make a firm conclusion.
After replacing samples I would stir the solution to mix it. With each case I cannot determine accurately the rate of stirring. This is could cause inconsistencies within my result as if I stir one solution more then I had others it would gain more kinetic energy, which as a result would cause more successful collisions between enzyme and substrate molecules therefore speeding up the rate of reaction. To overcome this I could use a mechanical stirrer that stirs each test solution at a steady rate.
When obtaining my results I took 3cm3 sample of the main solution to test for absorbency readings. This obviously will not produce an accurate representation of the whole experiment, as parts may not be mixed evenly, causing imprecise results. Additionally, due to removal and replacement of samples from the main solution some of the solution is lost which causes a decrease in volume as well as concentration. This could cause a change in the rate of reaction, as fewer particles would collide creating less successful collisions. Both limitations could be overcome by the experiment being done on a smaller scale so that samples are not necessary.
When carrying out my experiments I was only allowed to incubate my enzyme and inhibitor together for 2 minutes due to time restrictions. If I had sufficient amount of time to carry out all my investigation I would consider incubating the trypsin and copper sulphate for longer to make sure the all of the possible enzymes were inhibited before adding the casein.
Further Work
In order to provide further evidence for my conclusion, I would carry out further work to my investigation. I would firstly carry out more tests with a variety of different sources of trypsin, casein and copper sulphate, as the one source of trypsin may act differently to another. I would also use a larger range of copper sulphate concentrations to fully investigate in more detail how an inhibitor such as copper sulphate affects trypsin activity. In addition I would use a buffer solution to control the pH when carrying out experiments as this may add to accuracy to my results.
Bibliography
- Fig.1- http://www.blc.arizona.edu/courses/181gh/rick/energy/enzymes1.html
- Collins, Student Support material for Edexcel AS Biology, Unit 1: Molecules and Cells - Mary Jones & Geoff Jones.
- Nelson Thornes, Essential AS Biology, Glenn and Susan Toole
- Introduction to Advance Biology, CJ Clegg