Enzymes also have an optimum pH level. If it changes from the optimum pH level, the chemical nature of the amino acid can change. The tertiary structure will break down as the bonds will change. The active site will be disrupted if the tertiary structure breaks down and the enzyme will be denatured.
If the enzyme concentration is too low there is great competition for active sites and there are not enough to bind to, making the reaction rate extremely slow. If there are more enzymes, there are more active sites to bind to so that more enzyme-substrate complexes can be formed. Eventually increasing the enzyme concentration will have no effect as there is not enough substrate to bind to its active site, and the substrate concentration becomes a limiting factor. It may have an inhibitory effect as non-competitive inhibitors may destroy the active site of the unused enzymes. Low substrate concentrations means there are many unoccupied active sites, meaning the reaction rate is slow. If more substrate is added then more enzyme-substrate complexes can be formed, speeding up the rate of reaction. However, too much substrate means there will be no effect as the active sites will be saturated and no more enzyme-substrate complexes can form.
Variables: -
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Independent: temperature. The temperature will be changed by setting the water bath at six different temperatures, and also carrying out the experiment at room temperature.
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Dependant: activity of the enzyme, which will be measured using a stopwatch. I will time how long it takes for the solution to clear. It is inversely related, as the more time it takes to clear, the less enzyme activity there is. I will carry out a pilot experiment to determine what concentrations of casein and trypsin are best, and what kind of shaking regime is best to ensure the suspension doesn’t settle at the bottom.
- Control: keep the batch of casein and trypsin the same as you cannot ensure the concentration will be exactly the same in another batch. Use the same test tubes to ensure the experiment is fair, and wash them out with distilled water to get rid of contamination. Use the same syringes and thermometers so that the same fixed error is present throughout the experiment. Remember to wash out the syringes and the beakers with distilled water before using them. It is essential to wash out all glassware as they may be contaminated.
Apparatus: -
- 3 test tubes to mix the substrate and enzyme in.
- 2 syringes (one for casein, one for trypsin)
- Test tube rack (to carry out the experiment at room temperature)
- 105ml of 4% milk Marvel suspension.
- 105ml of 0.5% trypsin solution.
- 2 beakers (one to place the casein, and one to place the trypsin in)
- 3 thermometers to place in the test tubes in the water bath
- 3 stopwatches to time how long it takes for the solution to clear.
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Water baths at temperatures: 30oC, 40oC, 50oC, 60oC, 70oC, 80oC
- Distilled water to wash out the test tubes and emit any contamination.
Method: -
- Using distilled water; wash out the three test tubes, the two syringes and beakers, to emit any previous contamination.
- Place the test tubes on the test tube rack, as you will carry out room temperature first.
- Pour in 200ml of casein and trypsin into the beakers. Extra is needed in case there is any spillage, or if the batch runs out.
- Place a thermometer in the three test tubes and record the room temperature.
- Draw up 15ml of casein using one of the syringes, and put 5ml of it in each test tube.
- Using the other syringe, draw up 15ml of trypsin.
- Get the stopwatches ready and make sure you place them in front of the test tube so that you don’t get mixed up.
- Put 5ml of trypsin into one test tube and start the stopwatch immediately. To ensure that you accurately put 5ml, make sure the black part of the syringe sits exactly on the line.
- Put another 5ml of trypsin into the second test tube and start the stopwatch. Do the same for the third test tube.
- Use the thermometer to stir the solution from time to time to ensure the casein doesn’t settle at the bottom. Ensure you hold the thermometer from the rubber part, in case you heat up the experiment with your body heat.
- Stop the stopwatch when the solution goes as clear as possible. I will use the distilled water as a reference to compare the test tube with. The distilled water is good to use because it is not as clear as water, and due to the pilot experiment I know the solution doesn’t go as clear as water.
- Record how long it took for all three test tubes to clear, and then wash them out using distilled water.
- Draw up 15ml of trypsin using the same syringe. It is extremely important the casein syringe is not used as it will lead to inaccuracies in the experiment. 15ml is drawn up instead of 5ml because it is more efficient, as there are 3 test tubes needing 5ml of trypsin each.
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Place 5ml of trypsin in each of the test tubes. Place a thermometer in all three test tubes and place them in the water bath set to 30oC. The trypsin is placed in the water bath, not casein, because I am investigating enzyme temperature. The casein would also get denatured at high temperatures, as well as trypsin, because it is a protein.
- Get the stopwatches ready, and draw up 15ml of casein using the right syringe.
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Wait for the temperature to reach 30oC on the thermometer then immediately insert 5ml of casein to the test tube and start the stopwatch.
- Stir the solution using the thermometer from time to time.
- Do the same for the remaining two test tubes and use the distilled water again as a reference as to when to stop the stopwatch.
- Record the time taken and wash out the test tubes using distilled water.
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Repeat the procedure at temperatures of 40oC, 50oC, 60oC, 70oC and 80oC.
- The test tubes are put in the water bath three at a time at the same temperature because this repeats the results in exactly the same conditions. Therefore, my results will be more reliable and accurate.
Safety: -
- Keep the experiment away from the edge of the table to avoid spillage.
- Wash your hands if any enzyme gets onto it immediately.
- Tie back any loose objects (i.e. hair) as they may interfere with the experiment and loose objects can hook onto apparatus and tip it over.
- Tuck in stools and put away bags as they may cause you to trip over and injure yourself.
- Running is prohibited in the laboratory to minimise risk of accidents.
- If any glass is broken, inform the teacher immediately.
Implementing (skill C): -
Analysing Evidence and Drawing Conclusions (skill G): -
As you can see from the table, the enzyme trypsin worked best at 50oC, which I don’t understand, as 40oC is supposedly the optimum temperature for enzymes. When I encountered this anomalous result, I re-investigated it, but I still got similar results. I ended up repeating all the temperatures and ensuring I was using the same concentration of trypsin and casein each time, but it made no difference, 50oC was still the optimum temperature. As this is still quite close to 40oC, I am not overly concerned, as different enzymes may have different optimum conditions. However, it is scientifically proven that the optimum conditions for trypsin are: pH 7-9 and trypsin is not very stable above 40oC. This is very puzzling, and repeating my whole experiment did not solve this anomalous result.
Another strange fact about my results is that the trypsin did not denature at 80oC, it still broke down the casein, albeit slowly. Apart from the two anomalous results (40oC and 50oC), my results do generally fit the pattern as you can see below from my graphs. On the graphs, 40oC is not on the best fit curve, although it is not too far away. However, my results still fit the general pattern and they did form a parabola, which was expected.
I think my anomalous result is due to the amount of stirring I did for these two temperatures. I may have stirred the 50oC test tubes more then the 40oC. The stirring regime is a major inaccuracy in the procedure and it is the most likely explanation for my anomalous results. The optimum temperature should have been body temperature as trypsin is found in the body and is secreted in an inactive form by the pancreas.
At room temperature (21oC), the time taken for the trypsin and casein mixture to turn clear was extremely long. This is due to the fact that there is not enough thermal energy to convert to kinetic energy. Therefore, the particles of the substrate and enzyme cannot collide and form enzyme-substrate complexes as quickly. As the temperature is increased, more energy is supplied to the reaction and therefore more collisions occur as the particles have more kinetic energy. This means they collide more often and more enzyme-substrate complexes form as the substrate (casein) collides with the active site of the enzyme (trypsin).
However, above 50oC the reaction rate slows down. This is because too much heat energy is supplied and the tertiary structure of the enzyme breaks down, destroying the active site. The enzyme becomes denatured as it is permanently destroyed and will not function again even at lower temperatures. This did not happen to me during my experiment, but the rate of reaction did slow down as too much thermal energy was supplied, which caused some change in the tertiary structure of the protein trypsin.
Other than the anomalies, I think my results were quite accurate and reliable as they followed the expected pattern and I carried out my procedure extremely efficiently that I had time to re-investigate the anomalies that occurred. The most likely explanation for the resulting anomalies was the inaccurate stirring regime. It affects the distribution of the casein and trypsin molecules, and the number of enzyme-substrate complexes that form.
Evaluating Evidence (skill H): -
The main sources of error in the procedure were the stirring regime and the decision of when the mixture had turned clear. I think a magnetic stirrer should have been used to make the experiment more accurate. I also think a buffer should have been used, as the pH value changes and this makes the experiment unfair. There were fixed errors in the measuring devices, but they are cancelled out as they were present throughout the experiment. The use of stopwatches was extremely inaccurate as human error is a major factor that makes experiments inaccurate and the results unreliable. Time was lost between putting the casein in using a syringe and starting the stopwatch. I think specialist equipment should have bee used to trigger off the stopwatch as soon as all the substrate had left the syringe.
The temperatures were also inaccurate as some test tubes remained longer at a certain temperature than others, so they contained more thermal energy. Sometimes it was difficult to get at eye level and the temperature may have been 1oC above or below the intended temperature. This was because the water baths were not very good as they went over the temperature written on them. This made the experiment inaccurate as the test tubes containing trypsin were at higher temperatures than they were supposed to be. This may have increased or decreased the rate of reaction depending upon if it was above or below the optimum temperature. This makes the experiment unfair and unreliable.
Overall this procedure is quite inaccurate despite my efforts to measure the amount of casein and trypsin carefully by putting it at eye level and ensuring it is exactly at 5ml. I also washed out the test tubes with distilled water to avoid contamination from previous experiments. However, this may have diluted the trypsin slightly, therefore affecting the accuracy of the concentration. To avoid this problem, I should have used a new test tube, but there weren’t enough to carry out the experiment if I did that as I did obtain many results. I also used the same batch of casein and trypsin to ensure the concentration remained the same, making it a fair test. However, I think I shouldn’t have put 15ml of the substrate and enzyme in the syringe as I cannot accurately deliver exactly 5ml into the test tube by putting the syringe at eye level, ad then start the stopwatch at exactly the same time. I should have put only 5ml accurately beforehand, and carried out the experiment on one test tube at a time. Although my procedure was efficient and time saving, it led to unnecessary inaccuracies.
I also kept well away from the experiment to ensure my body heat did not affect the experiment, and I was extremely careful when using the thermometer to stir not to touch the glass part and not to stir vigorously and warm up the test tube. I kept the stopwatches in front of the test tubes to ensure they didn’t get mixed up and I used the distilled water bottle to determine when the test tubes had cleared. This was quite effective as I had done a pilot experiment to see how clear the mixture goes, and it was roughly the colour of the distilled water bottle.
The same syringes were used for putting in the casein and trypsin, and this ensured there was no previous contamination, and made my experiment more accurate. If I had used a new syringe, it may have had remains of other chemical substances that would have affected my experiment. I tried to keep the conditions the same as I used the same test tubes, the same syringes, the same thermometers, the same stopwatches, the same batch of casein and trypsin, and the same water baths. My experiment was quite reliable as I repeated the results three times, and I even re-investigated the anomalous results. I think the procedure was fairly accurate as I used mainly measuring devices containing fixed error, which was cancelled out. The main problem was human error. The only way to improve the procedure is to use specialist equipment to replace the job given to us, and therefore minimising errors. The number of experiments is obviously a limitation. If I had the opportunity, I would test more temperatures, and repeat the results more times to make the procedure more accurate and reliable.
I obtained two anomalous results due to my inaccurate procedure and they were 40oC and 50oC. They came about due to the stirring regime, which was to use the thermometer as a stirring rod from time to time if the mixture wasn’t clearing fast enough. This was obviously not a good idea for the optimum temperatures as they clear in a matter of seconds and you may not get time to stir it properly, or you may stir one test tube more vigorously than the other. A magnetic stirrer would make the procedure much more accurate. Other than that I think my procedure was quite good as I obtained quite good results and I followed the right safety precautions, ensuring my procedure was safe, efficient, accurate and reliable.