Procedure for Preliminary Test
Apparatus:
- Test tube rack
- 10 Test Tubes
- Ice for 0 degrees experiment
- Thermometer
- Water baths at 40, 60 and 80 degrees.
- Trypsin solution
- 10 pieces of photographic film
- 10 splints with cut ends
- Stopwatches
Method
Before beginning, we ensured the area was safe by wearing safety goggles and clearing the nearby area of books or obstacles. We placed 10 test tubes in the test tube rack. 5 were control tubes, so we added exactly 3ml of water to each of the 5. We then added 3ml of trypsin solution to the other 5. We inserted the photographic film squares, of 2mm squared in size, into each of the 10 splints. There were two tubes, one control and one test, at each temperature. We placed two test tubes, one filled with water, the other filled with trypsin, in the icebox. We placed two in a rack to stay at room temperature. Two were placed in the 40-degree water bath, two in 60 degree water bath, and two in the 80-degree water bath. Each one was allowed to acclimatise to the appropriate temperature in its water bath/environment for 10 minutes. This also ensured that the trypsin would denature if it were at too high a temperature, discussed in detail later. Stopwatches were started at the point when the splints were placed into the tube. Every 10 seconds the film would be examined. The timer was stopped only when the film was clear, so that all the film had to reach the same stage (eliminating the possibility of human error as to judging when the enzyme has completed it’s job). We then recorded all our results in a table, as shown below the ‘Fair Test’ Section. Finally, we all washed our hands to ensure any trypsin on them was washed off.
Fair Test
Ensuring that the experiment was a fair test was one of the most important parts of the experiment; if each test were not fair, then the results would be incorrect. The first thing we had to be sure of was that we did not contaminate the trypsin with dirt or bacteria that may have been on our fingers, as this may have affected the rate at which the enzyme works. We also made sure that all the test tubes reached their correct temperature and were allowed to acclimatise for 10 minutes. This is important for two reasons, the first being that if we did not ensure the test tube was at the correct temperature, then the results would not be a correct reflection of what we had hoped to achieve. Also, it is important to remember that at high temperatures, enzymes work at accelerated speeds for short periods of time before denaturing (when the enzymes lose their ‘key’ shape so they cannot fit in the ‘lock’ of the substrate), whereby they are useless. We can see this in commercial industry, where enzymes are used at extremely high temperatures when they work very quickly, and then denature and are removed for another batch of enzymes to work. It is also important we keep pH constant, as if the pH changes, the bonding of the enzyme would change, causing it to lose it’s active site. This could affect the results and therefore our final conclusion, so we used buffer to regulate the pH. We also decided to keep the photographic film size at exactly 2mm squared. If photographic film were at different sizes, then in some test tubes the trypsin would have to work for longer to break down the larger piece of photographic film, hence increasing the result time and making the test unfair. To ensure complete accuracy, we checked our stopwatch every ten seconds instead of twenty or longer, so that we could pinpoint exactly when the photographic film had become transparent.
Results from Preliminary Experiment
The results we obtained were tabulated in a table below:
As we can see, the control experiment showed no change, so I did not need to put the results into a graph. I then created a graph from my results and to ensure that a curve was made in the correct direction, I divided 1 by the time taken in seconds. This ensured that as the rate of reaction was faster, the value would be higher. Values were in seconds and degrees. The graph is shown below:
Preliminary Experiment Evaluation
As one can see from the above graph, it is very similar to my hypothesized graph. The slight difference may be due to human error, or the length of time in between each check of the tube. As we can see, up to 40 degrees, the rate of reaction steadily increases, and, as I hypothesised, after this point it begins to drop as the enzymes are denatured. We can also see that at 80 degrees the enzymes cannot break down the gelatine at all or very little as they have been completely denatured by the high temperature. It was important that we allowed the trypsin to acclimatise; else we may have found that the enzymes worked quickly on the photographic film and made it transparent before they became denatured. I saw no anomalies in my work; this is a sign of good planning, but I must continue to work effectively in the final experiment to ensure there are no anomalies then. However, after doing the experiment once, there are a number of changes I will make to my work. Instead of checking the tubes every ten seconds, I will check them every five seconds. This will ensure more accuracy, as my results will be more precise. We have obtained 4mm squared photographic film as opposed to 2mm squared photographic film, so that it would take longer for the enzyme to break down the gelatine, and therefore meaning that it give us a more accurate result; it is more easy to accurately measure a long period of time than it is a very short one. This will affect our spread of results, and ensured all tests were accurate. We will also triple the concentration of trypsin to speed the results, but at the same time, as a group, we have all decided we will not stop the stopwatch until the photographic film is completely transparent, without exception. This will regulate our results further as we are all stopping the timer at a predefined point. To ensure complete precision of results, in my next experiment I will use two test tubes filled with trypsin at each temperature instead of one. In this way we can cancel out anomalous results. In this way I hope I can further improve the accuracy of our results, so that my final table is a good reflection of the way that enzyme reaction changes.
Final Experiment
Method
Apparatus:
- Test tube rack
- 15 Test Tubes
- Ice for 0 degrees experiment
- Thermometer
- Water baths at 40, 60 and 80 degrees.
- Trypsin solution
- 15 pieces of photographic film
- 15 splints with cut ends
- Stopwatches
We ensured the area was safe by wearing safety goggles and clearing the nearby area of books or obstacles. We placed 15 test tubes in the test tube rack. 5 were control tubes, so we added exactly 3ml of water to each of the 5. We then added 3ml of trypsin solution to the other 10. We inserted the photographic film squares, of 4mm squared in size, into each of the 15 splints. There were three tubes, one control and two tests, at each temperature. We placed three test tubes, one filled with water, the other two filled with trypsin, in the icebox. We placed three in a rack to stay at room temperature. Three were placed in the 40-degree water bath, three in 60 degree water bath, and three in the 80-degree water bath. Each one was allowed to acclimatise to the appropriate temperature in its water bath/environment for 10 minutes. This also ensured that the trypsin would denature if it were at too high a temperature as explained before. Stopwatches were started at the point when the splints were placed into the tube. Every 5 seconds the film would be examined. The timer was stopped only when the film was clear, so that all the film had to reach the same stage (eliminating the possibility of human error as to judging when the enzyme has completed it’s job). We then recorded all our results in a table, and averaged out the results of the two trypsin test tubes at each temperature. Finally, we all washed our hands to ensure any trypsin on them was washed off.
Observations
Finally, I made an “S-1x1000” row, where 1 was divided by the average, and then the result was multiplied by 1000 for a more manageable number. This means that I had some values that correspond to the rate of reaction. For those results which showed no reaction, they were given a result of 0.
From these average results, we can construct a graph to display our information:
Analysis and Conclusion
The graph was constructed by taking the number of seconds the enzyme took to completely clear the photographic film, and called it X. The formula we used to plot each graph point was “1 divided by X times 1000” or S-1 x 1000. This was so as the time taken was shorter, the graph value was higher, hence reflecting the true rate of reaction. We can see a number of things from this graph. The basic observation, is simply that as the temperature increases, as does the rate of reaction, to a point round 42-44 degrees. As with my hypothesis, I believe this is because the heat energy the substrate has is converted into kinetic energy, moving it more quickly into the enzyme’s active site. Water molecules also move around at higher speeds, colliding with substrates, perhaps making them break apart. At this point, the optimum rate, it then begins to drop, and as the enzyme denatures, we can see the rate of reaction steadily drop until at 80 degrees it is 0. The high heat makes the enzyme, which is only a protein, lose it’s unique key shape, so it can no longer fit in the ‘lock’ of the substrate, as shown below.
As we can see, the high temperature has stopped the enzyme being able to fit into the substrate’s ‘keyhole’ shape as it is a protein and has denatured. This is why we saw no change at 80 degrees; we had allowed a short period of time for the temperature to denature the enzymes. This meant that when the photographic film was inserted, all the enzymes were denatured and useless. At 60 degrees, only some of the enzymes are denatured, leaving the others to remove the gelatine from the photographic film. We can clearly see that at around 40 degrees there is an optimum temperature. It is also interesting to notice that this is near body temperature, however body temperature is slightly lower as they body would need more energy input to maintain a higher temperature, so it is more energy efficient to work at a slightly lower temperature. At 20 degrees we can see the the rate of reaction still increases after this temperature, showing that the optimum enzyme temperature is obviously over 20 degrees. At 0 degrees there is no reaction. The reason why 20 degrees at 0 degrees ended up with lower reaction times or times about the cut off point is because the enzyme and substrate have less heat energy, therefore less kinetic energy, so the substrate will move into the enzyme’s active site more slowly. This means that it will take longer to break down the enzyme. The graph, and these descriptions support my hypothesis completely, with very little difference between my hypothesised graph and my real graph, bar minor differences. My hypothesis was that as temperature increased, as would rate of reaction, to a certain point whereby the enzymes would denature and the rate of reaction would start to drop till it reached zero. This is the exact same conclusion that I have drawn below. There is a very secure support of my first prediction, and this shows not only that I was correct, but at exactly what points the optimum trypsin level is.
I can also say that our results are very accurate, they are very similar to my planning results, and the two numbers from which I took my average were close together. I can put the fact that the planning experiment figures being lower than the final experiment figures down to the fact that we decided to stop the timer only when the photographic film was completely clear. I also found no anomalies in my results, which shows that my planning was effective and my experiment accurate.
We can therefore conclude from all of this, that the rate of reaction for trypsin is a curve, peaking at around 40 degrees, and dropping to zero around 80 degrees. We have firm support now for the kinetic and lock and key theories, and I have proved my hypothesis correct.
Evaluation
The experiment went very smoothly, and we worked efficiently and quickly from the beginning of the experiment. The results fit the prediction perfectly, backing up my hypothesis completely. We found no anomalies in our results, and they were all fairly accurate. We didn’t get any anomalous results because we were precise with our timing, we allowed the enzymes to acclimatise, and we ensured all variables, bar the temperature, were kept constant. We did not have to ignore any results, as they were all relatively accurate to what they should have been. We also know that none of the variables such as pH change or contaminations with fingertips occurred, since there were no anomalies. However, there is always the problem of human error, as they human eye is not a measuring device and works on what the brain deems to be correct rather than precisely measuring what is correct. We could have used a photosensor, which measures how much light passes through it. If we place this behind the photographic film, our results could have been accurate to a fraction of a second. There would also be no problems with human judgement. A light dependent diode could also have been used, and, when connected to a voltmeter, we could work out at how many volts the timer should be stopped (as the LDD works as when the light changes, as does the voltage), also giving us more accurate results, and would be viable to do in a school environment. More accuracy could also be employed with our range of temperatures; the main problem with our graph, is our curve is a hypothesis of what should be in between each number; we do not actually know what the curve looks like in between values. Therefore, for much more accuracy and a more accurate graph curve, we could have done tests every 5 degrees rather than every 20 degrees. This would ensure our graph’s line of best fit is the correct shape. We could also have extended our investigation by using different substrates and enzymes, examining how each substance breaks down, and which substrates break down more quickly. For further investigation, we could have even used pH as our independent variable (the one that did not change). However, under the circumstances, I believe that I did fairly well and produced an accurate table of numbers.