'Carry out an investigation to determine a factor affecting the rate of digestion of gelatin by the protease trypsin'.

Preliminary Work

For my preliminary experiment I used different concentrations of trypsin, 1%, 2% and 5%. I put them all in about a 40 degrees water bath and found that with the 1% solution I was waiting for too long and so would not get the results I wanted as quickly. As well as being expensive, 5% trypsin solution was too quick and would not leave me enough time during the experiment to record my findings and obtain my results. In the end I decided on 2% trypsin solution as this gave me enough time to get myself sorted out and gave me perfectly good results. There was also a slight error on the way I timed the duration of the experiment. A stopwatch did it all manually and I had to decide the end point for the experiment. By using the results from my preliminary experiment I was able to roughly tell where the end point would be for my real experiment. When this time neared I lifted the gelatine out every ten seconds, as this was adequate timing to collect my results and keep the stirring of the solution down to a minimum. I determined my end point when all the gelatine had completely gone off the film leaving it transparent. Here are my results from my preliminary experiment:

You will see that all my results are multiples of ten, this is because I was checking the gelatine film every ten seconds and so the couldn’t have been anything else. My preliminary experiment was very useful as it aided me a lot in what I was going to do in the real thing. It also confirmed any doubts I had previously about what to do. I realised that temperature would e the easiest variable as pH is easily controlled using a buffer and stirring was kept to a minimum due to useful results from the preliminary test. Also the concentrations of the enzymes and the gelatine were always constant so there was little to be done there. I was difficult to get the temperature at a exact degree which is why I chose it as once the points are plotted on the graph this doesn’t matter because you can see what the rate of reaction would have been at that temperature anyway.

Prediction and the scientific basis for making it

From a text book I obtained the equation Q10 = 2, this means that for every ten degree rise in temperature the rate of reaction will double. For example if the rate of reaction is X at 30o then at 40o it will be 2X. This is my prediction, I have predicted this because when the temperature rises the molecules move faster in the solution and therefore collide more quickly, which in turn speeds up the reaction.

Apparatus

• Water Bath  -  For heating the trypsin solution.
• Test tube  -  For containing the trypsin solution during the experiment.
• Syringe  -  To transfer the trypsin solution from the plastic beaker to the testube.
• Plastic beaker  -  Contained the trypsin waiting to be used in the experiment.
• Thermometer  -  To measure the temperature of the water bath and the trypsin solution in the test tube.
• Test tube rack  -  To hold all the used and unused testubes for the experiment
• Stopwatch  -  To time the length of the reaction.
• Splints  -  These held the gelatine film in place in the test tube.
• Gelatine film  -  Used for the experiment.
• Trypsin solution with buffer  -  used for the experiment.
• Scissors Used to cut the splint so that the film could be held by it.
• Plastic tray holding gelatine film  -  Kept the gelatine from touching anything which would affect the reaction time.
• Conical flask  -  This contained the trypsin before I transferred it to my plastic beaker in preparation for my experiment.

When set up properly the apparatus will look like this:

Method

1. Pour some trypsin out of the conical flask into the plastic beaker in preparation for the experiment.
2. Use the syringe to measure out 2cm3 of 2% trypsin solution and transfer is to one of the test tubes.
3.  Cut a small slit in a splint and slide a piece of gelatine film halfway into it. (Only slide it half way in so you can see the difference between the reacted and non-reacted gelatine.
4. Put the test tube into the water bath and let it heat up to the temperature of the water.
5. Test that both the temperatures are the same using the thermometer.
6. When both temperatures are the same put the splint with the film on the end into the test tube containing the trypsin solution while simultaneously starting the stopwatch.
7. Check the gelatine film every ten seconds to see if it has reacted.
8. Stop the stopwatch when the film becomes transparent.
9. Record the results in a table.
10. Throw the used gelatine film away and wash out the test tube.
11. Be sure to mark down the exact temperature of the trypsin solution, this is very important.
12. Wear safety goggles at all times to be safe and protect your eyes.
13. Take three readings for each temperature to eliminate any anomalies and be sure you are getting consistent results.
14. Take readings from 20o to 50o where the trypsin enzyme starts to denature but eliminate results where it is clear that the enzyme has denatured due to a slower rate of reaction. Do four different temperatures, 20, 30, 40 and fifty degrees.
15. .Put all your readings on a table and then find out the rate of reaction and then use the modal average for the graph, plot a graph in order to analyse them.                  

                                                                                                                                                      Results

Rate of reaction

To find out the rate of reaction I changed the time into seconds and divided it by one, then I multiplied the answer by 1000 to get a more practical number. Like the results I also took the modal average of the three readings.

Discussion

I am able to see clearly that as that temperature increases, so too does the rate of reaction.   I found the rate of reaction increases until it reaches 57o where it denatures due to excess heat, it denatures as the hydrogen bonds, ionic bonds, covalent bonds and sulphur bridges break, this causes the active site to be denatured making the enzyme inoperative as the substrate is no longer able to fit into them. By looking at my graph I can clearly see that in the beginning the rate of reaction more or less doubles, but then slows down considerably after that, this is shown by the decreased distance between the points at the higher temperatures. I eliminated my readings after 50o as these temperatures are where the enzyme starts to denature so effectively they are invalid results. The rate if reaction doubles in the beginning as there is little denaturisation going on in the enzymes, this means there are more to react and so the reaction happens faster. However, with the higher temperatures it takes longer for the trypsin to heat up and so even before I put the gelatine film in it is already partially denatured and continues to denature throughout the experiment. This denaturisation is fairly significant as looking at the graph it is clear that the rate of reaction decreases massively as we get to the higher temperatures due to the fact that too many active sites are misshaped by the time we get to about 50o, so the rate of reaction does not double as before, but only increases slightly and eventually becomes less than the temperature before at 57o (which I have not recorded here as it was too affected by denaturisation. Its rate of reaction was 9.09 though) the active sites are too denatured to produce valid useful results. The rate of reaction increases by so much at the beginning as it takes the trypsin less time to get the designated temperature and so the enzymes have less time to denature causing there to be a lot more active sites available than at 57o for example. The optimum temperature at which the trypsin works is 50o as after this the rate of reaction is less and therefore the reaction doesn’t happen as quickly as before. Also at higher temperatures due to the collision theory the reaction is able to work more quickly. The collision theory is that with increased temperatures the molecules in the solution move more quickly and gain more kinetic energy and therefore the trypsin collides with the active sites more often, speeding up the reaction time.

Conclusion

My results were similar to my prediction as looking at the lower part of the graph Q10 = 2 does apply as the rate of reaction seems to more or less double for every ten degree rise in the temperature. However, when the trypsin was placed in higher temperatures my prediction was not correct as the rate of reaction only increased by small amounts and eventually decreased due to denaturisation. I would say that my prediction is correct to an extent, as I did not anticipate the denaturisation of trypsin at higher temperatures, so  it is definitely true for the lower part of the graph and the first few results but after that due to the misshaping of the active sites it is not correct for the higher temperatures. Effectively these higher results are not as valid as the lower ones as the trypsin has denatured whilst it was heating up, and also during the experiment, whereas for the lower temperatures where it took less time for them to heat up the denaturisation was much less producing more valid results. If the trypsin were not to denature then I think that the rate of reaction would still be increased by double the amount for every ten degrees. However, this evidence clearly shows that this is not true but it is safe to say that overall my prediction supports my results to a certain extent and no further. I definitely feel very confident in making this conclusion as I carried out my experiment with precision and fairness so the results are perfectly valid and true. Therefore I can take my conclusion and results to be fair. Although the results for the higher temperatures are not compliant with my prediction, however, there is an excellent scientific reason for this and that is that the enzymes denature and became inoperative at these temperatures. I am unable to say that my results are definitely correct, but I think it is fair to presume they are and not just due to chance as I did each  result three times and got the same result or one very near to it.

Sources of Error

I think that the equipment was suitable for the experiment in its accuracy and fairness, in my method I was as accurate as possible and tried to get valid and fair results at every possible moment, I recorded my results in a table as soon as obtained them. Clearly there is the factor of human error and it is inevitable that there would be some errors in my experiment and results but overall I think that my results are fair and good enough to form a conclusion and analyse them.

1. Possibly the largest source of error was the lifting of the gelatine film in and out of the water bath. Here the time taken to look at the gelatine and see whether the experiment had finished was actually part of the ten seconds and not allowed for, so this decreased the time the gelatine was really in the trypsin. Also whilst out of the trypsin it would have cooled and then had to heat up again in the solution causing some difference to the real reaction time.
2. Also on doing the experiment I was the one who had to define the ‘end point’ of the experiment and it was not always entirely clear as to when this was. For some of the results a little bit of the gelatine remained on the film but most of it was clear, this is where I may have made a few errors in recording my results, taking them too early or too late adding or decreasing a whole ten seconds onto or off the time. This is where human error comes into it and must be allowed for or corrected.
3. When lifting the gelatine film in and out of the trypsin I unintentionally would have stirred it a little, which would have shaken of the saturated gelatine and trypsin on the surface of the film and allowed new unsaturated trypsin to reach the gelatine underneath and speed up the reaction.
4. The amount of gelatine in the trypsin would have also varied as I only put about half in so I would be easier to define the end point. This would have varied with each time I did the experiment and may have affected my end results.
5. Before beginning each experiment I had to let the trypsin solution heat up in the water baths prior to adding the gelatine film. The time it takes for the trypsin to heat up is called the incubation time and inevitably some denaturisation would have occurred here and so instead of being 2% the trypsin at the beginning of the experiment it may have only been 1.5% when the gelatine film was added.

I emitted one reading of 57o as this is where the enzyme starts to properly denature affecting the end result to a substantial degree as this would have definitely changed my graph and would not have been a good enough valid result to use. All the results I did use seemed to be adequate and behaved as I would expect. There were no anomalous results as far as I can tell and my graph shows no outliers this shows that the experiment went as well as it could have done. My method, plan and experiment were correct and performed to the best of my ability, so I can see no further sources of error. So I feel very confident about my earlier conclusion because as far as I can see my evidence is precise and reliable enough for me to know that it is perfectly sufficient for me to use. I am sure my earlier conclusion is correct as I checked each result three times and cam to within ten seconds if not the same as the one before. However there were errors with my results as they were not all the same for the same temperatures, these can be modified as follows.

Modifications

If I had a water bath with a glass wall, then I would be able to check my results all the time without having to lift the film out of the water, which otherwise disturbed it and definitely affected the rate of reaction. However near the end of the experiment the trypsin solution becomes cloudy and it would be extremely difficult for me to determine the end point so I would have to use a photometer to measure the cloudiness of the solution, I would know the correct reading by doing a preliminary experiment to find out. This method would eliminate the error of stirring the solution and also taking the film out to check so I would actually be in the trypsin solution for the amount of time recorded and not just most of it. I would have to trypsin heated up to the correct temperature more quickly in the water bath, as this would definitely prevent some denaturisation. Another method of doing the experiment is to not use gelatine film, but to use actual gelatine but just die it before putting it in the solution. Then you could see exactly where the end point was when all the die had disappeared. To extend the experiment I would do it with different concentrations of gelatine and trypsin to see how the rate of reaction is affected, this would tell me whether concentration of temperature had a larger effect on the rate of reaction. I would also later on change the variable to pH so to also determine which had the bigger effect on the rate of reaction. Here I could make different predictions and test them. I could use a larger range of temperatures and then test my current prediction with them. If I had further time I would conduct these experiments and then use them to test possibly my current prediction or a different one depending on what applied to the experiment.