The Effect of temperature on the activity of Trypsin


Introduction: Casein is a protein found in milk. When suspension of casein is hydrolysed, the suspension starts cloudy but becomes clearer as the products dissolve. This hydrolysis is catalysed by proteolytic enzymes such as trypsin.


Aim: The aim of the experiment is to investigate the effect of temperature on the activity of trypsin, using a suspension of casein as the substrate.


Hypothesis: It is expected that the higher the temperature, the higher the rate of reaction until the enzyme reaches its optimum temperature at which it functions most rapidly- which is expected to be around 45 C. After which the enzyme loses its activity and becomes progressively denatured.


Materials used:



Casein suspension,


4% (used Marvel milk powder, 4% solution)


Trypsin solution 0.5 %


Distilled water


Water baths (set at different temperatures)


Test tubes and rack


Graduated syringes


Thermometer


Black card


Stopwatch



Method:


Firstly, a water bath was set up at 35C.


Using a syringe, 5 cm of casein suspension was transferred into a test tube and 5 cm of trypsin solution into another tube.


Stood both tubes in the water bath and left them for several minutes to reach the temperature of the water bath.


Then the enzyme and substrate were mixed together and the tube was replaced in the water bath.


A stopwatch was started immediately.


Observed the contents of the tube carefully, checking against a piece of black card, and recorded the time taken for the suspension to become clear.


Repeated the same procedure for different temperatures. We carried out our experiment at 24C (room temperature), 35C, 45C, 60C and 75C.


P.T.O for Graph and Table of Results.


Conclusion: The table of results and the graph shows that the optimum temperature for my experiment was 75. Albeit the values of the rate of reaction were very small it still proves the hypothesis wrong and has shown that trypsin actually works faster in much higher temperatures than expected of most enzymes. However, when looking at the results for the experiment for the class as a whole, i.e. when an average is taken, it clearly shows there is a peak at 45 C, so there is an inconsistency between the 2 sets of results.


Evaluation: There are a number of factors which could have caused the errors in the experiments. Misjudgements could have been made on the temperatures of the reactants. By carrying out the experiments in haste, the substrate and enzymes may not have reached the temperature of the water bath and were mixed too early. So therefore the consequent results may not have been a true reflection of the actual rate of reaction.


When looking the graph of the rate of reaction of my experiment by itself, there were obvious inaccuracies or inconsistency. The line of the graph seems to be a slight peak at 45C, and then the rate goes down at 60C, only to come back up again at 75C. It was an anomalous result, and when compared to the results of others in the class, it further confirms that the time taken for the suspension to clear at 60C was rather higher than most.


There were some other anomalies in the experiments dome by others in the class, for e.g. in experiment No.11, at 45C, their time is a mere 5 seconds while the most of the class had times at least over 100 seconds. Again at 45C, experiment No.13, had a very long time of 776 seconds. These 2 results highlighted seem to be the extremes of one another.


The experiment could be improved if it was carried out at least 3 times by each person, so that we would more sets of our own results to compare with and with more practice of carrying out the whole experiment, our results would be much better reflection of the actual rate of reaction.


Using a control test tube next time with casein suspension plus distilled water in each water bath would let us compare the difference in reactions.


Finally, using a colorimeter to measure the transmission and/or absorption of light would give us a much more scientific measurement of clarity of the suspension after the enzymes and substrate have been mixed.