The effect of temperature on the activity of trypsin.

The effect of temperature on the activity of trypsin

Method:

To begin the experiment safety precautions were carried out, goggles and a laboratory coat was put on. A water bath had been previously prepared for us at 30c. Three test tubes were labelled ‘30c casein’, ‘30c trypsin’ and ‘30c control’. Using a pipette, 5cm of casein suspension was pipetted into each of the test tubes labelled ‘30c casein’ and ‘30c control’. Using another pipette to avoid contamination, 5cm of trypsin solution was pipetted in the test tube labelled ‘30c trypsin’. And again, using a different clean pipette, 5cm of distilled water was added into the test tube labelled ‘30c control’. The three test tubes were then placed into the water bath and left for several minutes to reach the temperature of the water (we tested this by placing thermometers in each test tube, we had to use three thermometers because if one thermometer was transferred from one tube to the other it would have contaminated and thus started the reaction). The enzyme (the trypsin) was then added to the substrate (the casein) and agitated to ensure that they were thoroughly mixed. This was then replaced in the water bath and a stopwatch was started immediately. The contents of the tube were then carefully observed (and every now and again the tube was agitated to keep the contents moving) and checking against a piece of black card the time taken for the suspension to clear was recorded.

We then repeated the exact same process at different temperatures (using different water baths), at temperatures 50c, 60c and room temperature.

Results:

Personal results (working with Francesca):

In order to work out the time taken for suspension to clear in seconds, the time in minutes was multiplied by 60.

In order to work out the reciprocal, 1 was divided by the time taken for the suspension to clear in seconds.

Class Results:

* We were group 1.

In order to calculate the reciprocal, 1 was divided by the time taken for the suspension ...

This is a preview of the whole essay

We then repeated the exact same process at different temperatures (using different water baths), at temperatures 50c, 60c and room temperature.

Results:

Personal results (working with Francesca):

In order to work out the time taken for suspension to clear in seconds, the time in minutes was multiplied by 60.

In order to work out the reciprocal, 1 was divided by the time taken for the suspension to clear in seconds.

Class Results:

* We were group 1.

In order to calculate the reciprocal, 1 was divided by the time taken for the suspension to clear.

In order to calculate the average of the 1/t, the 8 values were added together and then divided by 8.

From my results I can draw a graph, I will draw two lines: one for my personal results and the other for the average class results. I will put them on the same graph so that I will be able to compare them.

So that I can plot my graph easier I will change all the 1/t values into standard form values.

0.0048 = 0.48 x 10 -2

Analysis:

The two lines of my graph are similar and yet also different. At first both of the lines rise, this indicates that as the temperature is increased the time taken for the suspension to clear became less (the rate of reaction increases). But after a certain point they both differ; my personal line goes up, suggesting that the time taken for the suspension to clear became even quicker, but the class line goes slightly down, which suggests that the time taken for the suspension to clear was slower.

My line shows a pattern in which as the temperature increases the enzyme activity increases. The class line shows that the activity of the enzyme will increase if the temperature increases, but only within a certain temperature, after that it will then start to decrease.

Using my knowledge of the effect of temperature on enzymes I can understand why there are differences in my two lines.

If other factors such as pH, enzyme concentration and substrate concentration are kept constant then temperature has an effect on enzyme activity. At the beginning, a rise in temperature will increase the kinetic energy of the enzyme and the substrate molecules, and will therefore tend to increase the rate of a chemical reaction. On the other hand, an increase in temperature will tend to affect the stability of the enzyme molecule, they will vibrate and the chemical bonds that maintain the tertiary structure of the enzyme molecule creak. This will cause the shape of the enzyme to change, if the shape changes then the active site will also change and the enzyme will not be able to combine with any substrate molecules and therefore no enzyme-substrate complexes will be made. Therefore there is an optimum, which the enzyme will be able to work at its best and most rapidly; this is when the two factors are balanced. At temperatures above this optimum the enzyme rapidly loses is activity and becomes denatured, (unable to combine with the substrate molecules).

From this information I can say that this is what the class line showed me. So why is my personal line different? This is down to factors affecting the accuracy of the experiment (discussed in conclusion).

Conclusion:

Problems with the experiment is evident when looking at the class results, all of the results are very varied. This is due to problems with accuracy of human errors. There are several points to consider:

How clear is clear? – It is hard for one person to decide when the suspension is clear; lots of people have very different opinions and interpretations to what is clear. This is a very important factor because it alone could affect the results of the experiment.
Accuracy of measuring solutions – This might not affect the results as dramatically as some of the other factors but nevertheless needs to be considered. When measuring out the trypsin and the casein it is very hard for a human to get a perfect measurement of solution.
Temperature – It is very hard to maintain the temperature of the test tubes constantly. When the water baths was first set up, the temperature was set up to as needed. But as the lesson went on, as more and more people used the water bath the temperature went down. This is due to the fact that the lid was taken off of it and also more and more cold test tubes from each group were going into it so the energy went into heating the tubes up. A difference in temperature could affect the results of the experiment.
Some groups were treating the test tubes differently from others: after when the enzyme has been added to the substrate some groups remembered to regularly agitate them to ensure that they were still moving, other didn’t mix the tubes thoroughly and were left with a substance that was clear at the top but still cloudy at the bottom. Some groups also accidentally contaminated the test tubes by using one thermometer for the trypsin and the casein.

Considering all of these factors, it explains why my personal line was different from the class line.

If this experiment were to be carried out again I would try and make improvements:

A shared water bath is not ideal as the temperature of the water bath will drop and affect the results. Individual water baths should have been used. The temperature of the test tubes needs to be accurate in order to produce accurate results and a graph.
The test tubes need to be agitated regularly, as the milk is denser than trypsin and could sink to the bottom of the test tube and also contamination needs to be avoided at all costs.