Enzymes work better at higher temperatures, up to a certain point. This is to do with Kinetic Theory. Each atom has a bit of Kinetic energy so it moves around. When substances are heated up, the atoms gain energy. The more energy atoms have, the more they bump into each other. Atoms that react with each other are more likely to react quickly if they are heated together, because then they move around more, bump into each other sooner, and react (they can only react when they meet each other), so an enzyme is more likely to react with a substrate if they are at a higher temperature. However, if an enzyme molecule is heated too much, the atoms inside it move around too, and the shape of the enzyme is permanently changed, leaving it unable to do its job (the shape is vital, otherwise it won’t be able to latch on to the molecule it’s reacting with). This is called denaturing. Most enzymes are denatured at about 40ºC, and this is why normal human body temperature is about 37ºC. If your temperature rises too high, the enzymes inside your cells that release chemical energy and break down poisonous hydrogen peroxide molecules to keep your cells alive are denatured and you cannot stay alive, and this is why it is fatal for your temperature to rise to 44ºC or more.
Trypsin is a digestive enzyme that breaks proteins and peptides (simpler proteins) down into amino acids and it works in the duodenum, which is just before the small intestine, but it is produced in the pancreas. It works best in slightly alkaline conditions (pH 8.0).
A graph showing how the activity of enzymes in general are affected by temperature:
Diagrams of enzymes breaking foods up:
AN EXPERIMENT TO INVESTIGATE THE ACTION OF TRYPSIN
For my coursework I investigated how Trypsin is affected by temperature.
Preliminary work:
The first part was to investigate how long it took for test tubes of water to acclimatise to the temperatures of water baths. (This was done 3 times for each temperature). The second part of the work was to compare the time it took for 3 different concentrations of trypsin at 40ºC to digest the albumen, in order to decide which was the best concentration to use for the real experiment. If the concentration was too dilute, it would take too long, but if it were too concentrated it would happen so fast it would be difficult to measure.
To see how long it takes for water to heat up:
Average time (rounded) = 218 s. /3 min. 38 s.
Testing enzymes:
From these results, I can see that Enzyme A is the strongest concentration, followed by B, and then C. I decided to use concentration B for the experiment when I did it for real as C took far too long, and A was a bit too quick and it would be harder to get an accurate reading because of that.
Predictions:
I think that the rate of reaction will increase up until c. 40ºC, and then it will decrease, because the enzyme will work better at its optimum temperature, which is just under 40ºC, but after this temperature it will denature and stop working. Apart from temperatures higher than its optimum temperature, it will work better the higher the temperature gets because the heat will give the enzymes and albumen molecules more energy so they move around more and are therefore more likely to collide and cause a reaction. If I use the same concentration of Trypsin as in the preliminary work, I should get similar results for the reaction at 40ºC.
Method:
In one test tube, 2cm3 of suitable buffer (acidity/alkaline-regulating chemical) and 1cm3 of 0.25% albumen (egg white, rich in protein) are mixed together. Another tube contains 1cm3 of 1% trypsin solution. They are both placed in a water bath for approx. 4 minutes until the desired temperature is reached (the set temperature of the water bath). Then they are mixed together and the time it takes for the mixture to turn clear is recorded. To make it easier to tell, and to keep a standard, compare it to a test tube full of water.
Safety:
This enzyme can dissolve human tissues so safety is very important! Goggles and lab coats or overalls should be worn, and hair tied up if possible. Any spillages should be cleaned up as soon as possible. Also you should not run in the lab.
Results - Time:
I decided to do each of these three times, but some of the results weren’t quite what I expected so I did them more than 3 times.
Results - Rate of reaction:
(Here, the rate is 1 divided by the mean time, and then multiplied by 100.)
Conclusion:
The experiment is done three times minimum for each temperature, to get a good average, and also it might not go right the first time. Once the results have been written down in minutes and seconds, they have to be converted into seconds, and an average for each temperature is calculated. This average is what is used in the graph. The rate of reaction is worked out by dividing 1 by the average number for each average. This results in tiny numbers, so to make it a bit easier they are ALL multiplied by 100.
In the graph I have drawn of temperature and rate, the rate of reaction at 65ºC seems a bit high – this is probably an error – e.g. thinking it had turned completely clear when in fact the reaction hadn’t yet finished, comparing it to cloudy water, an error in writing down the time, or an error in timing.
General trends in the results graph: it is clear that the increase in the rate of reaction slows down around 50ºC and after this temperature the rate decreases. This is because at 50ºC the enzyme is denaturing – some enzymes will have already denatured, while others are still in the process of becoming denatured. By about 60ºC they have all denatured. The reason the reaction still happens at this temperature, even if it’s a lot slower, is because heat speeds up chemical reactions. Also heat itself can break down food particles – enzymes do not cause the reaction, they catalyse it. This experiment’s results are similar to what I predicted, but I thought the rate would decrease faster after 40ºC – instead the reaction worked faster at 50ºC than it did at 40ºC. (I thought it would be slower because the trypsin would have denatured by then.)
Q10: The Q10, or Temperature Coefficient, is an expression of the effect temperature has on a reaction. The formula for it is, using ‘t’ to represent the chosen temperature, is:
Rate of reaction at t + 10ºC
Rate of reaction at t ºC
In general, the Q10 doubles with every increase in 10ºC. This can vary with biological experiments like this, because of denaturing. Between 45 and 55 the Q10 increase is less than double, and I think this is because the rate of reaction is not increasing so much between those temperatures because of denaturing. The Q10 between 27 and 37 is triple, not double, and I think this shows the massive increase in rate between these temperatures as it is reaching the optimum temperature.
Evaluation:
Some of the results came out differently to what was expected – for example, after 40ºC the enzymes would have been denatured and so should have had a lower rate. This is because I took more than one lesson to complete the experiment, and the trypsin, like any enzyme, is slightly different each time it’s made, and these tiny differences can make a big difference to the results. The albumen could also have been different every time it was made.
Ways to improve:
I think that to make it more accurate next time, the experiment has to be completed in one day so that it is the same batches of chemicals that are used. More thermometers could be placed in the water baths to make sure that they are at the correct temperatures. There is another procedure to test trypsin but it uses photographic film instead of albumen, which is more difficult but gives more accurate results if done correctly.
Using photographic film: this method uses photographic film, which is covered in a layer of proteins, which can be digested by trypsin.