To investigate the affect of temperature on the enzyme trypsin.

The effect of denaturing is the reason why we must allow the trypsin to acclimatise to the temperature being tested. If not allowed to acclimatise, then no denaturing will take place at higher temperatures prior to the introduction of photographic film. Thus the highest temperature will show the highest rate of reactivity, which is only true until the trypsin reaches the stated temperature. This is similar to the use of enzymes in industry, where a great mass of enzymes is added to the substrate at a high temperature so that there’s a very fast (and short lived) rate of reactivity.

2) The kinetic theory.

This is theory describing how as temperature increases, so does the kinetic energy of molecules, and atoms. Therefore, the colder something is, the less it’s molecules move, and vice versa. In terms of the ‘lock and key’ hypothesis we can therefore see that as temperature increases, so will the rate of digestion, as the substrate will be entering the active site at a faster rate. However, there is a temperature boundary for enzymes, where denaturing begins to occur. This is usually at around 60°C when the temperature damages the enzymes active site, thus its work rate is lessened.

It’s for this reason that we observe temperature, as it is seen to affect the enzyme activity in a way that can affect its rate of reaction.

VARIABLES: 

1. Temperature
2. pH level
3. Substrate concentration/amount of photographic film

Dependent/Independent?

An independent variable is one that being changed throughout, and is independent of other factors. It is shown on the x-axis of the graph. In this case, it is temperature. The specific temperature being analysed will be maintained by a water bath. Also the enzyme will be allowed to acclimatise to the temperature being tested, so that the actual temperature is tested, and not the increase leading up to that temperature. The outcome of no-acclimatisation is seen on the predicted graph at point A.

A dependent variable is one that relies on a number of other factors. It should however only rely upon the independent to make it a fair test. In this experiment it is the rate/time of reaction.

The following variables are therefore acknowledged for their affect on the experiment, and controlled to allow a fair test to be conducted:

PH Level:

Proteins, including enzymes may be affected by pH. Each has an optimal pH level it requires to work at its best. An enzyme will work at most pH levels, providing they are not extremely different from their own optimum level. If the pH is vastly different from the optimum, then the enzyme may become denatured as the ionization of the hydrogen bonds between R groups is damaged, and the configuration of these side chains is changed. The optimal pH of trypsin is about 8.

To control pH, buffer will be used in order to maintain a constant pH. The presence of buffer has no adverse affect on the reaction.

Substrate Concentration:

There is a clear difference in reaction rate at different concentrations of substrate. At a low concentration, the collision of enzyme and substrate molecule is infrequent, thus the ‘lock, and key’ procedure takes place less frequently also. As the substrate concentration increases, so does rate of reaction rise proportionally, as the collisions are now more frequent. When the substrate concentration reaches a point where the enzyme is at it’s maximum activity, the effects of substrate concentration on reaction rate are none. At this point, the enzyme is saturated, and the reaction remains at the saturation level.

The substrate concentration is kept constant, as the same size and type of photographic film are used in each case. A piece 9mm² will be used.

APPARATUS:

• Test tube rack- to prevent the tubes rolling and smashing. Also, to hold the tubes being tested at 20°C (room temperature).
• 10 test tubes-to contain the trypsin, water, and photographic film.
• Trypsin, and buffer solution-to act as an enzyme in this experiment. Buffer maintains a constant Ph.
• Water-to create control.
• 15 spills-to hold the photographic film
• 15 pieces of 9mm² photographic film-to act as substrate for the trypsin.
• 3 water baths- to maintain one set temperature, for the test to work at.
• Stop watch-to measure the length of time for digestion.
• 5 thermometers-to ensure that each temperature is correct.
• Ice-for the test carried out at 0°C.

PRELIM PROCEDURE: 

We carried out preliminary tests, to understand how the test works, and discover any problems with our methods. Ultimately this will provide us with better results, as any problems are foreseen here, and then altered.

• Ensure a safe working environment, by clearing the entire area of all unneeded equipment.
• Add 5ml of trypsin, and buffer solution, to 5 different test tubes.

• Prepare control test tubes by adding 5ml of water to another 5 test tubes.
• Place 2 test tubes (one with trypsin, and one control) in the water bath, (or ice).
• Allow 5 minutes to acclimatise to the designated temperature.
• Use the thermometer to regulate the temperature of the water bath, adjusting the water bath dial where necessary.
• Add the photographic film to both test tubes, and begin timing.
• Examine the film every 30 seconds.
•  Record what changes (if any) occur in the tubes.

MAINTAINING A FAIR TEST: 

To ensure a fair test, not only do the afore-mentioned variables need to be controlled, but also the following procedures must be carried out, to create a fair test, and test results.

1. Ensure that the solution has been allowed to acclimatise, otherwise the temperature of the enzyme being tested will not be the same as that recorded, as we know that temperature affects enzyme activity due to the kinetic theory. Enzymes can work up until a certain temperature (in this case 60°C); thus in the experiment at 80°C, if we did not allow the solution to acclimatise, where no result is expected, we would see a result, as it would be working very fast until 60°C. This is seen in industrial use of enzymes, where many enzymes are added to a substrate at high temperatures; so they work very fast at first, denature, only to be replaced by more.
2. Use control to provide proof that no other factors are responsible for the any changes occurring in the enzyme experiment. If any other variables are discovered in the prelims, they must be accounted for, and controlled in the actual tests. If no changes are seen in the control, then we know that the changes seen are due to the action of enzyme.
3. Try to keep handling of photographic film to a minimum, and make sure hands are clean. This will prevent any other substances entering the test, and providing unaccounted for variables. This intern will lead to inaccurate results.

SAFETY PROCEDURES: 

In a work environment certain regulations must be followed to provide safety from accident in the lab.

• Goggles must be worn at all times, as the trypsin enzyme is harmful to eyes.
• If trypsin contacts skin, it must be removed, as trypsin is an irritant.
• All test tubes must be held in a rack, to prevent damage, breaking, and potential hazard.

PRELIM RESULTS: The results we obtained are recorded below.

(Anomalies would usually be highlighted, however there are none at this stage to highlight, as the results are in fitting with the prediction)

No change was noted in the control tubes.

*These results count as 0, as there would be no change, (or at least an insignificant change), over infinity.

ANALYSIS OF PRELIMS:

This graph is very similar to that seen in my hypothesis. However, some changes are evident these; are due to the period of time between the examining of the film. The time period was too long, and not accurate enough. For instance the actual time of change may have been at 1minute 10 seconds, but due to the checking period, is rounded up to 1 minute 30 seconds. Also, the time for 60°C was a little longer than expected, this is because we allowed the 5 minutes for acclimatization. Thus, the denaturing period was prolonged, before the enzyme was added. If instead, the photographic film is introduced as soon as the target temperature is reached, then the amount of denaturing before film introduction will be decreased. No change was seen in the control therefore, we know that we have accounted for all of the variables successfully.  

ALTERATIONS TO PROCEDURE:

From the analysis, the following changes are to be made, to improve the accuracy of the test:

1. This time, there will be three tests for each temperature. These are known as replicates, and will provide more accurate results, as an average may be taken, and a fairer result for each temperature provided.
2. The photographic film will be introduced as soon as the solution reaches the designated temperature, so that the substrate is introduced at the most accurate level of enzyme activity/denaturing.
3. Increase the accuracy of measurement, by examining the film every 5 seconds, as opposed to 30.
4. Controls are no longer necessary, as we saw last time, that no changes occurred.

FINAL EXPERIMENT

APPARATUS:

• (AS BEFORE)
• 20 test tubes- to enable the use of replicates (3x5=15) + control test tubes (1x5=5) = 20

PROCEDURE:

• Add 5ml of trypsin, and buffer solution, to 15 test tubes.

• Prepare control test tubes by adding 5ml of water to another 5 test tubes.
• Place 3 test tubes with trypsin, and 1 control test tube in each of the water baths, (or ice).
• Use the thermometer to regulate the temperature of the water bath, adjusting the water bath dial where necessary.
• Add the photographic film to test tubes, as soon as the temperature is reached, and begin timing.
• Examine the film every 5 seconds.
•  Record what changes (if any) occur in the tubes.

RESULTS:

 Again, no change was noted in the control tubes.

No anomalies noted.

* These results count as 0, as there would be no change, (or at least an insignificant change), over infinity.

ANALYSIS

In relation to my hypothesis, we see that it is proven correct by this experiment. The enzyme did increase in activity, until 60 o C, where it appeared to become denatured.

Analysis of graph

1. At this stage, the curve is similar to that predicted. It shows a low rate of reaction, as there is little thermal energy to provide both enzyme and substrate with enough kinetic energy to allow a significant number of collisions. Therefore, this is proving the kinetic theory, and the lock and key hypothesis.
2. A sharp increase is noticed here making the graph significantly steeper. The increase in thermal energy increases the kinetic energy of both substrate and enzyme, creating a considerable rate of reaction, due to the lock and key hypothesis. At this level, the amount of denaturing is insignificant.
3. The optimum temperature is reached, whereby the enzyme is working at its maximum level. At this temperature there is a high level of kinetic energy, allowing substrate and enzyme to move quickly, leading to a greater number of collisions, and a greater level of reaction. Thus the greatest amount of products produced. The amount of denaturing is certainly increased, but not to a point where it greatly affects the rate of reaction.
4. The affect of denaturing is so great here, that it is more substantial than the effects of increased kinetic energy. Therefore, we see the rate of reaction decline considerably from the optimum temperature. The enzymes do work momentarily at a very fast rate at the temperatures leading up to this temperature, however since acclimatisation has occurred, almost all enzymes are denatured.

These observations prove not only the hypothesis, but also therefore the theories applied to create the hypothesis. We can see that increased thermal energy increases kinetic energy, thus the molecules move at a faster rate. The increased rate of reaction, as a result proves the ‘lock and key’ hypothesis, as we see the increased kinetic energy responsible for the increased rate of reaction. Also, the unchanged control tubes prove that all variables have been accounted for making this a fair test.

EVALUATION

The experiment went generally very well, producing accurate, and efficient results. This is reflected through the similarities between both predicted graph, and actual graph. There were no anomalous results as we managed to keep all dependent variables, under control, and all other variables were measured precisely (i.e. time was measured within five seconds accuracy). The lack of anomaly proves the data to be very reliable, as it shows that it was a carefully planned, and controlled test. At each temperature the maximum difference between two test tubes was 10 seconds. Although a relatively large time period, this is a small percentage error, and is also made less significant by the use of average. There were very few procedural problems, as it went generally smoothly. The only slight problem noticed was a slight lack of time to perform this experiment. Had there been more time, and then we could have paid more attention to each test tube, and possibly increased the accuracy still further. Also had we more time, and greater resources then some type of light sensor could have been created using a LDD in a circuit, to measure the transparency of the photographic film. However, this method harbors its own problems, and is largely unnecessary in producing results to prove a hypothesis. A more considerable factor in the accuracy of our results is the temperature intervals in relation to our graphs, as we can only assume what lies between each temperature value. For example, we assume that 40o C is the optimum temperature, however the real optimum could lie anywhere between 40o C and 60o C. For further investigation into the enzyme trypsin, we could perform several other experiments: the affect of trypsin on other substrates; the exact affects of pH as an independent variable; the affects of a trypsin inhibitor, such as endogenous; as well as many other experiments related to the action of trypsin as an enzyme. However the results produced are more than sufficient in proving the hypothesis, and other predictions.