Enzyme Activity

Summary:   Experiment Aim: To determine the effects of substrate concentration variation on the activity of the enzyme pepsin. Also to determine the level of substrate concentration at which the enzyme pepsin functions most efficiently.

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Experiment Two - Substrate Concentration

AIM: To determine the effects of substrate concentration variation on the activity of the enzyme pepsin. Also to determine the level of substrate concentration at which the enzyme pepsin functions most efficiently.

HYPOTHESIS: The percent of substrate concentration that the enzyme pepsin will function at its optimum will be 10%.INDEPENDENT VARIABLE: The concentration of the substrate (egg albumin) used in the experiment.DEPENDENT VARIABLE: The efficiency of the enzyme in acting and breaking down the protein in the egg albumin, i.e. how much egg albumin is dissolved.CONTROL: The HCl solution without the enzyme pepsin.EQUIPMENT:☺46 large test tubes☺1 beaker☺2 thermometers☺ Pepsin powder (2g)☺ Distilled water☺ Hydrochloric acid (HCl-1 molar)☺ Egg albumin☺ Test tube rack x4☺ Data Logger + Light Probes

☺ Transformer

☺ Computer + Tainlab

☺ Light Globe

☺ Pipette

☺ Syringe

☺ Black Box Set-up (See Diagram 1)

☺ Bunsen

☺ Test tube holder

☺ Matches

☺ Retort stand

☺ Filter funnel

☺ Muslin

METHOD:

1.Both the pepsin and egg albumin solutions were prepared.

a) Pepsin solution- 10ml of HCl were added to 100ml of distilled water. This solution was halved and separated into two separate beakers. 2g Pepsin powder was added to one beaker and this beaker was labeled Pepsin/HCl solution. The other beaker containing only the HCl solution was labeled HCl solution.

b) Egg Albumin -

☺Pure albumin- 30ml of pure albumen was heated until the solution became opaque.

☺90% Albumin solution- 27ml of albumen were added to 3ml of distilled water and the solution was heated until it became opaque.

☺80% Albumin solution- 24ml of albumen were added to 6ml of distilled water and the solution was heated until it became opaque.

☺70% Albumin solution- 21ml of albumen were added to 9ml of distilled water and the solution was heated until it became opaque.

☺60% Albumin solution- 18ml of albumen were added to 12ml of distilled water and the solution was heated until it became opaque.

☺50% Albumin solution- 15ml of albumen were added to 15ml of distilled water and the solution was heated until it became opaque.

☺40% Albumin solution- 12ml of albumen were added to 18ml of distilled water and the solution was heated until it became opaque.

☺30% Albumin solution- 9ml of albumen were added to 21ml of distilled water and the solution was heated until it became opaque.

☺20% Albumin solution- 6ml of albumen were added to 24ml of distilled water and the solution was heated until it became opaque.

☺10% Albumin solution- 3ml of albumen were added to 27ml of distilled water and the solution was heated until it became opaque.

J 0% Albumin solution- 30ml of distilled water.

2. The data logger and light probe apparatus were set up at one end of the black box. A light globe, attached to a transformer, was set up at the opposite end, but left switched off. (See Diagram 1) The data logger was set-up to take readings ten times per second for sixty seconds. The transformer was set on "D."
3. 5ml of Pepsin/HCl solution were added to half of the test tubes. These were placed in a test tube rack and labeled.
4. 5ml of HCl solution were added to another twelve of the test tubes. These were placed in a second test tube rack and labeled.
5. 3 drops of Universal indicator were added to one test tube of Pepsin/HCl solution and to one test tube of the HCl solution and the pH was measured to ensure it was 2.5-3.5. The pH for each was recorded. These test tubes were then set aside from the others.
6. 5ml of each Albumin solution, from 100% to 0% were added to two test tubes.(Total of 22 test tubes). These were placed in a third and fourth test tube rack and labeled with the corresponding percentages.
7. One beaker was filled with 200ml water.
8. One test tube containing HCl solution and the test tube containing 100% Albumin solution were placed into the beaker.
9. One thermometer was placed inside each test tube in the beaker. The beaker was then heated in a water bath until the two solutions reached a desired temperature of 36.4°C.
10. The test tube containing 100% Albumin was placed into the black box via the opening in the lid and the HCl solution was poured into the 100% Albumin. The light globe was turned on. The light probe was then activated, and the amount of light allowed to filter through the solution was measured using the light probe ten times a second for sixty seconds using the data logger.
11. The graph, plotted by the data logger, was saved, and the light probe was then stopped and data logger reset. The test tube was removed from the black box, its contents disposed of, and the light globe turned off.
12. Steps 8-11 were repeated, but the test tube containing HCl solution in Step 9 was replaced with the test tube containing Pepsin/HCl solution and then heated to a desired temperature of 36.4°C before being tested.
13. Steps 8-12 were repeated. The solutions in Step 8 and 12 were replaced with the 90% Albumen solution.
14. Steps 8-12 were repeated. The solutions in Step 8 and 12 were replaced with the 80% Albumen solution.
15. Steps 8-12 were repeated. The solutions in Step 8 and 12 were replaced with the 70% Albumen solution.
16. Steps 8-12 were repeated. The solutions in Step 8 and 12 were replaced with the 60% Albumen solution.
17. Steps 8-12 were repeated. The solutions in Step 8 and 12 were replaced with the 50% Albumen solution.
18. Steps 8-12 were repeated. The solutions in Step 8 and 12 were replaced with the 40% Albumen solution.
19. Steps 8-12 were repeated. The solutions in Step 8 and 12 were replaced with the 30% Albumen solution.
20. Steps 8-12 were repeated. The solutions in Step 8 and 12 were replaced with the 20% Albumen solution.
21. Steps 8-12 were repeated. The solutions in Step 8 and 12 were replaced with the 10% Albumen solution.
22. Steps 8-12 were repeated. The solutions in Step 8 and 12 were replaced with the 0% Albumen solution.

Discussion

Because it was not possible to test every sample at precisely the same time, error may have occurred due to variation in environmental temperature. Whilst each test was cooled/heated to the desired temperature, the surrounding environmental temperature would have had an effect on the cooling rate on the test tube containing the Albumin/HCl or Albumin/Pepsin solutions. However this would not have had a significant effect on the results because the experiment was conducted over a relatively short period of time, in which the temperature did not vary greatly.

The absence of a Colorimeter led to the use of the Black Box apparatus to measure the amount of light transparency through the solution. While precautions were taken to ensure that this apparatus would take readings accurately, some errors may still have occurred. The box was painted black in order to absorb light rather than reflect it, which would have made the light probe readings inaccurate. A tunnel was set up to ensure that the light from the globe was directed directly through the test tube rather than spread throughout the box. Although these precautions were taken, there was still a fairly high margin for error. However these errors are difficult to avoid whilst using an apparatus such as the one used in the experiment. To obtain more accurate results, a Colorimeter could be used. However in this instance this equipment was not available for use.

One significant error did occur whilst conducting the experiment. When testing the pepsin on the 70% albumin solution, there was a data logging malfunction which led to the results only being logged for thirty seconds of the experiment. In order to correct this malfunction, the test would need to be repeated. Due to time restrictions this was not possible. An incorrect graph in this case may have led to an incorrect average light transparency, which would therefore impact, on the results showing average light transparency in comparison to substrate concentration.

Another source of error was through computer and data logging. While this apparatus is fairly accurate, it is possible that some errors may have occurred. The light probe readings may have been affected by sources of light other than the light globe. As mentioned, the only way to ensure that this did not happen would be to use a Colorimeter.

An advantage of using the Black Box/Data Logger apparatus was that, instead of a single reading as given by the Colorimeter which would have to be recorded and graphed manually, the data logger gave an accurate graph.

Another error, also associated with the Black Box/Data Logger apparatus was the placement and fixture of the test tube whilst the light probe readings were being logged.

It was important to have each test tube fixed in exactly the same position to ensure that the light transparency readings were not affected by the distance of the test tube from the light probe and light globe.

If these were not kept constant it greatly affected the readings on the graphs from one test to another. Whilst the test tubes were placed so that they were on level with both the light probe and light globe, and were fairly firmly fixed in the same position, there was still a margin for difference, and therefore a margin for error. To further unsure that these errors did not occur, a more fixture that did not allow movement of the test tube could be used.

One of the main sources of error in this experiment occurred due to the solidification of the high percentage albumin solutions. At high concentrations of albumin, when heated, the albumin solutions solidified. While the pepsin solution did still work to break down the substrate in some cases, it was found that "clumps" of albumin remained floating in the solution. Because of the use of sensitive data logging equipment this led to a high level of error. The light probe takes an accurate reading through only a small section of the test tube. If in this small section lay solidified albumin, then readings may be taken that would not reflect the results of the experiment. This led to many errors in the experiment, and was unavoidable when using high concentrations of albumin. However as the concentration decreased, and the solution no longer solidified when heated, this error was not present. This error did mean that the results gained in the experiment were not reliable or valid because it was an unfair test. In order to make the experiment valid, the amount of light transparency through the test tube and contents should be constant throughout. This was not possible to maintain when using high concentrations of albumin, which solidifies.

Originally, 1ml of albumin solution was to be used in each test. When tested, it could be seen that this was insufficient when trying to determine the effect of 5ml of pepsin solution on the albumin. The amount of albumin solution in each test was increased from 1ml to 5ml.

From the results of the experiment it can be seen that it was not a valid one. The results show no trends or patterns similar the results predicted, except with the exclusion of the 50%, 60%, and 70% tests. It can be deduced that possible errors in these tests led to inaccuracies in graphing which did not allow the expected trends to emerge in the results. However, when the averages of these tests were excluded, a pattern similar to that predicted can be observed. That is, with the decrease in substrate concentration came an increase in light transparency. While this pattern can be seen, due to the necessary inclusion of all tests in the results, it is found that the results obtained are not valid.

While the experiment did address the hypothesis in a real way, the high margin for error meant that any results obtained were not a fair representation of the effect of substrate concentration on the activity of the enzyme pepsin.

When observing the results, it must be taken into consideration that whilst the 100% solution was pure egg white, it was not pure albumin. The actual percentage of albumin in the white of a hen's egg is 58.9 %. Therefore, when analysing results this must be taken into consideration.

Whilst conducting the experiment, there were several safety precautions that were observed to ensure the safety of the experimenter. Whilst handling and measuring chemicals such as HCl care must be taken to ensure that spills do not occur. All beakers, flasks, and other glass equipment must be kept a safe distance from the edge of benches, and handled with care. When heating using a Bunsen burner, it was ensured that the Bunsen was kept at a safe distance from the edge of the bench, and a test tube holder was used to heat the contents to avoid burns. Safety goggles were worn to prevent eye damage in the case of spills. Care was taken when adding the contents of the Albumin/Pepsin and Albumin/HCl test tubes to ensure that spills did not occur which would lower the accuracy of the results obtained.

The results of the error were found to be invalid due to error by both the technological equipment used and unavoidable substrate concentration error. However, the predicted results through preliminary research, that is that with the decrease in substrate concentration came an increase in light transparency, can be explained.