Enzyme assay

Statement of the problem:

Enzymes are proteins produced by a cell, which act as biological catalysts by accelerating specific
biochemical reactions without themselves being altered. Enzymes are highly specific, and a particular
enzyme will catalyse only a single reaction. Their specificity stems from their mode of action, in which
the substrate fits into the active site in the enzyme molecule. Enzymes function within narrow limits of
temperature and pH. Most function best at around 37°C, and are inactivated on prolonged exposure to
temperatures over 45°C or by inappropriate pH.

In this experiment I aim to investigate the productivity of the enzyme; salivary amylase, when it is
subjected to different temperatures, concentrations and pH solutions.

Hypothesis:

I predict that the enzyme we are using: Salivary Amylase will work best if it is in a solution of 37°C or in a
solution with a pH of 7.

Apparatus:

 Six test tubes

 Test tube stands
 Buffer tablets (pH 4, 7 and 9)
 Funnel
 Droppers
 Beaker
 Iodine Solution
 Substrate (rice (boiled) or potatoes)
 Test tube holder
 Stopwatch
 Filter paper
 Bunsen Burner
 Tripod


Procedure:

1. Prepare the Iodine solution. Take of 3g iodine crystals and 6g potassium iodide. Dissolve
potassium iodide in about 200 cm3 distilled water and then add iodine crystals. Make the
solution up to 1 litre with distilled water. It is essential to prepare it 24 hours before it is
required, as iodine is slow to dissolve.
2. Prepare the substrate. Take 1 gram of substrate and mix it with 10 millilitres water. Then keep it
aside for a few minutes. Boil 90 millilitres of distilled waster and finally make up the volume to
100 millilitres. Filter the solution the following day and utilize the filtrate as the substrate.
3. Take six test tubes and label them A, B, C, D, E and F.
4. Let test tube A be a controlled experiment
5. Let test tube B be set for the effect of temperature.
6. Let test tubes; C, D and E be set for the effects of varied pH. 4, 7 and 9 respectively.
7. Let test tube F be set for the effect of the substrate when it is in the concentration of 0.5.
N N
8. Take Iodine solution as an indicator. ( = normality)
10 10
9. Observe the suspension formed and the solution colour.
10. Take 10 millilitres of distilled water in a clean beaker and then dissolve a pH tablet completely
then pour into tube C and decant.
11. Likewise prepare the other two buffer solutions.
12. Transfer 3 millilitres of substrate to each to each test tube.
13. For test tube F add an equal quantity of water (3 millilitres).
14. Then add two drops of Iodine solution to each test tube.
15. Shake each test tube well.
16. Then add 2 millilitres of saliva or salivary amylase to each test tube.
17. Transfer test tube B into a water bath.
18. Maintain the water bath at 45°C
19. Then for the test tubes C, D and E add the buffer solutions with the pH’s 4, 7 and 9 respectively.
20. Leave for a day or for a set amount of time and observe the results.

Observation:

All the test tubes contained clear solutions except for test tube C. Test tube E has a clearer solution
than test tube D. Test tube B had a completely clear solution. Test tube A had what seemed like a
slower rate of reaction than the other samples.

Conclusion:

After conducting this experiment, I can reject my hypothesis. Salivary Amylase worked best when it
was in an alkaline solution and when it was in temperatures of approximately 45°C. However the
fact that it works best at 45°C either means that the enzyme does not catalyze at it’s productive
potential in our body, which remain at 37°C, or that there was an unknown factor which altered the
factors which affect it’s rate of reaction.

Increasing the substrate concentration increases the rate of reaction (enzyme activity). However,
enzyme saturation limits reaction rates. An enzyme is saturated when the active sites of all the
molecules are occupied most of the time. At the saturation point, the reaction will not speed up, no
matter how much additional substrate is added. The graph of the reaction rate will plateau. In which
another factor would have to be altered, to increase the rate of reaction. In this case we saw that it
didn’t catalyse the reaction of the substrate because the substrate was in a diluted solution.

There are factors which could also have affected the experiments as they were left for a day. For
example the room temperature was not constant during both day and night so it could also have
affected the solutions in which the enzymes were being kept in. Therefore fluctuating and de-
fluctuating temperatures could have slowed down or sped up reaction. Test tube B was not under
total observation during the whole 24 hours in which it was left alone. So whether it was in a slightly
colder solution during the night remains unknown and hence questions whether it was actually
maintained at 45°C or not.