Investigating the effect of pH on the activity of phosphatase enzymes

My aim in this experiment is to see how well an enzyme (phosphatase in this case) reacts under a controlled temperature but a varying pH.

Enzymes are known to be effected by pH and temperature. Both of these change how quickly the enzyme can process a substrate, so perfect matches must be found for each enzyme. At a low temperature, the enzymes reaction is so slow that any product is hardly noticeable. At a high temperature, or an extreme pH, the active site of the enzyme is damaged, so the substrate cannot be processed.

I predict that the optimal pH for the reaction to take place will be more acidic when the temperature is set at 25o c and the length of incubation is 10 minutes. A suitable pH would be between 3 - 5oc.

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I conducted preliminary experiments and chose to incubate at 25o c instead of the higher temperatures for the simple reason that I knew that at a higher temperature (around 35o c), the reaction would go at its fastest, and I ran the risk of high magenta values (I wanted to keep them all under 1 so they could be easily compared). I therefore wanted to see what would happen at lower than 35o c as far as reactions were concerned, so I chose 25o c.

My method was adapted from a worksheet on varying the temperature in the same reaction, keeping pH constant.

1. Label a microfuge tube with your initials.

2. Place two mung beans into the labeled tube.

3. Add 0.5ml distilled water into the tube containing the beans.

4. Crush and macerate the beans with a small glass/plastic rod.

5. Take a second microfuge tube and add water to the same level as the one containing the mung beans. (TO BALANCE THE CENTRIFUGE RACK)

6. Place the tubes into opposite holes of the centrifuge rack and spin for 5 minutes at maximum speed

7. After spinning, draw off as much of the clear supernatant above the pellet as possible and place into a clean microfuge tube. This solution now contains the enzymes for the experiment.

8. Using a graduated pipettor, add 100?l of sodium carbonate (the buffer solution in this experiment).

9. Then add 20?l PPP substrate to each of the eight microfuge tubes. Wash the pippettor thoroughly.

10. Finally, add 20?l enzyme solution into it.

11. Repeat steps 8 through 10 as quickly as possible, to collect all the microfuge tubes. Now insert them into a Styrofoam float and place this on the surface of the water bath for 10 minutes, timed with a stop clock.

12. Now add 100?l Sodium Carbonate to stop the reactions.

13. Estimate the colour of the magenta using the magenta filters provided.

The possible variables in this method are the volumes of substrate, enzyme and sodium carbonate along with the time in the water bath and the temperature of the water bath. The volumes will be measured as closely as possible with a micropippettor.

Results:

The number in the test tube column is the magenta filter that corresponded to the colour of the completed reaction. The higher numbers mean more reaction, lower means less reaction.

Every time that I added the sodium carbonate to cancel the reaction, the colour change to magenta was sudden and with a small amount of shaking, the whole liquid was tinted purple.

I managed to take 2 readings for each pH, and therefore average them. Without doing the preliminary experiment, I would have never known what temperature to try.

This graph shows clearly how good my results were. They fit with my prediction that the optimum pH for a Phosphate enzyme is around pH 3-5, and therefore we can say that it requires a more acidic pH than an alkaline one.

My conclusion, using this graph as evidence, is that a Phosphate enzyme works at its maximum speed at a lower pH, in this experiment pH 4, taking into account the other variables in the experiment. For instance, at a different water temperature, the pH required may vary.

As mentioned before, as the temperature raises, so does the probability of denaturation. From the results, I assume this is beginning to happen before pH 5. But these results are not precise. I have no way of knowing which side of pH 4 the reaction is faster, i.e. if pH 3.9 is faster than pH 4, or pH 4.1. The pH4 that I got as being the fastest speed may not be the pinnacle of the reaction curve.

Huge accuracy errors could have been made, for instance:

* Was the precise equal amount of liquid put in each of the tubes? Probably not, the micropipette was hard to use and had very small scales.

* Some reactions began before others when preparing to put the microfuge tubes into the water bath. You had to work incredibly quickly to prepare all of the tubes in as fast a time as possible.

However, seeing how precise my results were, either I made the same mistakes over and over, therefore giving a whole set of incorrect results, or I did them all very well. This is the risk in using this method. If I were to change the method, I would get far more precise pipettes and find a way of adding the enzyme into the solution as quickly as possible, like getting 8 micropipettes filled and ready, then using one for each microfuge tube in quick succession.

If this experiment was to be taken further, I would get people to work together and double check their accuracy as they go, so that they can do the final step before incubation in half the time or less. Instead of changing the pH, they could change the variable concerning the temperature of the water bath to be incubated in. Another possibility is that the different volumes could be changed to see how the results vary, of course only one at a time. For example, change the amount of enzyme to be put into the mixture, continue the experiment with other set variables and see what type of results you get.

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