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Investigating the effect of pH on the activity of phosphatase enzymes

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Introduction

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. 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 ...read more.

Middle

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. ...read more.

Conclusion

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. Charles Amick 4Alpha : Biology SC1 18/8/2001 1 ...read more.

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