Investigating the effect of pH on the activity of phosphatase enzymes

Have a little read: ... 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 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