Investigating the effect of pH on the activity of phosphatase enzymes

My aim in this experimentationation is to gibe how well an enzyme (phosphatase in this case) reacts to a humble ready a controlled temperature but a varying pH.Enzymes argon known to be effectuate by pH and temperature. Both of these change over how readily the enzyme send away process a substratum, so perfect matches must be found for apiece enzyme. At a low temperature, the enzymes reaction is so thudding that any product is hardly noticeable. At a high temperature, or an natural pH, the active site of the enzyme is damaged, so the substrate cannot be processed.I predict that the optimum pH for the reaction to take place will be more vitriolic when the temperature is specify at 25o c and the space of pensiveness is 10 minutes. A fitted pH would be between 3 5oc.I conducted preliminary experiments and chose to incubate at 25o c instead of the higher temperatures for the unbiased reason that I knew that at a higher temperature (around 35o c), the reaction would go at its fas examination, and I ran the risk of high chromatic values (I cherished to keep them e truly last(predicate) under 1 so they could be easily compared). I therefrom wanted to see what would bef every at lower than 35o c as far as reactions were concerned, so I chose 25o c.My mode was change from a deedsheet on varying the temperature in the same reaction, keeping pH constant.1. tag a microfuge tube with your initials.2. Place twain mung beans into the labeled tube.3. Add 0.5ml distilled piddle into the tube containing the beans.4. Crush and macerate the beans with a sm on the whole glass/plastic rod.5. precede a second microfuge tube and imbibe urine to the same level as the one containing the mung beans. (TO BALANCE THE separator RACK)6. Place the tubes into opposite holes of the centrifuge roulette wheel and spin for 5 minutes at maximum stronghold7. After spinning, take a shit off as much of the unsnarl supernatant above the pellet as possible and pl ace into a modify microfuge tube. This origin now contains the enzymes for the experiment.8. Using a graduated pipettor, add blow?l of sodium carbonate (the buffer solution in this experiment).9. Then add 20?l PPP substrate to to severally one of the eight microfuge tubes. Wash the pippettor thoroughly.10. Finally, add 20?l enzyme solution into it.11. Repeat locomote 8 through 10 as quickly as possible, to collect all the microfuge tubes. Now insert them into a Styrofoam bollix up and place this on the surface of the water clean for 10 minutes, timed with a throw overboard clock.12. Now add 100?l Sodium Carbonate to stop the reactions.13. Estimate the falsify of the magenta employ the magenta filters provided.The possible multivariates 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 calculated as closely as possible with a micropippettor.ResultsThe number in the test tube column is the magenta filter that corresponded to the colour of the completed reaction. The higher poetry mean more reaction, lower gist 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 meter of shaking, the whole facile was tinted purple.I managed to take 2 readings for each pH, and therefore average them. Without doing the preliminary experiment, I would do never known what temperature to try.This represent shows clearly how good my results were. They fit with my prescience that the optimum pH for a orthophosphate enzyme is around pH 3-5, and therefore we can say that it requires a more bitter pH than an alkaline one.My conclusion, victimisation this chart as evidence, is that a Phosphate enzyme turn tails at its maximum speed at a lower pH, in this experiment pH 4, taking into account the new(prenominal)(a) variables in the experiment. For instance, at a contrastive water temperature, the pH required may vary.As mentioned before, as the temperature raises, so does the luck 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 swift, 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 circus tent of the reaction curve.Huge trueness errors could have been made, for instance* Was the precise equal amount of liquid range 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 di d them all very well. This is the risk in victimization this method. If I were to change the method, I would get far more precise pipettes and strike 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. other 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.