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Enzyme studies using acid Phosphate. This experiment will investigate the effect of pH on enzyme activities.

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Enzyme studies using acid Phosphate Introduction: This experiment will investigate the effect of pH on enzyme activities. Enzymes are biological catalyst, made of protein and held together by peptide bonds. Catalyst accelerates the rate of reaction by lowering the activation energy. The rate of reaction is dependent on several factors which include pH, temperature. Another dependent factor which is worth considering is the product and substrate concentration, which become a rate limiting factor at certain point during the catalytic process. Acid phosphatase is the name given to a general enzyme activity which is present in most eukaryotic cells. This enzyme hydrolyzes a phosphate group from a variety of natural and artificial substrates. The substrate for an acid phosphatase reaction must contain a phosphate functional group attached to another molecule through a chemical bond involving the phosphate and a hydroxyl (-OH) or alcohol functional group. The products of the reaction are the free phosphate group and the rest of the molecule with a free -OH group. more.


Using a 10ml pipette measure out different amount of standard 200µM p-nitrophenol solution. It is important that all volumes are made up to 6mls with 0.1M of NaOH. The content of the test tubes should mix gently and after mixing, place it in the spectrometer and read at 405nm. After taking all the reading, a table should be drawn and a standard curve. In part B of the experiment, a total of sixteen test tubes is needed. A total of 8 tubes containing 5.5 ml of 0.1M NaOH should be set aside for a later stage of the experiment. Label the remaining 8 test tubes pH 3 to pH 8. Then Add 2.0ml of 2.5 mM p-nitrophenyl phosphate to each tube. At this stage the test tubes should be placed in a water bath for 4 minutes, and to allow it to equilibrate at 37?C. After the stipulated time add 1.0 ml of distilled water to test tube one labelled pH 3 blank, this tube will used as the blank. more.


0.0 0.196 3.82 41.21 40.78 4.54 3.09 8.46 Equation: y = 0.0138x + 0.1473, by using this equation I so able to substitute y which is the absorbance and find the p-nitrophenol concentration which is x. For example: y = 0.0138x + 0.1473 0.15 = 0.0138x + 0.1473 Therefore: x = 0.15 - 0.1473 / 0.0138 x = 0.196 µM Optimum pH = 5.0 (41.21µM) Discussion and Analysis: The experiment went according to the pre-set method, and the overall objectives have been achieved. In term results and data, when compared with other in class data, it seems that the general aims of the experiment have been achieved. A structural change in the enzyme was inevitable as the pH would alter the enzyme shape as well as the substrate. There was not much complication with the experimental methods; the only source of error identified is timing. A delay in the second part of the experiment could affect the results. The optimum pH was the main indicator of success or completion of the experiment; it is observable from the smooth curve graph that the optimum activity has been achieved. ?? ?? ?? ?? more.

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