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In this experiment, you will measure the absorbance every 30 seconds instead of 10 min. Therefore, do each of the reactions independently. Table 2: Assay Mixtures for determining the optimum pH for LDH enzyme activity pH 5 6 7.4 8 9 3 1.8 1.8 1.8 1.8 1.8 Citrate buffer - 50 MM, PH 3 (mL) Citrate buffer - 50 mM, PH 5 (mL) Phosphate buffer - 50 mM, pH 6 (mL) Phosphate buffer - 50 mM, pH 7.4 (mL) Tris buffer - 50 mM, pH 8 (mL) Carbonate buffer - 50 mM, PH 9 (mL) Pyruvate - 3 mM (ML) NADH-4.5 mM (mL) Absorbance at zero time LDH - 5 units/mL (mL) 1.8 1.0 1.0 0.1 1.0 0.1 1.0 0.1 1.0 0.1 1.0 0.1 0.1 0.1 0.1 0.1 0.1 1) Set the wavelength to 340 nm 2) Zero the spectrophotometer using 50 mm phosphate buffer as the reagent blank 13) Set up the assay as described in Table 2 and mix ONLY the buffer and pyruvate at this stage. Do not add the 0.1 mL aliquot of NADH or LDH at this stage 3) Add 0.1 mL of NADH, mix and determine the absorbance of the solution. Record these as zero-time values 4) Add 0.1 mL of the LDH solution to the tube, mix quickly and place the cuvette in the spectrophotometer (Make sure you do this quickly as the reaction is fast) 5) Read absorbance at 30 sec intervals for 5 min (30 sec reading is from the time you add the LDH solution) 6) Using the extinction coefficient provided, determine the enzyme activity in each case (easy way is to use Abso min -- Absimin for calculations) 7) Plot enzyme activity vs. pH 8) Determine the optimum pH for LDH Effect of pH on the activity of Lactate dehydrogenase enzyme Table x. table caption pH 3 5 6 7.4 8 9 Absorbance at zero min 0 0.8 0.67 0.87 0.78 0.75 Absorbance at 30 s 0.79 0.54 0.71 0.64 0.64 0.52 Absorbance at 60 s 0.76 0.48 0.66 0.52 0.47 0.39 Absorbance at 90 s 0.72 0.41 0.58 0.41 0.36 0.21 Absorbance at 120 s 0.70 0.36 0.49 0.33 0.23 0.09 Absorbance at 150 s 0.69 0.26 0.41 0.23 0.14 0.05 Absorbance at 180 s 0.68 0.22 0.34 0.18 0.09 0.02 Aomin - Almin Enzyme activity (umoles pyruvate/min) Answer the following questions 1) What is the optimum temperature for the LDH enzyme activity used in these studies? 2) Why do you think the enzyme activity changes at higher temperatures e.g. 60 C? Explain your answer. 3) What is the optimum pH for the LDH enzyme activity? 4) Most proteins have a relatively stable conformation at pH 6 or pH 8. Assuming that LDH has a stable conformation in this pH range, explain what factors you think might contribute to changes (if any) in the activity of the enzyme at the different pH's tested. 5) From your studies where you determined rate of change in absorbance every 30 seconds (pH experiments), is the rate of reaction linear over the 5-minute period? If not, why do you think the rates are not linear over this period of time? 6) Why do we calculate initial velocity for determining the activity of natural enzymes