answer the first page form3-9 Standard enzyme assay procedure for pyruvate kinas
ID: 148321 • Letter: A
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answer the first page form3-9
Standard enzyme assay procedure for pyruvate kinase 1. You are provided with a "stock solution", which contains lactate dehydrogenase (the coupling enzyme), Mg2", K', fructose-1,6-bisphosphate (an activator of pyruvate kinase), and NADH-this should be kept on ice. You are also provided with the substrates for the pyruvate kinase reaction, ADP and phosphoenol pyruvate (PEP) also kept on ice Set up the spectrophotometer assay cuvettes as follows: Stock Solution ADP (150 mM) Phosphoenol pyruvate (150 mM) Water Total volume 720 uL 20 L 220 L 980 2. Incubate the assay solution at room temperature for 10 min. This allows the assay to come up to temperature. IMPORTANT: The rate of a catalysed reaction is dependent on temperature so this needs to be constant, i.e. do not start the reaction until the assay solution reaches room temperature . Blank the spectrophotometer with distilled water. 4. Start the reaction by adding 20 uL of appropriately diluted enzyme (see note below). Mix rapidly but thoroughly by inverting the cuvette, and replacing it in the spectrophotometer as soon as possible. Record absorbance (A340) at 15 second intervals for as long as required. Record readings for up to 3 min (or until the reaction is complete). The maximum rate should be no more than 0.05 absorbance units per 15Seconds. A quick plot of your readings will give you a better idea of the course of the reaction than a table of numbers. IMPORTANT: Get a demonstrator to check your first graph before you carry on NOTE: It will be difficult to initially gauge how to dilute your enzyme so that you get meaningful results. If you add too much enzyme, the rate of reaction will be too fast to get an initial rate. If you do not have enough enzyme, the rate will be very slow. For the crude lysate, initially try a 100x and 200x dilution. Record your data in the table on the following page, and plot your data with the absorbance measurement on the Y-axis and time (in seconds) on the X-axis. Repeat the experiment with different dilutions of enzyme until you have a useable rate of change of absorbance. Check with your demonstrator to confirm that your data are useful. When you have identified a concentration of enzyme that is appropriate, make duplicate measurements.Explanation / Answer
Considering the value for actual concentration in 20l of protein used to initiate the reaction as 0.52mg/mL (The image resolution is really very low to determine wheteher its .52mg/mL or .052mg/mL).
3) Actual concentration of protein in cuvette= 0.01mg/mL
4) slope of the line 1 (for 100x)= A/min = (y2-y1)/(x2-x1)= (0.684-0.725) / (90-80) min= - 0.0041
5) slope of the line 2 (for 200x)= (0.886-0.893)/ (90-80) min = - 0.0007
Negative line only denotes that the slope of line is downwards towards x axis.
6) Average slope change= ((-0.0041)+ (-0.0007)) /2= - 0.0024
7) Change of concentration of NADH in M/min = Row 6/ 6220 x 106 = (- 0.0024/6220) x 106
= - 0.39 M/min
Negative sign implies that the concentration of NADH decreases with time.
8) Amount of NADH consumed in 1 min mol/min = 0.39M/min x 10-3 = 3.9 x 10-4 mol/min
9) Specific activity = activity of an enzyme per milligram of total protein
= (3.9 x 10-4 mol/min) / (0.01mg) = 3.9 x 10-6 mol/min/mg
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