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Question 3 (15 marks) A phosphatase assay was performed on a sample of CFE (cell free extract) containing GST- TC45-349 using pNPP as a substrate. The CFE was diluted in TBS (50mM Tris-HCI pH 7.5, 150mM NaCl) prior to being assayed, as shown in column 1 of Table 2. A 20 ul aliquot of diluted CFE was added to 20 ul 50 mM pNPP plus 10 uL 5x assay buffer (50mM Tris-HCI pH 7.2). The reaction was incubated at 37 "C for 30 min. A control reaction was also set up with 20 ul water. After 30 min, 1 mL of stop solution (0.2M NaOH) was added to each reaction. Average absorbance at 405 nm of each 180 ul sample (measured in triplicate) was recorded by a platereader (Table 2). Table 2: Ammanm measurements of the dephosphorylation of pNPP by GST-TC45-349 CFE dilution A 405 nm PNP formed TopNPP consumed Control 2.78 162.17 16.22 1/2 6.95 405.42 40.54 1/5 2.78 162.17 16.22 1/10 1.39 81.08 8.11 1/15 0.93 TBD* TBD* 1/20 0.69 40.54 4.05 1/30 0.46 27.03 2.70 * TBD (to be determined) - values will be calculated as part of Q3 (iii) (i) Looking at the results obtained in Table 2, what can you conclude about the control reaction? (3 marks) (ii) Based on the information in Table 2, explain if pNPP is a good substrate for this reaction. Include in your answer the steps you would take to determine this. (5 marks) (iii) For the 1/15 dilution of CFE in Table 2, the last two columns are incomplete. Calculate the pNP formed and %pNPP consumed, including all necessary steps in the calculation. The molar extinction coefficient (Egos) of p-nitrophenolate is 1.8 x 10* M-1 cm-1