Effect of Inhibitors (Phosphate and Urea) on Alkaline Phosphatase (Week 2) Inhibitor - Phosphate (Week 2) (working in pairs; but each student submits their own report) Solutions Reaction Buffer (0.1 M

Tris-HCl, pH 9) ● 5.4 mM p-nitrophenyl phosphate (PNPP) in 0.1 M Tris-HCl, pH 9 ● 25 mM sodium phosphate (monobasic dihydrate) in 0.1 M Tris-HCl, pH 9 ● 0.002 mg/ml Calf Intestinal Alkaline Phosphatase (AP) ● 3 M NaOH Procedure 1. Make the following tubes according to the table below. Cover your tube with a piece of Parafilm and invert 3 times to mix. Phosphate Concentration #1: Reaction Buffer Phosphate PNPP Tube Blank* 1 2 3 4 5 6 3.350 mL 70 UL 100L 3.320 mL 70 4 45.0 Reaction Buffer (mL) 3.360 3.350 70 ut + 3.320 3.300 3.270 3.185 3.010 3.300 mL 60 BUL 3.270 mL 70 UL 900 vl 3.185 mL TQ UL + 178 ul. 25 mM Phosphate (μL) 70.0 70.0 70.0 70.0 70.0 70.0 70.0 3.010 mL 70 ul. 310 ul 5.4 mM pNPP (μL) 0.0 10.0 40.0 60.0 90.0 175 350/n2. To make the blank*, add the following reagents to a fresh tube: 0.875 mL 3M NaOH (add before AP) 0.070 mL 25 mM phosphate 3.360 mL Reaction Buffer (pH 9) 70.0 μL AP 3. Make the following tubes according to the table below. Cover your tube with a piece of Parafilm and invert 3 times to mix. Phosphate Concentration #2: Reaction Buffer Phosphate PNPP Tube Blank* 1 2 3 4 6 3.280 mL 140 L 10,0 UL 3.250 mL 7 140 L 40.0 Reaction Buffer (mL) 00 3.290 3.280 3.250 3.230 3.200 3.115 2.940 3.230 mL 140 L 111 8 400 9 3,200 m 140 ut + 90.04. 10 3.116 mL 1401 + 175 25 mM Phosphate (μL) 140 140 140 140 140 140 140 11 2940 ml 140 ut 350 12 5.4 mM pNPP (μL) 0.0 10.0 40.0 60.0 90.0 175 350/nCalculations: The Effect of Inhibitors (Phosphate and Urea) on Alkaline Phosphatase To calculate reaction volumes, include volumes of all reactants except for the 3 M NaOH. The tables and figures must have appropriate titles and captions (see Figures/Tables, page 15). For each step show a sample calculation using Tube 1 for both inhibitors. Record your results in the table below. You may create one table to include both inhibitor concentrations used. 1. Using Beer's Law, convert the absorbance in each tube to concentration (UM). The molar extinction coefficient of p-nitrophenol at 410 nm is 16.2 mM¹¹cm¹. The path length is 1 cm. Note: the concentration must be corrected because of the addition of 3 M NaOH. 2. Calculate the velocity (μM/min) for each tube. 3. Calculate the final substrate concentration (μM) in each tube. -. Tabulate the results from Steps 1-3 - Use Excel to create a Michaelis-Menten plot of velocity (μM/min) (y-axis) vs. substrate concentration (UM) (x-axis). This plot should include the KM data (from last week) and the two different inhibitor concentrations. Use Excel to create a Lineweaver-Burk plot for the inhibitor used. The plot should include the KM data (from last week) and the two different inhibitor concentrations. Include in your caption: the equation of the line and the R2 value for each line. Determine the K, value(s) for the inhibitor by re-plotting key points from the Lineweaver-Burk plot in a new graph. Refer to your lecture notes for information on what to re-plot and how to interpret the plots. You will need to determine the type of inhibition occurring in order to do the re-plot. Include in your caption: the equation of the line and the R2 value for each line.