Effect of Inhibitor (L-Phenylalanine) on Alkaline Phosphatase (Week 1) (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 ● 50 mM L-phenylalanine 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. Phenylalanine Concentration #1: Tube Blank* 1 Reaction Buffer PNPP 2 345 6 Phenylalanine 3.070 mL 350 ul. 100 ul 3.040 mL $50 ul 400 ut Reaction Buffer (mL) - 3.080 3.070 3.040 3.020 2.990 2.905 2.730 3.020 mL 350 + 60.0u 2.990 mL 950 ul. 90.0 ul 50 mM Phenylalanine (μL) 350 350 350 350 350 2.906 mL 350 ul + 175 350 350 2.730 mL 350 A 350 L 5.4 mM PNPP (μL) 0.0 10.0 40.0 60.0 90.0- 175 350 0.0 Abs Blank - O 1-0.012 2-0.085- 3 -0.164 4 -0.148./n2. To make the blank*, add the following reagents to a fresh tube: 0.875 ml 3M NaOH (add before AP) 0.350 ml 50 mM phenylalanine ✓ 3,080 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. Phenylalanine Concentration #2: Tube Blank* 7 8 9 10 11 12 Reaction Buffer Phenylalanine PNPP 2.720 mL 700 UL 10.00 u 7 2.690 ML 700 1 40.0 8 Reaction Buffer (mL) 2.730 2.720 2.690 2.670 2.640 2.555 2.380 2.670 mL 700 μL 60 0 ut 2610mL 700 g 90.0 50 mM Phenylalanine (μL) 700 700 700 700 700 700 700 2.656 mL 700 UL 175 ]] 10 11 2.360 mL 200 UL 350 ut 12 5.4 mM PNPP (μL) 0.0 10.0 40.0 60.0 90.0 175 350 Abs Blank - 0.0 7- 0.075 8- 0.090 90.5 9-0.055.2 10 - 0-085. 11-0088 12 -0.102./n4. To make the blank*, add the following reagents to a fresh tube: Abs Conc 0.075 4.63 5.56 3.40 0.875 mL 3M NaOH (add before AP) -0.700 mL 50 mM phenylalanine 2.730 mL Reaction Buffer (pH 9) 70.0 μL AP 1 NaOH 875 uL NaOH 875 uL BANG & OLUFSEN 1 0.090 |||| 2 3 1 1 1 delete 5 Coretad 5.79 6.95 4.25 veloa tu 1.16 1.39 0.85 IN 0.862 61719 1176 0.763/nCalculations: The Effect of Inhibitor (L-Phenylalanine) 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. 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 mM ¹cm¹. 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. 4. Tabulate the results from Steps 1-3.