BCEM2201-Kinetics of Alkaline Phosphatase-Instructions

BCEM2201 Kinetics of Alkaline Phosphatase Purpose ● Explore the kinetics of alkaline phosphatase acting on p- nitrophenyl phosphate ● Determine the number of “units” of alkaline phosphatase in a solution Introduction Enzymes are protein catalysts that can alter the rate of a reaction, but not its equilibrium. A highly negative free energy does not correlate to any specific rate or speed at which a reaction reaches equilibrium. Phosphatases catalyze the removal of a phosphate ester through the following general hydrolysis reaction: Upon inspection, this reaction is possibly a second-order reaction with the following rate equation/law: However, for all intents and purposes, this can be treated as a pseudo-first-order rate equation/law: Para -Nitrophenylphosphate (PNPP) is a popular substrate for identifying and quantifying phosphatase activity. Upon PNPP hydrolysis, the resulting p -nitrophenol leads to a quantitative increase in the absorbance centered around 405 nm. The extinction coefficient is approximately 1.8×10 5 M -1 cm -1 but strongly depends on pH. In addition to the familiar units of moles and milligrams (mg), the quantity of an enzyme can also be “functionally” measured. One enzyme unit (U) is defined as the amount of enzyme that can 1 of 5

BCEM2201 Kinetics of Alkaline Phosphatase catalyze the reaction of 1 micromole (µmole) of the substrate into a product in 1 minute at 25°C and under otherwise “ideal” conditions. Because U is an activity-based measurement, two samples of the same enzyme with an equal number of units can have different physical amounts of the enzyme (i.e., moles, mg, etc.) One sample may have a greater fraction of the enzyme in a degraded/inactive state than the other sample. Although this may seem strange, natural gas used to heat homes is sold analogously. In your monthly energy, natural gas is billed by the gigajoules of energy and not by cubic meters (or any other unit of physical quantity). This is because the amount of energy release depends on other factors that can vary such as temperature and gas composition. It makes “sense” to bill consumers by the amount of actual energy used versus the amount of gas delivered. Enzyme units function in a similar manner. When you purchase an enzyme, you want to pay for the amount of activity and not the physical amount of protein. Example: Q: How many units of an enzyme do you need to transform 4 µmole of substrate into product(s) in 15 seconds? A: 1U = 1 µmole/min. We want to transform/convert 4 µmoles in 15 seconds (or 0.25 minutes). Therefore: To measure the activity of an enzyme, one performs an assay which measures the production of product (or disappearance of reactant) as a function of time. A typical result of an assay is shown in the graph below. 2 of 5

BCEM2201 Kinetics of Alkaline Phosphatase 3) The remaining 20 mM Tris solution will serve as the “blank” to use to zero the spectrophotometer before measuring the “Control” sample. 4) To prepare the “control,” add the calculated volume of 4 mM PNPP solution to reach 150 μM final PNPP concentration to a 13×100 mm glass tube. Add 0.4 mL 0.2 M Tris buffer (pH 8) and enough water to a final volume of 4.0 mL. (Note that the concentration of Tris buffer in this sample is diluted to 20 mM.) 5) Briefly vortex and measure the A 405nm of the PNPP solution and record both the absorbance and the time at which you measured the absorbance. 6) Save both the Blank and the Control tubes to be re-measured at the end of the lab. III. Enzyme Assay Part A Prepare a table in your notebook, similar to what is shown below, indicating the volume of 4 mM PNPP, 0.2 mM Tris and water you need to mix to make 4 assays containing the ¼ dilution of alkaline phosphatase. Also, have a table prepared to record the absorbancy change over time. Assay # PNPP, conc. μ M Volume of 4 mM PNPP, mL Volume of 0.2 M Tris pH 8, mL Volume of H 2 O, mL Volume of 1/4 enzyme solution, mL Total volume, mL 1 150 0.4 0.100 4 2 75 0.4 0.100 4 3 37.5 0.4 0.100 4 4 18.75 0.4 0.100 4 1) Make 0.5 mL of a 1/4 dilution of the provided stock alkaline phosphatase (AP) solution using the 20 mM, pH 8 Tris buffer (you saved 1 mL of this in step I.) This enzyme solution will be enough for 4 assays of different PNPP concentrations. 2) Keep the Enzyme in an ice bath 3) Set up the enzyme assay #1 (150 μ M PNPP) in a 13x100 mm glass test tube. Measure water, 0.2 M pH 8 Tris buffer, and 4 mM PNPP. Briefly vortex or mix by inverting the solution and put the test tube with the mixed sample into the spectrophotometer to zero it (@ 405nm). 4 of 5

BCEM2201 Kinetics of Alkaline Phosphatase 4) Remove the tube from the spectrophotometer and add 100 μL (0.1 mL) of ¼ enzyme solution. Take note of the start time of the experiment and get ready to monitor the time for the subsequent steps. 5) Quickly vortex/mix the tube again and put it back into the spectrophotometer. Immediately begin recording the absorbance (@ 405nm). 6) Take measurements every 20 s for the first 2 min and then every minute for the next 5 min (7 min of data in total) . 7) For Calibration graph: After the experiment, save the solution (in the tube) so that you can later measure the final concentration of PNP, and use it to construct the calibration curve. Make sure you label the tube! Using fresh 13×100mm tubes, repeat the above steps with assays #2-4, to have a total of 4 assays with the 1/4 enzyme dilution. Part B 1) Make 0.5 mL of a 1/8 dilution of the provided stock alkaline phosphatase (AP) solution using 20 mM, pH 8 Tris buffer . Note that this solution was made as “ blank ” in the previous step and you can use a little bit of that. This is your working solution (1/8 dilution). This will be enough for 4 assays of different PNPP concentrations. 2) Repeat steps 2-6 in Part A using the 1/8 enzyme working solution. Note, for part B, you do not need to save your sample till the end of the lab. Question: Why are we only saving the test tubes until the end of the lab from Part A, and making only one control sample? In total, you should have collected 8 different datasets. Your lab book must contain all tables that you need to record data when you come to the lab! IV. Wrapping Up the Experiment After all the enzyme assays are done, measure the final A 405nm from the tubes that you have saved from the 1/4 dilution experiment (i.e., first run). At least ~30 minutes is needed to have passed before you take this measurement. This data will be used to determine the p -nitrophenol calibration curve . Finally, use your saved tubes from Part II (Control Sample) 5 of 5

BCEM2201 Kinetics of Alkaline Phosphatase 9 0.28 10 0.29 Lab Report ● Follow the instructions on the worksheet to analyse the data, and report your calculated results. ● Attach relevant images (pictures) of your lab notebook in the supplementary session. 7 of 5