E10_ Acid Phosphatase Enzyme Assay

E10: Acid Phosphatase Enzyme Assay BIOL 2281-306 April 18th, 2023

Post Lab Report (20 pts total) – Submit in eLearning Title Page : Include your name, section number, date, the title of the experiment. (-0.5 if no title page) Include the questions in your report. (-0.5 if questions are not included) 1. Write a short summary paragraph of the experiment outlined in this protocol that describes the purpose, the chemical reaction, and how enzyme activity was measured. When discussing how enzyme activity is measured, think about how the measurement is presented/described in addition to the general methodology for determining it. (4 pts) - The purpose of the experiment is to determine the rate of the reaction of acid phosphatase with nitrophenyl phosphate. Enzymes which are proteins that are produced by living cells that act as catalysts. A catalyst is an agent used to speed up chemical processes. This is very important because the process is responsible for performing essentially all changes associated with life processes. In wheat germ-acid phosphatase the acid phosphatase catalyzes the removal of phosphate groups from macromolecules and smaller molecules that are stored on the wheat seed. By doing so it creates a free phosphate which is then used by the growing embryo. In our experiment, nitrophenyl and H2O are catalyzed by acid phosphatase to produce phosphate and nitrophenol. In alkaline solutions nitrophenol becomes yellow in color to indicate how much product has been formed. The darker the shade of yellow indicates that more product is found. In this lab, we started a serial dilution using KOH and nitrophenol to create different concentrations. The tubes were labeled numbers one through six where one was considered the blank test tube, no solution was transferred into the vial after initial transfer. The absorbance was set to 410 nm throughout the experiment because that is the absorbance of nitrophenol. After performing serial dilution we used a water bath at 5-minute intervals for 25 minutes at 37 degrees Celsius. The reaction from placing a tube into the water bath was the KOH stopped the reaction and a color change was observed from the nitrophenol. After using the water bath for 25 minutes we obtained data of the absorptions at 410 nm and created a standard curve, where we can clearly see a linear trend. This means that as the reaction time increased the absorbance of the nitrophenol solution increased as well, more levels of product were formed and this can be proven by the yellow color of the tubes darkening at higher reaction times. In order to measure enzyme activity we prepared a standard curve of nitrophenol. 2. Copy/paste your completed Table 1 into your report. Use Excel to generate a standard curve of OD 410 vs nmole/ml of nitrophenol. Make sure you include the equation and R 2 value on your graph. If the R 2 is less than 0.95 , you need to drop some outlying data point and recalculate. Be sure to set your intercept to zero. Failure to do so will result in loss of points. Paste the graph into your report. (4 pts)

3. What is the purpose of the standard curve generated in question 2? (1 pt) - The purpose of the standard curve generated in question 2 is to extrapolate concentrations (nmole/ml) of nitrophenol based on absorbance values. The data is based upon the relationship between the absorbance (y-axis) and the concentration of nitrophenol (nmol/ml) (x-axis). Using a standard curve helps to indicate the total nitrophenol produced in nmol from the absorbance values without needing to convert between different units. 4. Based on the equation generated by the standard curve (question 2), convert the OD 410 values in Table 2 into nmole of nitrophenol produced . Remember that the standard curve allows you to extrapolate concentrations (nmole/ml) of nitrophenol based on absorbance values. You need to calculate the amount (in nmole) by multiplying the concentration of nitrophenol by the volume measured (in the cuvette). The volume measured at each time point was 1 ml. (3 pts) Tabel 2: Acid Phosphates Assay at 37 degrees celsius. Reaction time min OD410 A1 0 0 A2 5 0.207 A3 10 0.330 A4 15 0.551 A5 20 0.757 A6 25 0.908 Reaction Time (min) Original OD 410 Reading from Table 2 nmole/ml based on Standard Curve Nitrophenol Produced in nmole (= nmole/ml x 1 ml) 0 0 y=0.0096x 0=0.0096x = 0 0 5.0 0.207 y=0.0096x 0.207=0.0096x =21.5625 21.5625

10.0 0.330 y=0.0096x 0.330=0.0096x = 34.375 34.375 15.0 0.551 y=0.0096x 0.551==0.0096x =57.3958 57.3958 20.0 0.757 y=0.0096x 0.757=0.0096x = 78.8541 78.8541 25.0 0.908 y=0.0096x 0.908=0.0096x =94.583 94.583 5. Use Excel to plot a scatter diagram of nitrophenol produced (nmole) over time (min) for the reaction using data in the above table in question 4. Perform linear regression analysis and determine the slope of the line. (You need to keep as many data points as possible in generating the linear relationship). a. Paste in the graph with the equation and the R 2 value displayed. (2 pts) b. Based on this graph, what is the reaction rate (V 0 ) for the acid phosphatase? (2 pts) - The reaction rate (V 0 ) is 3.8204 nmol/min 6. Include scans or images of your two hand-drawn graphs (completed during the lab) and the two data tables on page 11 in the file you upload to eLearning. You WILL NOT receive credit for them if they are not attached to your report! (4 pts) Tabel 1: Standard curve of Nitrophenol