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BIOL 3380 Experiment 2 - Lab Report Characterization of Phosphatase Activity in a Crude Alfalfa Sprout Extract Name: Rania Mohamed Circle your lab section: T–AM W-AM R-AM F-AM T–PM W-PM R-PM F-PM Instructor: Jing Pan Graduate TA: Rafah Falah Date: 09/20/2023 Partner: Varshini Experiment 2 - Lab Report Characterization of Phosphatase Enzyme Inserting your answers after each question/space. Please denote your answers by using bold , italic s, or colored font. Do NOT renumber/reorganize the questions. “Showing your work/calculations” can be achieved by typing it out, pasting in a clear image of your handwritten work, or using handwritten annotation on the document (if you have the technology to do so). Insert graphs (handwritten on graph paper or electronically constructed with horizontal/vertical gridlines) as a clear image with appropriate titles, labeled/scaled axes, and standard deviation bars when appropriate.

1 . (6pts) Construct two separate bar graphs depicting your results for the effect of pH and temperature on phosphatase activity. All figures, graphs, and tables must include appropriate titles, legends, labeled/scaled axes, and standard deviation bars. Do not include a figure caption. * If you experience problems adding custom error bars with Excel/Google sheet graphs, hand-drawn error bars are acceptable.

2. (2pts) Show all the calculations for the pH 5 data point that was plotted on your pH graph. 3. (3pts) Based upon your data, a) What was the temperature optimum of the phosphatase assay? pi erature Aug : 907+1 - 751= 1 3 - Ang : 00 . 60370 . 613 = 0 . 603 28(2(1 - 901-1 . 918) =(1 . 751 - 1 . 918) 3 - 3 20 spat3(2+10 . 605 -10 . 6130 . 60312 Sa e 3 - 0 . 123 - = 0

45 degree celsius b) What was the pH optimum of the phosphatase assay? pH= 4 c) How would you classify the phosphatase activity found in your extract? It would be acidic phosphatase activity 4. (4pts) Write an abstract for this experiment. An abstract is a brief paragraph (three to five sentences) that communicates the purpose, the experiment conducted, and the specific conclusions of the experiment. Abstract should be concise without going into details about the protocol. Each sentence should convey required, non-redundant information. The aim of the experiment was to find the optimum temperature and pH which are unique to the phosphatase enzyme from Alfalfa Sprout. From the data obtained, it can be concluded that the optimum pH and temperature for This enzyme is 5 and 65°C respectively. 5. (3pts) A student added sodium carbonate to a reaction tube and the color remained clear. Propose three specific reasons why the student saw no color change. - they forgot to add PPP solution - the sample present in the tube maybe a breaking buffer instead of reaction buffer - no crude extract was present 6. (3pts) Increasing the temperature of a chemical reaction generally favors product formation. Propose a hypothesis for why the 75°C reaction conditions did not produce the most activity. - since 75 deg Celsius is past the optimal temperature for this enzyme, it is possible that at this temperature, most of the proteins would have been denatured, resulting in low activity.

10. (4pts) A vegetative storage protein (VSP2) from the model plant Arabidopsis thaliana (At) was purified as a wild-type (AtVSP2) and a recombinant protein (Nus-AtVSP2). D119E is an aspartic acid to glutamic acid substitution at amino acid position 119. The phosphatase activity was measured under different pH conditions (adapted from Liu et. al. Plant Physiol . 2005;139:1545-1556). a) What observation can you make about adding the Nus recombinant tag to AtVSP2? Propose a reasonable scientific hypothesis for the observed effect. Upon adding Nus recombinant tag, there is a slight reduction in phosphatase activity. Progression of the reaction could imply the protein structure was not lost. b) What can you conclude about the effect of the D119E mutation on the phosphatase activity? The D119E mutation induced significant structural modifications in the protein, leading to the loss of solubility and complete inactivation of its phosphatase activity, possibly by disrupting the substrate-binding site.