Slide 1 / 28 Slide 2 / 28 New Jersey Center for Teaching and Learning Progressive Science Initiative AP BIOLOGY This material is made freely available at www.njctl.org and is intended for the non-commercial use of students and teachers. These materials may not be Investigation #13 used for any commercial purpose without the written permission of the owners. NJCTL maintains its Enzyme Activity website for the convenience of teachers who wish to make their work available to other teachers, participate in a virtual professional learning community, and/or provide access to course Summer 2014 materials to parents, students and others. www.njctl.org Click to go to website: www.njctl.org Slide 3 / 28 Slide 4 / 28 Pacing/Teacher's Notes Investigation #13: Enzyme Activity Click on the topic to go to that section · Pacing/Teacher's Notes · Pre-Lab · Guided Investigation - Procedure 1 · Guided Investigation - Procedure 2 · Independent Inquiry Return to · Spectrophotometer Use Table of Contents Slide 5 / 28 Slide 6 / 28 Pacing Teacher's Notes General Reference Lab procedure adapted from College Board AP Biology Day (time) Activity to Unit Plan Notes Description Investigative Labs: An Inquiry Approach Teacher's Manual Day 1 (40) Pre-Lab Pre-lab MP Day 17 questions If you have a spectrophotometer, Click here for CB see instruction in presentation or lab Day 2 (40) Procedure 1 Setting baseline MP Day 18 manual. Prepare for tomorrow: pH AP Biology buffers Teacher Manual Guided Practice - Affect of pH Procedure 2 Day 3 (40) and review MP Day 19 experimental design Conduct independent Independent investigation Day 4 (80) MP Day 20 Investigation and share results and discuss Assessment Day 5 (20) Lab Quiz MP Day 21
Slide 7 / 28 Slide 8 / 28 Pre-Lab Question/Objectives How do abiotic or biotic factors influence the rates of enzymatic reactions? In this lab we will: · Understand the relationship between enzyme structure and function. · Make some generalizations about enzymes by studying just one enzyme in particular. · Determine which factors can change the rate of an enzyme reaction. · Determine which factors that affect enzyme activity could be biologically important. Return to Table of Contents Slide 9 / 28 Slide 10 / 28 Safety Pre-Lab Questions Follow general laboratory safety procedures. Wear proper Read the background information and answer the following questions footwear, safety goggles or glasses, a laboratory coat, and in your lab notebook. gloves. Use proper pipetting techniques, and use pipette pumps, 1. Describe the structure of enzymes and relate this structure to an enzyme's function. syringes, or rubber bulbs. Never pipette by mouth. Dispose of any broken glass in the proper container. 2. Graph free energy (G) vs. time of a exergonic biochemical reaction with and without an enzyme present. Since the concentrations of the reactive materials in this laboratory are environmentally friendly (0.1% hydrogen peroxide and 0.3% guaiacol), they can be rinsed down a 3. Explain what is meant by "induced fit." standard laboratory drain. The concentrations used here are deemed safe by all chemical standards, but recall that any compound has the potentiality of being detrimental to living things and the environment. Slide 11 / 28 Slide 12 / 28 Materials - Procedure 1 Guided Investigation - · Turnip peroxidase Procedure 1 · 0.1% hydrogen peroxide · Guaiacol · Distilled (deionized) water · 3 test tubes and test tube rack · Timer · 1, 5, and 10 mL graduated pipettes · Camera · Spectrophotometer (if available) · Laboratory notebook Return to Table of Contents
Slide 13 / 28 Slide 14 / 28 Procedure 1: Determining a Baseline Procedure 1: Determining a Baseline Step 1 Using two 16 x 150 mm test tubes, mark one "substrate" Step 3 Combine the contents of the two tubes (substrate and and the other tube "enzyme." To the substrate tube, add 7 mL of enzyme) in another 16 x 150 mm test tube, cover the tube with distilled water, 0.3 mL of 0.1% hydrogen peroxide, and 0.2 mL Parafilm, invert twice to mix, and place the tube in a test tube rack. guaiacol for a total volume of 7.5 mL. Cover the test tube with a IMMEDIATELY begin timing the reaction. piece of Parafilm and gently mix. Step 4 Observe the color change for the next 5 minutes. Rotate Step 2 To the enzyme tube, add 6.0 mL of distilled water and 1.5 the tube before each reading. Record the observed color at 0, 1, mL of peroxidase for a total volume of 7.5 mL. Cover the test tube 2, 3, 4, and 5 minutes using a digital camera or cell phone. with a piece of Parafilm and gently mix. Slide 15 / 28 Slide 16 / 28 Procedure 1: Determining a Baseline Analyzing & Evaluating Results Step 5 Use the color palette/chart to help you quantify changes in Analysis Questions: color over time. Graph your data in your laboratory notebook. · You measured the color change at different times. Which time will you use for your later assays? Why? (The time change that you select will serve as your baseline for additional investigations.) · When you use this assay to assess factors that change enzyme activity, which components of the assay will you change? Which will you keep constant? Slide 17 / 28 Slide 18 / 28 Materials - Procedure 2 Guided Investigation - · Turnip peroxidase Procedure 2 · 0.1% hydrogen peroxide · Guaiacol · Buffers with range of pH · Distilled (deionized) water · 18 test tubes and test tube rack · Timer · 1, 5, and 10 mL graduated pipettes · Camera · Spectrophotometer (if available) · Laboratory notebook Return to Table of Contents
Slide 19 / 28 Slide 20 / 28 Procedure 2: The Effect of pH Procedure 2: The Effect of pH Step 1 Using clean 16 x 150 mm test tubes, make six sets of pairs Step 2 Combine the substrate and enzyme tubes for all six pairs of substrate and enzyme tubes for a total of 12 tubes. You will (total volume 15.0 mL per pair), cover with Parafilm, gently mix, substitute a different pH buffer for the distilled water used in the and place the tubes back in the test tube rack. IMMEDIATELY original enzyme tubes. Prepare the tubes as follows and be sure to begin timing reactions. label them. · For each substrate tube in a pair, add 7 mL of distilled water, 0.3 Step 3 Record the observed color for each tube at 0 minutes and mL of hydrogen peroxide, and 0.2 mL of guaiacol for a total again at the time you chose based on your results in Procedure 1. volume of 7.5 mL. · For each enzyme tube in a pair, add 6.0 mL of a specific pH Step 4 Use the palette/color chart to help you quantify the changes solution and 1.5 mL of peroxidase for a total volume of 7.5 mL. you observe. Graph your data as color intensity vs. pH · Cover each test tube with a piece of Parafilm, and gently mix. Slide 21 / 28 Slide 22 / 28 Analyzing & Evaluating Results Independent Inquiry Analysis Questions: · What conclusions can you draw from your results? Return to Table of Contents Slide 23 / 28 Slide 24 / 28 Designing & Conducting Your Investigation Designing & Conducting Your Investigation You now have the basic information and tools needed to Design and conduct an experiment to investigate an answer explore enzymes in more depth on your own. In this part of to one of the questions above or another question that might the lab, you will do just that. You will have the chance to have been raised as you conducted Procedures 1 and 2. develop and test your own hypotheses about enzyme activity. Remember, the primary objective of the investigation is to To help you get started, read the following questions, and explore how biotic and abiotic factors influence the rate of write your answers in your laboratory notebook. enzymatic reactions. · In Procedure 1, was the limiting factor of your baseline reaction the enzyme or the substrate? How could you modify Complete the following investigation proposal for teacher the procedure you learned to answer this question? approval. · What are three or four factors that vary in the environment in which organisms live? Which of those factors do you think could affect enzyme activity? How would you modify your basic assay to test your hypothesis?
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