Lab 8: Assay Development, Day 3 Today you will conduct another round of assays. This week, the assays will focus on the activity of an enzyme called peroxidase. Hydrogen peroxide (H2O2) is produced in cells as a byproduct of cellular respiration. Since H2O2 is toxic, cells must rid themselves of it rapidly. Within cells there are enzymes called peroxidases that bind to H2O2 and break it down into water and oxygen. The overall reaction equation is: hydrogen peroxidase 2 H 2O 2 2 H2O + O 2 reactant (substrate) products [hydrogen peroxide] [Water] + [oxygen] Continued on next page 69 Activity 8a Testing Samples for Hydrogen Peroxidase Activity Purpose In this activity, we will test several plant and animal tissues for the presence of the peroxidase enzyme. Background In order to determine whether a tissue sample has peroxidase activity, we need a way of monitoring the reaction. Therefore, we need to detect either the disappearance of H2O2 or the formation of one of the reaction products, water or oxygen. Since oxygen is formed as a gas that bubbles out of solution as it is formed, the peroxidase enzyme activity can be easily monitored by measuring the amount of oxygen gas (bubbles) produced as the reaction proceeds. Procedure 1. Cut duplicate small (roughly 1 cm3-size) pieces of each sample to be tested. It’s important that your samples are about the same size and have the same amount of surface area exposed. You are testing each sample in duplicate to minimize any error. 2. Place each sample into a glass test tube from your drawer. Label the test tubes with tape so you know which sample is which. 3. Set up a negative control test tube containing 1 mL of deionized water. 4. Add 2 mL of 3% H2O2 to each test tube and allow the enzymatic reaction to occur for exactly 60 seconds. Observe the results at 60 seconds, and record the relative levels of reaction occurring in each tube. 5. Record the amount of peroxidase activity in each sample in a data table similar to Table 8.1 (see next page). Use a numerical scale of 0 to 5, w/ 0 = no bubbles and 5 = enough bubbles to overflow the test tube). Table 8.1 Peroxidase Activity in Tissue Samples Tissue sample Peroxidase Activity (0-5 scale), trial #1 70 Peroxidase Activity (0-5 scale), trial #1 Activity 8b Isolation of HRP from Horseradish Root Purpose The purpose of this activity is to isolate an active form of horseradish peroxidase (HRP) from horseradish root. Background Proteins in plants are potential biotechnology products. To examine proteins for their commercial or research value, researchers must purify them from their source cells. Procedure 1. Weigh out 5 g of grated horseradish root. 2. Mix the 5 g of horseradish root with 15 mL of 25 mM NaH2PO4 buffer. I will make this buffer for you in order to save time, but for practice, do the calculation as if you were going to prepare 100 mL of 25 mM NaH2PO4 buffer. The molecular weight of NaH2PO4 is 120. 3. Homogenize the sample, grinding with a mortar and pestle until the sample appears as a relatively uniform suspension. 4. To remove large cellular debris, filter the homogenate through three layers of cheesecloth into a 15 mL plastic conical centrifuge tube, placed on ice. 5. Centrifuge the sample tubes for 5 minutes in a 3000X-g centrifuge. Make sure to balance the tubes. Have your instructor help you use the large tabletop centrifuge.* 6. After centrifuging the tube, discard the supernatant and save the pellet. The pellet is cell debris that contains a large amount of proteins. 7. Place the tubes on ice to chill them for at least 5 minutes. 8. Add prechilled acetone to the horseradish extract at a concentration of one part extract for every three parts of acetone (a 1:4 ratio). To determine how much acetone to add, use a micropipet to measure the volume of horseradish extract that you have. Then, multiply this volume times 3 to determine the volume of acetone to add. After you have added the acetone, finger-flick to mix, then invert to mix 3-4 times. 9. Centrifuge the sample for 15 minutes in a microcentrifuge at maximum speed (you will need to transfer the mixture from step 8 into microcentrifuge tubes in order to do this). Make sure that the tubes are balanced. 10. After centrifuging, you should see a pellet containing HRP at the bottom of the tube(s). Gently, decant (pour off) the supernatant into a waste container. 11. Redissolve the protein-containing pellet in 2 mL of cold 25 mM NaH2PO4 buffer. The sample is now approximately 10-20 times more concentrated than the starting sample. It is by no means a pure HRP extract, but it is highly enriched in the HRP enzyme, which should be active in the NaH2PO4 buffer. * Note: Depending on how well our tabletop (large) centrifuge is working, we may need to experiment with other ways of pelleting the HRP-containing pellet. We will troubleshoot this together in lab if we need to. 71 Activity 8c Testing for the Presence of Peroxidase using TMB Purpose In this activity, you will test the HRP extracts that you made in Activity 8b for peroxidase activity. This will give you an idea of how effective your HRP purification was, as well as introduce you to the technique of using an indicator to monitor a chemical reaction. Background In this assay, we will use the chemical indicator TMB (3,3’,5,5’-tetramethylbenzidine). As H2O2 is broken down by the peroxidase enzyme, electrons are transferred from H2O2 to TMB (an oxidation-reduction reaction). As TMB accepts electrons from H2O2, the TMB turns a blue color. A stop solution is then added containing acid, which turns the TMB from blue to yellow. The degree of yellow color is directly related to the amount of peroxidase activity in the sample. 1:10000 1:1000 1:10 • your full-strength extract • a 1:10 dilution • a 1:100 dilution • a 1:1,000 dilution • a 1:10,000 dilution 1:100 Procedure 1. Prepare 1:10 serial dilutions of your HRP extract so you have: 2. Get a 96-well plate and label the wells to show where the HRP extracts will be placed. Include negative controls using the NaH2PO4 pH 7.0 buffer (not water). Test each sample in triplicate (3 times) (in total, you should have 18 wells being used on your plate). Have everything ready (extracts & dilutions, ~2 mL of diluted TMB, ~300 µL of 0.5 M HCl, micropipets and tips) before proceeding further because the next steps need to be performed quickly and on time. 3. Add 100 µL of diluted TMB to each of the 18 wells that will be receiving sample. 4. Add 100 µL of the appropriate HRP extract or the negative control to each well. Mix thoroughly by pipetting up and down with your tip. 5. Let stand for 10 minutes. 6. Add 10 µL of 0.5 M HCl to each well. This is the “stop” solution. 7. Look for a color change from blue to yellow. 8. Dilute the extracts until there is no evidence of a positive TMB assay (if necessary). 9. Record the data, including a rating system for the degree of yellow color visible. If there are differences among the triplicate samples, take an average. Draw some conclusions as to the success of your HRP purification procedure in Activity 8b. 72 Lab 8 Homework Name: ________________________ 1. Why do cells make the peroxidase enzyme? 2. In Activity 8a, how could we tell that the peroxidase enzyme was present in a sample—what reaction product did we observe being formed? 3. Why did we set up multiple samples of our peroxidase assays? (In Activity 8a, we set up duplicate samples; in Activity 8c, we set up triplicate samples). 4. You want to make a serial dilution of an amylase solution using water as your diluent. Describe how you would perform your serial dilution so that you end up with 1 mL each of: full-strength amylase, 1:10 dilution, 1:100 dilution, and 1:1000 dilution. 73 5. How would you make a 1:100 dilution of a chemical solution (using water as your diluent) so that your final volume was 5 mL? 6. In the peroxidase assay in Activity 8c: a) What was the purpose of TMB? b) What was our negative control, and what was our expected result for the negative control? 74