Enzyme Lab Dr. Ippolito BIO121 section MC, SD

Materials Test Tubes Sulfuric Acid 3% Hydrogen peroxide Liver homogenate pH 3 buffer pH 7 buffer pH 11 buffer*

Goals Understand and quantify enzyme activity Practice the Principles of the scientific method Develop laboratory skills Develop inductive / deductive reasoning skills

Revised Data Table please copy this exactly Tube #ConditionsRel. Activity (cm of bubbles) Rx Time (s) NOTES 1Positive Control 2+ Strong Acid 3pH 3 4pH 7 5pH 11 6Temp 45 C 7Temp 4 C 8Temp 100 C 90.1x Substrate

Background

Catalase: more accurately, hydrogen peroxidase, this enzyme catalyzes the breakdown of hydrogen peroxide to water and molecular oxygen. Because it was the first enzyme truly characterized, it has a historical name that simply means “catalyst” enzyme. Reaction: 2 H 2 O 2  2H 2 O + O 2 (gas) September 2004 Molecule of the MonthMolecule of the Month

About Catalase Catalases are some of the most efficient enzymes found in cells. Each catalase molecule can decompose millions of hydrogen peroxide molecules every second. The cow catalase shown here (PDB entry 8cat) and our own catalases use an iron ion to assist in this speedy reaction.8cat The enzyme is composed of four identical subunits, each with its own active site buried deep inside. The iron ion, shown in green, is gripped at the center of a disk-shaped heme group. Catalases, since they must fight against reactive molecules, are also unusually stable enzymes. Notice how the four chains interweave, locking the entire complex into the proper shape.

Why the liver? The liver detoxifies dangerous molecules. What is a ‘dangerous’ molecule? –Some are dangerous because they have a lot of free energy. –The liver carefully extracts this energy, like diffusing a bomb. –How? It oxidizes! It removes the high-energy electrons.

Why the liver? One of the side-products includes a lot of Hydrogen Peroxide, which while dangerous, is much less dangerous than the toxic molecules the liver processed. The final step in diffusing this energy is to convert hydrogen peroxide into harmless water and oxygen. So the liver has tons and tons of catalase, because it has tons and tons of H202 to deal with.

Get into groups and grab your gear!

Control Reaction Preparation –9 tubes labeled 1-9 –Mark them at 1 and 2 cm Protocol –Step 1: Substrate Preparation Add 1 ml water to Tube 1 Add 1 ml sulfuric acid to Tube 2 Add 1 ml of 3% H202 to each tube –Step 2: Enzyme addition (and reaction start) Add 3 drops of catalase (from crude liver preparations) to Tube 1, then tap the bottom of the tube GENTLY. Immediately after this, record initial time. Measure bubbles with ruler and measure time; we will be calculating reaction rate with these numbers, so be as accurate as you can. Repeat this for Tube 2. Record Data.

Discussion What are some good reasons to do this control? Why are we only doing a positive control? Why did I change the table in the book?

Experiment 1 Background Enzymes are proteins; what effect with changing pH have on catalase activity? –We have 3 buffers to work with, but pH 11 was made incorrectly. State our Hypothesis State our Predictions

Experiment 1 Setup –Use tubes 3, 4, 5… Buffer Preperation –Add 1 ml of pH 3 buffer to tube 3 –Add 1 ml of pH 7 buffer to tube 4 –Add 1 ml of pH 11 buffer to tube 5 Substrate Addition –Add 1ml of 3.0% H202 to each tube. Enzyme Addition (and reaction start) –Add 3 drops of enzyme to tube 3 and mix. Quantitate reaction similar to control experiment. REPEAT FOR TUBE 4, THEN 5. RECORD ALL RESULTS.

Experiment 1 Discussion Did you get your expected results? Why or why not? Why isn’t everyone getting the exact same data (or are they)? What would be a good next experiment to conduct?

Experiment 2 Background Enzymes are proteins; what effect with changing temperature have on catalase activity? –We will look at cold, warm, and hot conditions (and compare these to our room temperature control) State our Hypothesis State our Predictions

Experiment 2: Temperature Preparation –Make sure there is an icebath, a 45 C waterbath, and a boiling waterbath. –Use tubes 6, 7, 8 Add 1ml of water and 1 ml of H202 to each tube. Buffer Prep: 20 minutes of boiling will evaporate; let’s do 5 minutes in each with a gentle swirl every 2.5 minutes. –Tube 6: 45 C –Tube 7: ice –Tube 8: boil Enyzme addition (and reaction start). You know the drill.

Experiment 2 Discussion Did you get your expected results? Why or why not? Why would a weak buffer not work? What would be a good next experiment to conduct?

Experiment 3 Background Another variable that alters enzyme reaction rates is how much substrate the enzyme has to work with. What effect will reducing substrate levels have on the reaction rate? State your hypothesis State your prediction

Experiment 3 Preparation and reaction start (this one’s easy) –We’ll be using tube 9 only. –Add 1 ml of water to tube 9. –Add 1 ml of 0.3% H202 (a dilution factor of 1:10) –Add 3 drops of enzyme –Quantitate the reaction as you’ve done previously

Experiment 3 Discussion Did you get your expected results? Why or why not? What would be a good next experiment to conduct? What effect would adding 10x more substrate have? What kinds of variables would you expect to impact an enyzmatic reaction rate besides pH, temperature, and substrate concentration? –(we usually write concentration like this: [substrate])

Expectations In two weeks: Hand in a packet that contains: –A completed data table from each group –The answers to questions 1-13 on page 29 of your lab manual –A written summary of the control and each of the 3 experiments. Do not exceed 2 pages double-spaced. One paragraph / experiment is sufficient. –A brief discussion on your results Hand this in and you will receive a maximum of 20/20 points. Points will be given based on effort and final presentation of the lab report. –This means that well-thought out wrong answers will not hurt your grade. For an additional 5 points, download this powerpoint and answer the discussion questions for each “Discussion” section