Name ______Date ______Per ______

ENZYME CATALYSIS LAB PROTOCOL (PROCEDURE)

Background

The enzyme, catalase (or peroxidase) is found in all aerobic organisms to prevent the accumulation of hydrogen peroxide produced as an end-product of metabolism.

2H2O2 → 2H2O + O2

In this lab, the rate of the reaction will be determined by measuring the disappearance of the substrate, H2O2. Sulfuric acid (H2SO4) will be used to stop the reaction at various time intervals, followed by a titration procedure using potassium permanganate, KMnO4. KMnO4 may be used to quantify the amount of H2O2 in solution because as long as H2O2 is present, KMnO4 reacts with it to form a colorless product. Once all of the H2O2 is used up, continued addition of KMnO4 will produce a purple-colored solution.

Set-Up:

  • Goggles, gloves, aprons are required
  • Catalase
  • Obtain a Styrofoam cup.
  • Fill it with ice until it is approximately ¾ full.
  • Locate the plastic cup labeled Catalase at your lab station. Take your plastic cup to the side counter where the stock solution is located and pour approximately 15 mL of catalase into the cup.
  • Immediately place the cup on ice.
  • Buret
  • Orient buret so that it is secure and readable. Make sure the valve is closed.
  • Use the funnel to add enough KMnO4 to fill the buret.
  • Practice titrating KMnO4 into the cup marked “Waste”.
  • After you have practiced, record the initial buret reading in the Data Table.
  • Solutions
  • H2SO4is in the small beaker (labeled).
  • 1.5% H2O2is in the plastic cup (labeled).
  • dH2Ois in the plastic squirt bottles, and Wash Wateris in plastic cup labeled “wash water”.
  • Syringes
  • Use the Sharpie provided to label the three syringes as follows:
  • 5 mL syringe Transfer
  • 10 mL syringe H2O2
  • 10 mL syringe H2SO4
  • Cups
  • There are two sets of cups:

One set of cups should say: baseline, 10sec, 30 sec, 60 sec, 90 sec, 120 sec, and 180 sec.

The second set of cups should say the same thing, but they should be marked with a “T”. These cups will be used as the titration cups throughout the procedure.

Part A – Establishing a Baseline

  1. Use the H2O2syringe to add 10 mL of H2O2 to the cup labeled Baseline.
  2. Use the plastic pipette to add 2 mL of dH2O to the Baseline cup. This is in place of the catalase you will add in Parts B and C.
  3. Use the H2SO4 syringe to add 10 mL of H2SO4 to the Baseline cup.
  4. Gently swirl the cup to mix the contents.
  5. Use the Transfer syringe to remove 5 mL of solution from the Baseline cup and put it in the second Baselinecup, marked with a “T”. This is your Baseline Titrationcup. Rinse the Transfer syringe by drawing up water from the Wash Water cup and squirting it into the sink.
  6. Make sure you have recorded the initial volume of KMnO4 in the Data Table.
  7. Place a piece of white paper underneath the Baselinetitration cup. Titrate with KMnO4, adding a drop at a time until a persistent pinkish-brown color is obtained. Be sure to continuously swirl the cup or mix with the glass stir rod. Record the final burette volume in the data table.
  8. Determine the base line by subtracting the final reading from the initial reading.
  9. Record your group’s baseline on the white board so that we can determine a class baseline average.

Part B – Determining the Rate of H2O2 Decomposition

  1. Use the H2O2syringe to add 10 mL of H2O2 to each of the cups labeled 10 sec, 30 sec, 60 sec, 120 sec, and 240 sec.
  2. Before beginning the timed tests, pre-measure 10 mL of H2SO4 in the H2SO4 syringe so you can stop the reaction at precisely the desired time.
  3. Use two transfer pipets to add 1 mL of catalase and 1 mL of dH2O to the cup labeled 10 sec.
  4. Swirl the cup to mix the contents.
  5. At exactly 10 seconds, add 10 mL of H2SO4 to the 10 sec cup.
  6. Repeat steps 2-5, as above, but allow the reactions to proceed for 30, 60, 120, or 240 seconds before adding the H2SO4.
  1. To titrate . . . For each test, use the Transfer syringe to remove 5 mL of solution from the test cup and put it in the second labeled Titration cup, marked with a “T”. Rinse the Transfer syringe between tests by drawing up water from the Wash Water cup and squirting it into the sink.
  2. Make sure you have recorded the initial volume of KMnO4 in the Data Table.
  3. Place a piece of white paper underneath the titration cup. Titrate with KMnO4, adding a drop at a time until a persistent pinkish-brown color is obtained. Be sure to continuously swirl the cup or mix with the glass stir rod. Record the final buret volume in the data table.
  4. Do not discard solutions until all titrations are completed.

Part C – Determining the Effect of a Variable on the Rate of H2O2 Decomposition at 0-30 seconds

  1. Using table data from Part B, calculate the Rate of Reaction from 0-30 seconds. Record.
  2. Rinse out the cup labeled 30 sec
  3. Repeat steps 1-10 as listed above for the 30 second test ONLY, making the necessary adjustment for the variable being tested.

2X Enzyme (1:5 ratio of Enzyme to Substrate)

Add 2.0 mL catalase and no dH2O in Step 3.

2X Substrate (1:20 ratio of Enzyme to Substrate)

Use 3.0% H2O2 in Step 1.

  1. Calculate the Rate of Reaction for 0-30 seconds for doubling the enzyme concentration and doubling the substrate concentration.
  2. Post class data.

**Class Data will be posted on my web page**

Clean-Up

  • Please place lab handouts, Lab Notebook in lab chairs
  • Use the remaining H2O2 and dH2O to clean the buret…
  • Empty KMnO4 from buret into plastic cup labeled KMnO4.
  • Close buret valve and flush buret with remaining H2O2.
  • Discard buret contents into large plastic cup labeled “Buret Flush”.
  • Flush buret with distilled water.
  • Empty contents of Buret Flush cup in sink and rinse the cup.
  • Discard all remaining solutions into sink.
  • Rinse and dry all plastic cups.
  • Rinse all syringes, plastic pipets thoroughly.
  • Wipe down table top.
  • Remove safety equipment and wash hands.