Enzymes

 

Quick Introduction

 

Enzymes are globular proteins that selectively speed up (catalyze) the rates of many chemical reactions. The determining factor as to whether a reaction rate will be increased is if the chemical reactant(s) or substrates bind to the surface of the enzyme in a specific location called the active site. (Thus, any environmental influence or other factor that changes the shape of the active site will alter enzyme activity.)

 

Today’s lab will examine the enzyme catalase. The reaction it catalyzes is as follows:

 

2H2O2 -à 2H2O + O2

 

This reaction normally occurs at a very slow rate, but in the presence of catalase, the reaction is greatly accelerated as the substrate, hydrogen peroxide (H2O2), binds to the active site of catalase, producing water and oxygen gas.

 

What is a rate?

 

Remember that rates are quantities expressed per unit time. In the diffusion lab just completed, diffusion rates were expressed as μS/cm/sec. Chemical reaction rates are typically expressed as the amount of reactant (substrate) utilized per time or the amount of product synthesized per time. For chemical reactions that are catalyzed by an enzyme, the reaction rate is considered equivalent to the enzyme activity. For catalase, enzyme activity will be estimated as the amount of oxygen gas produced per second.

 

How do you determine catalase activity?

 

We will measure the amount of oxygen liberated over a fixed time period in a closed chamber containing catalase and hydrogen peroxide. The oxygen content will be determined indirectly by measuring the increased pressure in the reaction chamber using a pressure probe linked to your computer workstation. The units for pressure are mmHg.  If the oxygen concentrations (dependent variable) are plotted against the time they were measured (independent variable), the slope of the resulting line is the catalase activity expressed as mmHg/sec.

 

Where is catalase found?

 

Catalase is found in all living cells. It has the essential role of breaking down the toxic metabolic by-product, hydrogen peroxide. In today’s lab, we will study catalase extracted from the common garden turnip.

 

What To Do Today

 

Extraction of catalase from turnip

 

1.     Peel a small piece of turnip

2.     Using a vegetable grater, remove ~30 g of tissue. Record this weight in your notebook.

3.      Transfer the tissue to a plastic homogenizing vessel and add 40 ml of cold buffer. Homogenize the tissue with the “boat motor” homogenizer until it reaches the consistency of applesauce.

4.       Filter this mixture through cheesecloth into a cold graduated cylinder and record the volume in your notebook. Calculate the g plant tissue/ml buffer concentration of your homogenate. This is your enzyme solution. Label it as 100% catalase. Leave this mixture on ice until you need it.

 

Pressure probe set up

  1. Make sure the pressure probe is connected to the LabPro interface and the computer.
  2.  Make sure the tip of the probe is in air before you continue.
  3.  On the right hand of the screen, click on the “Logger Pro” icon.
  4. Check the boxed display. It should read approximately 750 mmHg. You are ready to begin readings. The pressure probe requires no calibration.

 

How to run a catalase assay

  1. You need a tall (20 X 150 mm) test tube for each control and experimental group.
  2. To each tube add 3 ml 3% H2O2.
  3. To begin an assay, add 1 ml of your catalase solutionA. Briefly shake the tube to mix the contents.
  4. Firmly insert the 1-hole stopper assembly (attached to the pressure probe) into the test tube. Gently shake the tube throughout the assay. If you place the tube back in the test tube rack, the activity will be lower. .
  5. Click on “collect” on the Logger Pro software. It has been preset to collect pressure data for 100 seconds.
  6. Once data gathering is complete, hold down the left hand button on the mouse and select an interval on the plot that represent a linear change.
  7. Select “analyze” on the menu across the top of the screen and then chose “linear fit”. A box should appear on the graph with the equation of the line and the correlation coefficient. Record the slope in your data collection table. This is the catalase activity. What are the units?

ANOTE: If the top pops off the reaction tube during the run, dilute your original enzyme solution with a known amount of buffer and try the assay again. Repeat this step if it pops off again.

 

How to save data after a run:

  1. Select "experiment" on the menu across the top of the screen and then chose "store latest run". 
  2. If you want to hide the data from a run, select "data" from the top menu and chose "hide data set".  NOTE: Always choose “run 1” (or whatever number is appropriate) on the menu that appears. DO NOT CHOOSE “LATEST RUN”. If you do, on the next assay, this will cause the program to lock up and give the message, “waiting for data”. (If you make this mistake anyway, go to the “data” menu and select “show data set” and the problem will be resolved.)

 

 

How to save your Logger Pro file and close the Logger Pro program when the lab is completed:

  1. Go to the "file" menu and select "save as". 
  2. Make sure to re-name your file and save it in the appropriate student folder.
  3. Using the horizontal menu (at the top left of the screen) select "Logger Pro" and choose "Quit Logger Pro".

 

Experiment 1 (Research Question): Does the concentration of enzyme affect catalase activity? 

1.      Pose a hypothesis from this RQ and design an experiment to test your hypothesis. Answer the following questions: What are the controls? What are the experimental groups? Independent variable? Dependent variable? Replications? Remember to construct an appropriate data collection table in your lab notebook and check with the instructor before you proceed. HINT: When designing your experiments, add the same amount of hydrogen peroxide (3ml) and the same volume of enzyme (1ml) to each tube. Vary the amount of enzyme in a sample by diluting the stock enzyme preparation (100%) to a desired amount (e.g. 20, 40, 60, 80%) before it is added to the tube.

2.      Run each sample and record the catalase activity (mmHg/sec/ml). FOR MORE OBJECTIVE RESULTS, DO NOT RUN THE ASSAYS IN ORDER, BUT SELECT THEM RANDOMLY FROM THE RACK.

3.      Construct a final figure of the results and bring it with you next week.

 

Analysis:

1.      What was the relationship between catalase concentration and oxygen evolution in your experiment? Was it linear? Nonlinear? Explain.

2.      Did the results support your hypothesis?