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