Enzyme Reactions in Living Organisms DAY 1: DEMONSTRATION and LAB

GUIDED QUESTIONS 1. Give an overview of the function of an enzyme. Pull in specific examples of enzymes found in the human body and their function.   2. Describe the general structure of an enzyme. List and describe the following parts: amino acids, protein folding, substrate, product, and active site. 3. Explain hydrogen peroxide and its role in cell health. How is it produced in the body and explain its purpose and/or lack of purpose (review article if needed). Image courtesy of: http://www.biologycorner.com/APbiology/intro/notes_ch6-metabolics.html

DAY 1: DEMONSTRATION

OVERALL REACTION

Is Catalase the same enzyme as Peroxidase? Catalase is a member of the peroxidase family that specifically uses hydrogen peroxide as a substrate, hence it could be called "Hydrogen Peroxidase": however, so does Peroxidase (also called Myeloperoxidase). The major difference between Catalase and Peroxidase is that Catalase generates water and Oxygen, while Peroxidase generates water and an another activated donor molecule. So "Hydrogen Peroxidase" could actually refer to more than one enzyme without identifying the more important aspect of the reaction: how our enzyme is measured.

PROCEDURE (STEP 1) Measure approximately 5 mL of hydrogen peroxide into a clean test tube. Is the hydrogen peroxide bubbling?

PROCEDURE (STEP 2) Measure 5 mL of liver solution into the other clean test tube. Pour the test tube with the hydrogen peroxide into the test tube holding the liver solution. Notice that this reaction produces bubbles. What gas do you think is being released and why? How could you test for the identity of the gas?

PROCEDURE (STEP 3) Recall that a reaction that absorbs heat is endothermic; a reaction that gives off heat is exothermic. Now feel the temperature of the test tubes with your hand. Has the test tube gotten warmer or cooler? Is the reaction endothermic or exothermic?

REFLECTION What do you think is happening in the test tube to cause this reaction to happen? What do you think would happen if you added more hydrogen peroxide?

Let’s Make Our Model How do enzymes work?

ADD THE FOLLOWING TO YOUR RESEARCH JOURNAL: The Research Question: Do all living organisms perform a similar reaction when combined with hydrogen peroxide? Null Hypothesis: There is no significant difference in the rate of reaction (mL/sec) between the peroxidase found in different living organisms. Alternate Hypothesis 1: (If/Then/Because): Think about ranking the four different tissues (potato, yeast, liver, and spinach) Independent (experimental groups)/dependent variables (units): Control and constants

MATERIALS: Stop watch Tubing Thistle tube Stopper (2) 10 mL graduated cylinder (1) 100 mL graduated cylinder (1) pneumatic trough or shallow pan (1) 250 mL beakers Dropping pipette Premade solutions of varying tissues H2O2 solution

PROCEDURE Make sure your trough and gas production apparatus are properly set up. Fill a pneumatic trough ¾ full of water place the overflow nozzle towards the sink. Completely fill and submerge a 100 mL graduated cylinder in the water upside down in the trough. Measure 10 mL of hydrogen peroxide (diluted with water by 50) in a graduated cylinder. In a second graduated cylinder measure 10 mL of tissue solution. Remove the stopper and pour the 10 mL of tissue solution into the thistle tube to add it to the flask. Replace stopper, release pinch clamp. Place the mouth of the inverted submerged graduated cylinder (keeping the mouth of the graduated cylinder under the water) over the air inlet. Make sure the cylinder is full of water (with limited to no air pockets).

PROCEDURE Get ready to start the timer. Pour the 10 mL of the hydrogen peroxide. Gently swirl the contents of the flask. Begin the timer when the first bubble appears in the cylinder and stop the timer then the bubbles cease. Collect the amount of water displacement (mL) over time (sec) in the chart below. Replace the pinch clamp Dump the contents of the flask into the labeled container. Wash the flask and repeat steps 1 through 11 using each tissue. Repeat the experiment again for each solution and share your data with the class.

Lab Set Up with Pneumatic Trough

Lab Set Up with Cylinder in Place Over The Air Inlet Hole for Air Bubbles

Lab Set Up with Water Displacement – measure the difference in mL

Amount of O2 Produced (mL/sec) DATA TABLE Does the type of organism affect the enzymatic rate of reaction (mL/sec)?   Amount of O2 Produced (mL/sec) Trial Potato Yeast Liver Spinach 1 2 Your Average: Class Average:

INVESTIGATION Students should be in groups of four and collect two trials for each sample. Data will be shared with the class by placing it in an Excel spreadsheet provided by the teacher. Create a bar graph in your research journal which includes a title, labeled x axis and y axis, and a key that represents the class and your calculated averages for each pair of tissues sampled.