1. Biology 120 Enzyme Function Exercise 4 www.celltech.com/resources/vt/enzymes.html

2. What you need to Know Understand the experiment being preformed today What is: Enzyme / Catalyst / Active Site / Substrate Answer all questions in the lab handout Understand the effect of pH, temp, and concentration has on an enzyme Understand the scientific processes that took place

3. Terminology Metabolism –Definition: The sum total of all biochemical activity that takes place in a living organism Catabolic Metabolism – break down –AB = A & B Anabolic Metabolism – build up –A + B = AB In vitro – out of the body (or in a tube) Substrate - The material being acted upon

4. Introduction Catalyst – speeds up the rate of a reaction Enzyme – a protein that is a organic catalyst –Hint: Most of the time ends in –ase Active Site – a pocket at the outside of the enzyme specific to a substrate (here is where it is taken apart or put together) 2H 2 O 2 2H 2 O +O 2 Catalase (organic) (Hydrogen peroxide) Substrate (Water & Oxygen) **Oxygen will bubble in vitro** Bubbles Catalase H2O2H2O2 H2O2H2O2

5. How Enzymes Work Biological Catalysis (usually proteins) – Make a reaction faster Enzymes are Proteins – built to work “active site” Proteins are Happy’ist in their natural Temp and pH –Usually ~37 0 C or 98 0 F & Usually a neutral pH (Function) –What would happen if you boil it? Guesses -___________ –What would happen if you put it in acid? ________________ –What would happen in the cold?________________ Enzymes can only work so fast…….. –If you keep adding substrate will the speed of the Rxn Catalase H2O2H2O2 O 2 & H 2 O Catalase or a)b) Poison What does cyanide do? Arsenic?

6. How Enzymes Work Biological Catalysis (usually proteins) – Make a reaction faster Enzymes are Proteins – built to work “active site” Proteins are Happy’ist in their natural Temp and pH –Usually ~37 0 C or 98 0 F & Usually a neutral pH (Function) –What would happen if you boil it? Guesses -____denature________ –What would happen if you put it in acid? _______denature_________ –What would happen in the cold?________inhibit________ Enzymes can only work so fast…….. –If you keep adding substrate will the speed of the Rxn Catalase H2O2H2O2 O 2 & H 2 O Catalase or a)b) Poison What does cyanide do? Arsenic?

7. Temperature Denature: enzyme unravels/unfolds and loses structure and function COLDHOT Low activity High activity -cold temperatures inhibit (prevent) enzyme activity -hot temperatures denature enzymes Best temperature Normal enzyme Extreme heat Denature d enzyme p. 35?

8. pH Acidic –less than 7 Basic (alkaline) – greater than 7 Neutral = 7 Enzymes have a certain pH at which they work best -above or below optimum pH inhibits activity pH = 0pH = 14 p. 35?

9. Your Experiment The reaction: Catalase: enzyme (organic catalyst) that converts hydrogen peroxide into water and oxygen 2 H 2 O 2 Hydrogen peroxide water + oxygen 2 H 2 O + O 2 What’s the substrate? Product? Catalase H2O2H2O2 catalase H 2 O + O 2 (gas)

10. The Experiment Close the buret valve & fill with water (bucket also) Invert the buret and clamp (make sure the water level is blow the 50 ml mark) Add 3 Grams of plant tissue and stopper the flask ***Put the tubing into the buret*** Put the rubber stopper on the flask an put the hose into the buret (careful not to lose the water) Add 3 ml 2H 2 O 2 to the flask (do NOT remove pipette) Record water level and Time!! Swirl the flask for 30 sec. Record the Level (Table 1)

11. To Remove – take out the hose and then uncork Add Tap Water to the plant tissue (Clean Flask) Repeat with Blood (animal) Test the Effects of Temperature (Hot and Cold) –Do Not Leave The Flask Unattended when on the hot plate.*** –Follow the instructions – pg 43 & Record on Table 2 The Effects of pH (3 different flasks) –Follow instructions on pg 47&48 – Record on Table 3 –You MUST ware safety equipment when near the HCL & NaOH The Effects of [Concentration] –Read the instructions Carefully –Be as accurate as possible –No exact time limit (until oxygen is no longer produced) Water Level at: T 0 = Time Start T f = Time Finish Pre-incubate

12. When ever you remove the Stopper…… –Where should the tube be? What would happen? Calculate the amount of oxygen produced –Oxygen will displace the water (water level will go ) –Simple subtraction (bigger # from the smaller #) Put answers on the board THE CLEAN UP!!! Clean all Flasks and other glass stuff completely (Tap water) Empty all water Wipe up the counter and any spills Check the center counter – Wipe up Put equipment all on the side counter Points can (and will be) deducted for messes left

13. Buret tube Flask Rubber stopper Tubing Ring Stand Water trough Experimental Setup -Set up apparatus as described on p. 36. -Place tissue in flask -Add hydrogen peroxide -Swirl flask 30 seconds -Record exact water level after 30 seconds -Record exact water level in buret tube Why does the water level change? What specific molecule caused it to change? Is this a substrate or product? What was in the tissue that caused the reaction to occur? -Test under other conditions 2 H 2 O 2 2 H 2 O+ O 2 p. 36-37?

14. Activities: 2) Activity #2: Effect of Temperature 3) Activity #3: Effect of pH 1) Activity #1: Presence of catalase 4) Activity #4: Effect of Enzyme concentration and Substrate concentration Once you’ve completed the tables, answer questions on p. 42 and understand the objectives in the lab on p. 35 At ~4:20, we’ll stop to discuss your observations & take roll Plant tissue Animal tissue For #2, #3, #4 use plant tissue only!!! IceHot plate HCl BufferNaOH -Let sit 10 min with HCl or NaOH before running the experiment -Remove once it bubbles or it will burn! -Keep on ice entire time! -To keep the swirling consistent, designate one group member to swirl gently for all activities!

15. Critical Thinking Take a look at the board –Do all the results match or show a trend? –Are there a few results that don’t make sense (why do you think this happened?) –What do the results lead you to conclude? –Why is it good to replicate experiments? Hummm What to do …. But think Brain in a jar