Biology 211 - Lab Three: Membranes & Enzymes
Reminder: Bring your textbook, a calculator, and goggles!
Pre-Lab Homework:
1. Today, you will use the following: I2KI (iodine solution), glucose, and starch.
Which is largest, which is smallest? How will you detect starch and glucose?
2. Fully understand DIFFUSION and OSMOSIS. How do cells respond to solutions
that are Hypotonic? Isotonic? Hypertonic?
3. Using your text (8.4) and this handout, describe enzymes, name the TWO
enzymes you are testing today, and explain what reaction each carries out.
4. Using your text (8.4) and this handout, compare and contrast competitive vs.
non-competitive inhibition. What is the name of the inhibitor you are testing in
lab? What enzyme does it inhibit?
In-Lab Activity Overview
a. Investigating Selectively Permeable Membranes
b. Viewing Osmotic Behavior in Plant Cells
c. Catechol Oxidase Activity*
d. Catechol Oxidase Inhibition*
e. Amylase - Two Temperatures, Three pH’s*
*For enzyme activities, we have adapted labs by Helms et al. (Biology in the
Laboratory, 1998) and Morgan & Carter (Investigating Biology, 2008).
Investigating Selectively Permeable Membranes - DIFFUSION
All cells are surrounded by selectively permeable membranes. Today, you will build a
"fake cell" made from dialysis tubing. The inside of your dialysis bag will contain starch
and glucose; it will be placed in an iodine solution. Your goal is to determine WHAT
your dialysis membrane is permeable to: starch, glucose, and/or iodine.
Set-Up Procedures – Work In Pairs
1. Obtain a piece of dialysis tubing from the fingerbowl at the front of class
2. Dialysis tubing is open at both ends so you need to close the bottom to fill
3. Do this by folding the tubing and clipping it with a provided barrette
4. Now add 5 ml glucose and 5 ml starch to the bag – rinse outside of bag
5. Fill a beaker with 300 ml water and add 2 full droppers of I2KI
6. Place the bag in the beaker and secure top to beaker rim with a rubber band
7. Wait 30-45 minutes before performing follow-up tests below
Follow-Up Tests AFTER Diffusion – Work in Pairs
Starch and Iodine Diffusion
What do you know happens when starch meets iodine? Use this logic to figure out
whether starch moved OUT of the bag, and whether iodine moved IN!
Biology 211 Lab Manual, Lab Three, page 1
Glucose Diffusion
Today, you will learn a new assay that detects simple sugars like glucose. This test
uses a light blue Benedict’s Solution and heat. If simple sugars are present, the
solution turns anything but blue (green, yellow, orange, or red – depending on the
amount of sugar). Set up the following test tubes to determine glucose diffusion.
Control Test Tube
Bag Test Tube
Beaker Test Tube
Add 3 ml of
Water
Bag Solution
Beaker Solution
Add 1 dropper of
Benedict’s
Benedict’s
Benedict’s
Heat all test tubes in hot water bath for 3-5 minutes
Viewing Osmotic Behavior in Plant Cells - OSMOSIS
For this exercise, you will study osmosis in plant cells (Elodea), determining whether
“mystery” solutions are hypertonic or hypotonic.
Procedures – Work In Tables
1. At the front of the lab are Elodea in solution A, and Elodea in solution B
2. One pair will prepare a wet mount of Elodea/A, the other Elodea/B
3. View using your microscope, examining at the 10X and 40X objectives
4. Compare to Elodea in isotonic pond water, which is set up for class viewing
Catechol Oxidase Activity
Catechol oxidase is an enzyme found in plant cells. Its activity explains why many
fruits and vegetables turn brown after you cut them. Today, you will use catechol
oxidase from potatoes. Catechol oxidase carries out the following reaction:
Catechol + O2 yields Benzoquinone + H2O
Catechol (substrate) has no color; benzoquinone (product) is brown. Your first task
will be to view this reaction, establishing basic observations you need to interpret the
next experiment about enzyme inhibition.
Tube Set-Up Procedures – Work In Pairs
Tube
1
2
3
Water
Catechol (SUBSTRATE)
Potato Extract*
5.5 ml
0.5 ml
None
5 ml
0.5 ml
0.5 ml
5.5 ml
none
0.5 ml
*Potato Extract CONTAINS catechol oxidase, the ENZYME
Catechol Oxidase Inhibition
There are TWO kinds of enzyme inhibitors: COMPETITIVE mimic the substrate, bind
the active site, and can be relieved by adding more substrate. NON-COMPETITIVE do
not mimic the substrate, bind at a different site (allosteric!), and CANNOT be relieved
Biology 211 Lab Manual, Lab Three, page 2
by adding more substrate. In this experiment, you will determine whether PTU is a
COMPETITIVE or a NON-COMPETITIVE inhibitor.
Tube Set-Up Procedures – Work In Pairs
Tube
1
2
3
Water
5.5 ml
5 ml
6 ml
Potato Extract
0.5 ml
0.5 ml
0.5 ml
PTU
0.5 ml
0.5 ml
none
Catechol
0.5 ml
1 ml
0.5 ml
Assaying Amylase Activity - Two Temperatures, Three pH’s
Amylase, an enzyme made in the saliva of many animals (including your own!), carries
out the following reaction:
Starch + H2O yields Simple Sugars
Because neither the substrate nor the products are colored, you will have to use other
tests (e.g. starch/iodine and simple sugars/Benedict’s) to indirectly detect enzyme
activity. As with all enzymes, amylase activity is optimal in conditions that reflect its
native environment. The following two experiments will allow you to compare amylase
activity in both native and non-native conditions.
Two Temperatures Procedures – Work In Pairs
1. Set up the following TWO tubes (see table below)
2. Place each tube at the stated temperature for 5 minutes
3. After 5 minutes, add 1 ml starch to each tube and wait 2 minutes
4. After 2 minutes, add 1-2 drops of I2KI and observe, interpret
ADD IN ORDER
Water
Buffer (pH 7)
Amylase Enzyme
Test Tube - 25°C
2 ml
0.5 ml of pH 7
0.5 ml
Test Tube - 80°C
2 ml
0.5 ml of pH 7
0.5 ml
Three pH’s Procedures – Work In Pairs
1. Set up the following THREE tubes (see table below)
2. Incubate all test tubes at room temperature for 5 minutes
3. After 5 minutes, add 1 ml starch to each tube and wait 2 minutes
4. After 2 minutes, add 1-2 drops of I2KI and observe, interpret
ADD IN ORDER
Water
Different pH Buffers!
Amylase Enzyme
Tube - pH 4
2 ml
0.5 ml of pH 4
0.5 ml
Tube - pH 7
2 ml
0.5 ml of pH 7
0.5 ml
Tube - pH 9
2 ml
0.5 ml of pH 9
0.5 ml
Biology 211 Lab Manual, Lab Three, page 3
Lab Three: Membranes & Enzymes - Turn In As Pairs At End of Lab
Pair Member Names:
1. 2 pts. Complete the following table that summarizes your RAW data about the
dialysis tubing/diffusion experiment.
Bag
Starch/Iodine
Beaker
Color Before Diffusion (T=0 min)
Color After Diffusion (T=40 min)
Control
Glucose
Color Before
Heating
Color After
Heating
Bag
Beaker
2. 1 pt. The dialysis membrane was permeable to:
3. 1 pt. Solutions A was:
4. 2 pts. What kind of inhibitor is PTU? How do you know?
5. 1.5 pts. Complete the following table about your amylase T° and pH assays.
T° Studies
80°C
25°C
pH 4
pH Studies
pH 7
pH 9
Final Tube Color
Amylase Active?
6. 1 pt. Describe the biological source of amylase and use this to interpret data above.
Biology 211 Lab Manual, Lab Three, page 4
7. Enzyme PROBLEMS
a. 0.5 pts. The adjacent graph shows the
activity for an enzyme that makes black fur
pigment in Siamese cats. If Siamese kittens
were raised at 5°C, what color(s) would you
expect them to be?
b. 1 pt. PCR (Polymerase/Pol Chain Reaction) is a powerful DNA-copying technique
that was made possible after scientists discovered heat-stable DNA Pol from bacteria
that live in hot springs. Although humans make our own versions of DNA Pol, our
enzymes denature during a boiling step required during PCR. Using this information,
draw TWO simple graphs (e.g. like in problem 7a) that represent predicted DNA Pol
reaction rates as a function of temperature from hot spring bacteria vs. humans.
Graph 1: Hot Spring Bacteria DNA Pol
Graph 2: Human DNA Pol
Biology 211 Lab Manual, Lab Three, page 5