Bio 151 Macromolecules, IB
Macromolecules in food
We metabolize food in order to release energy to produce the ATP needed for cellular work.
We also break down food molecules in order to use their subunits as raw material for
synthesizing our own macromolecules. In this exercise, you can investigate certain foods to
learn which macromolecules are present in each.
Objectives:
1. explain how the components of butter are distributed into fractions by the process of
clarification
2. define the macromolecular components of fractionated butter
3. explain how to use an immuno-chromatographic test strip to detect the presence of a
variant enzyme commonly found in plants that are genetically modified, including the
significance of
a. CP4 EPSP
b. shikimate pathway
c. glyphosate
d. antibody-capture assay
 components
 mechanism
Activity A: Separation of butter to make clarified butter (ghee)
Introduction





Most foods are complex mixtures of substances.
Butter, for example, may appear to be solid fat, but it is actually a mixture of proteins,
carbohydrates and lipids.
o It is an emulsion, which means that the lipids occur in very small droplets dispersed
throughout the water-soluble portion.
The lipid can be separated from the water soluble, protein-containing part of the butter in
a process called clarification.
Butter is often clarified for use in cooking.
Once the water-soluble part has been removed, the lipid that remains can be used to fry
at higher temperatures than for whole butter because it is the protein in butter that
scorches first. It is also the protein that spoils most readily, so clarified butter keeps longer.
1
Activity B: assessing plant material for RoundUp® herbicide resistance
Introduction
(Background reading: pp 835-838)
 The engineering of transgenic crops resistant to the broad-spectrum herbicide glyphosate
has greatly improved agricultural efficiency worldwide.
 Glyphosate-based herbicides, such as RoundUp®, target the shikimate pathway enzyme 5enolpyruvylshikimate 3-phosphate (EPSP) synthase, the functionality of which is absolutely
required for the survival of plants.
 RoundUp Ready® plants carry the gene coding for a glyphosate-insensitive form of this
enzyme, obtained from Agrobacterium sp. strain CP4.
o Once incorporated into the plant genome, the gene product, CP4 EPSP synthase,
confers crop resistance to glyphosate.
o A synthetic gene coding for CP4 EPSP synthase was cloned and characterized using
kinetics and crystallography.
- The CP4 enzyme has unexpected kinetic and structural properties that render it
unique among the known EPSP synthases.
- Glyphosate binds to the CP4 EPSP synthase in a condensed, non-inhibitory
conformation.
- Glyphosate sensitivity can be restored through a single-site mutation in the active
site (Ala-100–Gly), allowing glyphosate to bind in its extended, inhibitory
conformation.
A simple test for RoundUp Ready®plants
 Reveal for CP4 (Neogen Corporation) is a single-step, lateral flow Immunochromatographic assay.
 The assay:
(Background reading (antibodies as tools): p 967)
o a water extract of a sample being tested is prepared.
o the sample is wicked up the test strip by capillary action through a reagent zone,
which contains antibodies specific for the CP4 EPSPS protein (CP4), conjugated to
colored particles.
- if CP4 is present in the test sample, it will be captured by the conjugated
antibodies;
- if no CP4 is present, there will be no reaction.
o if CP4 is present, the CP4-antibody-particle complex is then wicked upward on the test
strip into a zone that contains antibody specific for CP4.
- this zone captures the complex, allowing the particles to concentrate and form
a visible line; if no CP4 is present, no line is formed.
2
Protocols for Bio 151 Macromolecules, II
Activity A: Separation of butter
1.
2.
 Cut ~ 1 tablespoon of butter into smaller chunks and put them into a 50-ml beaker.
 When the water boils in the larger beaker on the hotplate, remove the beaker from the
hot plate with a potholder and set it on a paper towel on the lab bench. Choose a
spot where it will not be disturbed.
3.
4.
5.
6.
 Put the 50 ml beaker into the 250 ml beaker (“water bath”).
 Leave butter undisturbed for ≥15 minutes. While you wait continue with steps 5-7.
 Get ~1 teaspoon of butter and put it into a 13 x 100 test tube: “whole butter” sample.
 Put the test tube in a warm water bath to melt the butter.
(Note: If you use the water bath the 50 ml beaker is in - don’t disturb the beaker).
7.
 When the butter in the test tube has melted, perform the four tests that were introduced
previously and record your results in the data section table.
8.
 After 15 minutes, gently pick up the 50 ml beaker; and, note the layers,
carefully skimming off the top foamy layer (which contains water and some proteins)
and setting aside; and, then setting the beaker carefully in ice. Leave it undisturbed for
about 10 minutes. Work on Activity B while the lipid (upper) layer solidifies
9.
 The upper, yellow layer (clarified butter) should now be solid or semisolid.
Remove it with a spatula and put it on a paper towel. Pat the bottom of the butter dry
with a paper towel to remove any contaminants from the lower layer.
10.  Place the clarified butter in a test tube and melt it in a warm water bath.
11.  Perform the four tests on the melted clarified butter and record the results in the data
section table.
12.  Perform the four tests on the lower (white) layer of the clarified butter; record results in
the data section table.
3
Activity B: A simple, immunochromatographic test for RoundUp Ready® (genetically
modified) plants
1.
2.
3.
4.
 Using a mortar and pestle, crush some of the experimental sample (e.g., corn chips)
 Weigh out 2 g of each of three corn samples:
a.  GMO corn (positive control)
b.  Non-GMO corn (negative control)
c.  crushed, ground-up potato chips or similar (experimental test) from step 1
 Place each sample into its own 16 x 125 mm test tube (one tube for each sample).
 Using a serological pipette, add 6 ml deionized water to each tube;
cover/seal with Parafilm.
5.
 Shake tube vigorously for ≥10 seconds (and/or vortex at high speed for 10 seconds);
let particulates settle.
6.
 Using a Transpette, draw off ~ 1 ml of the supernatant (fluid above particulates/pellet)
place into three separate 13 x 100mm test tube (one tube for each sample).
7.
8.
9.
 Carefully place one CP4 Reveal® test strip into each test tube.
 Incubate at room temperature for 10 minutes.
 Read results
a. a positive result: two bands (one control band, CP4 gene positive band)
b. a negative result: one (control only) band
4