Lab 3 Membrane structure and function
Objectives
1. Investigate effects of stressful experimental
treatments on living membranes
2. Investigate concepts about membrane structure
3. Learn basic principles of spectrophotometry and gain
experience using spectrophotometer
The Membrane: Overview
The Membrane: Overview
The Membrane: Overview
Phospholipids
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Phospholipids contain hydrophobic and hydrophilic
regions (amphipathic)
The heads are polar which makes them hydrophilic
(water loving)
The tails are non-polar which makes them
hydrophobic (water hating)
This molecular structure is what allows phospholipids
to form membranes
Phospholipids form a bilayer
Membrane is embedded with proteins
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
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Membrane contains
many proteins
Peripheral proteins
are bound to the
surface of the
membrane
Integral proteins
penetrate the
hydrophobic core
These proteins provide
a wide variety of
functions for the cell
Fluid mosaic model


The membrane is FLUID
 Lateral movement of
phospholipids is rapid
 Fluidity of the membrane is
important to its function
 Fluidity changes with
temperature
 Fluidity depends on the
composition of the membrane
 Ex: some fish live in extremely
cold environments. How do
they keep their membranes
fluid?
The membrane is a mosaic: many
different proteins
Membrane permeability:
“Are you on the guest list?”


Plasma membranes are selectively permeable
Permeable to non-polar molecules



hydrophobic molecules that can enter the lipid bilayer
Ex. O2, CO2
Non permeable to polar molecules (charged molecules)


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hydrophilic therefore cannot enter the lipid bilayer and remain in
the aqueous environment.
Ex. Na+, glucose, amino acids.
(there are, however, mechanisms the cell has developed to allow
import/export)

 what do you think the transmembrane proteins do?
Hydrophilic VS Hydrophobic
Hydrophilic
- Polar (charged)
- Water loving
- H-bonds to H2O
O 3.5
H
H
Hydrophobic
- Non-polar (non charged)
- Water fearing
- Binds to other non-polar
molecules
C–H
2.5 2.1
2.1
(close  non-polar)
Solvents used today
Polar
Non-Polar
(Ratio of polar : non-polar groups)
2-propanol
Methanol
Size
SUMMARY:


Polar molecules are hydrophilic will stay in the water (Hbonds with water) and not enter the lipid bilayer of the
membrane
 Do we expect them to cause damage to a cell membrane?
Non-polar molecules are hydrophobic therefore will enter the
lipid bilayer
 Do we expect them to cause damage to a cell membrane?
 Does the size of a non-polar molecule influence the extent of
damage?
Today’s lab

Investigate how different temperatures and solvents
can cause membrane damage
Temperature stress
Organic solvent stress
Beta vulgaris
Measure betacyanin leakage as a way
to quantify membrane damage
Betacyanin: red pigment
• Betacyanin is found in
vacuole (enclosed in
membrane)
Betacyanin
• If vacuole membrane is
damaged, betacyanin
will leak out (red pigment)
•How can we measure this?
Spectrophotometer
525nm
(the wavelength
absorbed
by betacyanin)
•Betacyanin absorbs
light at a wavelength of
525nm
•The spec will be set to
shine 525nm light on
your tubes
• the amount of light
being absorbed will be
measured.
•The more betacyanin in
the tube, the ___?___
absorbance reading.
Spec 20s


Follow the instructions
in Appendix F
Betacyanin absorbs
light maximally at
525nm, so you need to
set the Spec to shine
light of 525nm. Make
sure filter lever is
turned to the right
range
Experimental design

Hypotheses


Specific, logical
Independent variable
 This
is the part that you are controlling
 ________ (Temperature)__________

Dependent variable
 This
is the part that you measure
 _________ (absorbance)___________

Controls
 Best

control is the removal of the independent variable
(ie: the controls for this experiment are room temp OR water)
Hypothesis:
A good hypothesis must:



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

explain how or why: provide a mechanism.
be compatible with and based upon the existing body of
evidence.
link an effect to a variable.
state the expected effect.
be testable.
have the potential to be refuted.
Hypothesis: make your own!
“I hypothesize that …”
“The rationale for the hypothesis is…”
Remember

You will find that certain temperatures and
solvents will damage the membrane more than
others. MAKE SURE YOU CAN EXPLAIN WHY
certain temps/solvents damage the membrane
What is happening to:




the phospholipids?
the proteins?
Why does this result in betacyanin leakage?
Graphs
 Temp
treatment is a line graph because the data is
continuous
 Independent
variable on the X axis
 Dependent variable on the Y
 Solvent



treatment is a bar graph
Follow guidelines in Appendix
Graph should be full page
Include your data AND class data