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Transport Across Membranes
Selectively permeable membranes define the boarders of all cells and many
organelles. Because everything entering and leaving the cell must pass through the
membrane, their function is essential in maintaining homeostasis. Desirable molecules in
the environment must be transported into the cell across the membrane while undesirable
molecules must be kept out. Waste molecules must be allowed out while valuable
molecules must be kept in. Transportation of molecules across biological membranes can
occur in three different ways: 1) Passive diffusion which requires no energy on the part of
the cell an allows molecules that can naturally cross the membrane across, 2) Facilitated
diffusion in which molecules that would not naturally cross the membrane are allowed to
cross as a result of special protein channels found in the membrane, and 3) Active
transport which is the energy consuming activity of pumping molecules across
membranes, usually against their concentration gradient.
There are three factors that govern the direction and rate of diffusion across
membranes in the absence of channels or pumps: 1) Size (and to a certain extent shape),
large bulky molecules tend to diffuse slower and to have greater difficulty crossing
membranes; 2) Charge, biological membranes tend to not let charged ions across or very
polar molecules; 3) Concentration, the higher the concentration on one side of a
membrane, relative to the other, the greater the rate of diffusion across the membrane.
When thinking about diffusion, there is a rule of thumb that if kept in mind will
help avoid confusion: "Every molecule will diffuse down its concentration gradient. That
is to say, molecules tend to go from where they are in high concentration to where they
are at a low concentration. When dealing with selectively permeable membranes like
biological membranes, this rule holds true as long as the membrane will allow the
molecule in question to cross it.
The term osmosis is used to describe the movement of water across membranes.
As water is usually thought of as a solvent, it may seem a little difficult to apply the rule
given in the last paragraph to osmosis, but it does hold true even here. If a solute is
dissolved in a solvent like water, the molecules of the dissolved solute take up space that
can not be occupied at the same time by water molecules. The net result is that there are
less water molecules per unit volume so the concentration of water goes down. Solutions
with more solute dissolved in them compared to another solution are called hypertonic (or
hyperosmotic). The antonym to hypertonic is hypotonic meaning having a lower solute
concentration. The prefixes hypo and hyper are used commonly and mean less than or
below and more than or above respectively. It is important to remember that these are
relative terms and that, for example, a solution that is hypertonic compared to one
solution may be hypotonic compared to another. Two solutions having the same solute
concentration are called isotonic, iso meaning the same. Keep in mind that these terms
refer to the solute concentration and not water concentration. If a solution is hypertonic,
having more solute than another solution on the other side of a membrane, the
concentration of water will be lower than the hypotonic solution on the other side. The
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net result here, if a selectively permeable membrane only
permeable to water is used, will be the net flow of water from
the hypotonic solution down its concentration gradient across
the membrane and into the hypertonic solution.
In this laboratory, you will be looking at both artificial
and biological membranes.
Exercise 1: Measurement of osmotic pressure
When a hypertonic solution is placed on one side of a membrane and a hypotonic
solution is placed on the other side, there is potential to do work by harnessing the energy
in molecules of water molecules as they diffuse across the membrane. This potential
energy can be thought of as analogous to the energy of water behind a dam that has the
potential to do work if allowed to flow through a hydroelectric plant. This energy across
the membrane can be measured in terms of pressure called osmotic pressure. Osmotic
pressure may be important in helping cells maintain their shape. For example, if a plant
is not watered it will wilt due to the decrease in pressure within each cell.
In this exercise, pressure will be calculated by measuring the height of a column
of water as water moves across a membrane and up a tube. This exercise will be done
once for the entire class, but it will require periodic checks over the next few days to
measure the height of the water column.
1.
Measure off 4 cm of 2 cm diameter dialysis tubing (yes, this is the same stuff they
use when doing kidney dialysis) and soak it in distilled water for 2 to 3 min.
2.
Open up the tube and cut it so that you now have a sheet of membrane 1 layer
thick and approximately 4 cm square.
3.
Measure the diameter of a thistle tube at both the wide and the narrow end, then
place 5 ml of molasses in the thick end while blocking the thin end and cover the
thick end with the dialysis membrane. Hold the tubing in place with a rubber
band wound around many times. It is essential that no molasses can leak out this
end of the tube.
4.
Turn the thistle tube so that the wide end is facing down then quickly rinse the
membrane with distilled water.
5.
Wait until all the molasses has flowed down into the thick end of the thistle tube
then place the membrane end into a 1 L beaker containing 1 L of distilled water.
Hold the thistle tube in place with a ring stand and clamp then attach at least 2 m
of glass tubing of the same diameter as the thin end of the thistle tube to the tin
end. Tubes may be connected using rubber tubing. This glass tubing will need to
be vertical and it may require some ingenuity to figure out how to do this. In the
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past, I have found the stairwell to be a good place to set this up as more tubes can
be added if the water goes higher than 2 m and the janitor gets to clean up the
sticky mess if the contraption breaks under the pressure!
6.
Because dialysis tubing is permeable to water, but not to the sugars and other
molecules found in molasses, osmosis should occur across the membrane and
water should flow into the thistle tube and up the glass tubing. Measure the height
of the water every few hours during the first day. If the water column is still
rising at the end of the day, come back and keep measuring it the next day and so
on until the water stops rising. Make sure that you carefully record the times and
heights so that you can graph them in your results section.
7.
The pressure across the membrane can be measured as a function of the height of
the water column. First of all, you will need to calculate the surface area of the
membrane on the end of the column using πr2 where r = 0.5 x the diameter of the
wide end of the thistle tube. Next you will need to calculate the volume of water
that traveled up the water column. This can be done by first calculating the
surface area of one end of the column and then multiplying by the height. As the
density of water is 1 g/ml multiplication by 1 gives the mass of water. Finally the
pressure can be calculated in terms of mass per unit area, g/cm2. Be sure to
convert this into the standard scientific unit of pressure the Pascal (Pa). To do
this, you need to first convert your calculation of force into newtons (N), the
standard unit of force that will accelerate a mass of 1 kg 1 m/s2. As the
acceleration due to gravity is 9.8 m/s2, multiplying the mass of the water column
by the acceleration due to gravity should give the force in N. If you let M
represent the mass of the water column and F represent the force in N you can use
the following formula:
Mx
9.8m
s
2
=F
Pa are measured in N/m2 so dividing F by the area in m2 of the membrane at the
end of the thistle tube gives the osmotic pressure in Pa.
Exercise 2: Properties of an artificial membrane
1.
Cut 2 8 cm lengths of dialysis tubing and soak them in distilled water until they
can be opened into tubes.
2.
Seal off one end of each tube approximately 1 cm from the end using a dialysis
tube clamp.
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3.
Using a pipette, fill one tube to 1.5 cm from the top with 0.2 % starch solution
then seal it off with a second clamp being careful to remove any air in the tube.
Do the same thing with the second tube, only use Lugol's solution (IKI) instead of
starch.
4.
Rinse both tubes with distilled water then place the starch containing tube into a
100 ml beaker containing 50 ml of Lugol's solution. Place the Lugol's solution
containing tube into a 100 ml beaker containing 50 ml of the 0.2 % starch
solution.
5.
Make observations every 15 min. for the next 90 min. You may want to recall the
results obtained when Lugol's solution reacted with starch in the enzyme
Laboratory. Which molecules are able to move through this membrane?
Knowing what you know about starch, how do you explain this? Are there any
other observations that you can make based on this experiment?
Exercise 3: Osmosis in a living cell
Many plant cells have a large central vacuole. In red onion cells, this vacuole
contains a red pigment that gives the onions their red color. Changes in the size and
shape of the vacuole can be easily seen under the microscope because of this pigment. If
water leaves the cell, the cytoplasm becomes hypertonic relative to the vacuole so water
leaves the vacuole, but the pigment stays behind. In a hypertonic solution, red onion cells
should have vacuoles that shrivel up. In a hypotonic solution, the vacuole should expand
to fill almost the entire cell.
1.
The segments that make up an onion are actually specialized leaves. On the
surface of each leaf is the epidermis and it is the epidermal cells that contain the
red pigment. Cut a 1 cm square of onion then carefully peel off the epidermis and
make a wet mount using distilled water. Set this slide aside and do the exact same
thing, but use the 3 M salt solution instead of distilled water. A third unknown
solution will be available, make a slide using this solution. Make sure that you
clearly mark each slide to avoid confusion about which slide used which solution.
2.
After 5 min. examine each slide under the microscope. If there is no difference
between slides, wait another 5 min. and examine again. Be sure to include the
results obtained with the unknown solution and state in your discussion if it was
hypotonic, hypertonic, or isotonic and why you came to this conclusion. Why do
you think that salt was used as the solute and not sugar? Remember the rules for
handling the microscopes.
Exercise 4: For A students only
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Design your own experiment dealing with osmosis, diffusion, or semipermeable
membranes. Here are a few ideas to get you thinking:
1.
Try placing red blood cells in different solutions and observing the results. Make
sure that you get help from your instructor when pricking your finger.
2.
See how differences in osmolarity of solutions result in differences in rates of
absorption or excretion in humans by drinking different solutions (ie salt water,
distilled water, pop, . . .) and measuring the rate at which it comes out!
3.
Try finding the isotonic point of onion cells by soaking epidermis in solutions
with different concentrations of salt.
Materials:
Equipment
Beakers, 1 1000 ml and 2 100 ml/student
Clips for dialysis tubing
Compound Microscopes
Cover slips
Pipettes, 10 ml
Rubber bands
Slides
Thistle tubes
Glass tubing
Stands
Chemicals
Distilled water
Lugol's solution
Molasses
3 M NaCl
Starch solution, 0.2 %
Supplies
Dialysis tubing
Red onion