15.

6/20

EXPERIMENT 2
THE PERMEABILITY OF CELL MEMBRANES
CARANDANG, CRUZ, PASUMBAL, SALEM, TOLENTINO
Institute of Biology, University of the Philippines, Diliman

ABSTRACT

This experiment deals with the effects of different solutions on the hemolysis of red blood
cells. 2 drops of a 2% red blood cell suspension were added to varying concentrations of sodium
chloride, glucose, potassium chloride, calcium chloride and sodium sulfate to ascertain the effect of
concentration on cell permeability. The procedure was repeated with solutions containing large
molecules, varying alcohols, and nonionic polar compounds to observe the effects of molecular size,
partition coefficients, and polar groups on cell permeability respectively. Hemolysis readings were
done with comparison to distilled water and 0.2 M sodium chloride controls. Data gathered from the
experiment indicated that low molecular size, high partition coefficients, and less polar groups would
allow faster transport of the solute through the membrane while the counterparts suggest otherwise.
With this it is proven that the cell is membrane is composed of a hydrophobic interior that has small
spaces to allow simple diffusion to take place.
INTRODUCTION concentrations inside and outside of the cell of
nonpenetrating solutes, while tonicity indicates
Cell membrane which separates the the effect of nonpenetrating solutes in the
internal components of the cell to the outside external environment on cell volume.
medium depends on the properties of the lipids Hemolysis of erythrocytes occurs in a
to form bilayer. The formation of bilayer hypotonic solution where the nonpenetrating
allows specialized properties of the membrane solute concentration outside of the cell is
to arise such as a receptor for external signals, lower than the concentration of solute inside
a medium for the electron transport and the cell. Hypotonic solution causes the
oxidative phosphorylation and the selective swelling of cells as the water enters the cells
transport of solutes across it. The selective through osmosis to the point of cell lysis or
permeability of the membrane allows small cell rupture. [2] Hemolysis is a parameter that
nonpolar molecules are soluble in the lipid can be measured to assess the permeability of
bilayer that allows the cell to control and the cell with respect to the different reagents.
maintain its internal composition. [1] Factors such as the effect of electrolyte and
Materials pass through the cell membrane nonelectrolyte, molecular size of the
through passive and active transport. compounds added and the relative value of the
Relatively small nonpolar molecules carrying partition coefficients are often observed to
no charge (oxygen, alcohol, steroids, and fatty study the permeability of the membrane with
acids) can easily pass through the lipid bilayer, respect to hemolysis.
whereas charged molecules such as ions (Na +
and K+) and polar molecules such as glucose In this experiment, the researchers
and proteins have difficulty in passing observed the permeability by comparing the
permeating the membrane without channels time for hemolysis for each of the compounds
and carriers. [2] added in the red blood cells (RBCs) to
establish and correlate the different factors that
Osmosis is the movement of water affect the permeability of the membranes
through a membrane due to differing including (1) concentration gradient of

1
substance, (2) molecular size, (3) partition plotted versus the molecular weights of each
coefficient, and (4) presence of polar groups. substance.

Different alcohols (methanol, ethanol,

MATERIALS AND METHODS isopropyl alcohol, and n-butyl alcohol) with
different partition coefficients were mixed
with two drops of RBC suspension. The
To observe the effect of different hemolysis time was observed and recorded;
compounds on the hemolysis of human the hemolysis time was plotted against
erythrocytes, various electrolyte and partition coefficient. Another set of substances
nonelectrolyte solutions (Sodium chloride (chloroform, hexane, ethylene glycol,
NaCl, glucose, potassium chloride KCl, isopropyl alcohol) were used to observe the
calcium chloride CaCl2, sodium sulphate effect of polar groups on membrane
Na2SO4) of different concentrations were permeability.
prepared. The erythrocyte cell suspension was
prepared by adding ten drops of blood sample
to ten milliliters of 0.9% NaCl solution. RESULTS
Sodium chloride solution and distilled water
were used as controls. Five drops of red blood Different compounds were used to observe the
cell (RBC) suspensions were added to five hemolysis of erythrocytes indicated by a clear
milliliters of each solution and mixed the solution. As shown in table 1, electrolytes
contents. The solution is observed for at most require lower concentrations than non-
ten minutes or until the solution turned clear electrolytes to reach hemolytic point due to its
indicating a complete hemolysis. The duration dissociation into ions.
for each solution until complete hemolysis was
recorded. The isotonic coefficient (i) of each Table 1. Experimental data for isotonic
reagent was calculated using the following coefficient and degree of dissociation of
formula: different compounds.
Complete
hemolysis Isotonic Degree of
Solution (M) coefficient dissociation
sodium 0.055 1.818 0.8181
The isotonic coefficient was used to obtain the chloride
degree of dissociation (a) where k is the glucose 0.1 1 0
potassium 0.075 1.33 0.33
number of ions present in a salt:
chloride
calcium 0.05 2 0.5
chloride
sodium 0.035 2.857 0.9285
sulfate
Other factors, such as molecular size,
One of the factors that could affect the
partition coefficient, and the presence of polar
membrane permeability of the cell membrane
groups were also observed. On the effects of
is the molecular size of the solute present in
molecular size on membrane permeability, the
the solution. Large molecules would have
hemolysis of erythrocyte to urea, ethylene
more difficulty in entering the cell, while
glycol, glycerol, and glucose were observed
smaller ones would have more ease, as
and compared, and the hemolysis time was
depicted in Figure 1.

2
Figure 1. Hemolysis time vs. Molecular Figure 2. Hemolysis time (s) vs Partition
Weight. The Effects of Molecular Size on Coefficient (logP). Correlation of Permeability
Membrane Permeability with Partition Coefficients

The solubility ratio of a compound in oil to its
solubility in water, known as the partition
coefficient, was also taken into account in
analyzing the erythrocytes’ membrane
permeability. A higher partition coefficient
indicates that the compound has a high
solubility in lipids, which correlates to a
longer carbon chain and therefore higher
molecular weight.
A separate graph, Figure 3, was plotted in
order to show the relationship of the cell
permeability and the molecule’s carbon chain
length.
* MEASURES OF POLARITY->
DIELECTRIC CONSTANT (VALUES)

Table 2. Correlation of Permeability with

Partition Coefficients
Reagent Partition T0 Tf Time
Coeff. (s)

0.6 M -0.77 0 51.7 51.7

methyl
alcohol Figure 3. Hemolysis time (s) vs. Length of
Carbon Chain. Correlation of Permeability
0.6 M -0.31 2.4 51.7 49.3
with Length of Carbon Chain
ethyl
alcohol
Polarity was also analyzed in terms of its
0.6 M 0.34 0 27.5 27.5 effects on the permeability of the erythrocytes.
isopropyl When subjected in an electric field, polar
alcohol molecules would orient themselves along with
0.6 M n- 0.88 2.3 27.5 25.2 it while non-polar ones would have more
butyl mobility to enter the cell.
alcohol
Figure 2 shows the inversely proportional Table 3. The Effect of Polar Groups on
relationship of the permeability of the cell to Membrane Permeability
the partition coefficient of the solute. Reagent T initial T final Time (s)

0.3 M 0 14 14
chlorofor
m

3

0.3 M 11 32.6 21.6
hexane
0.3 M 9.5 26.2 16.7
ethylene
glycol
0.3 M 18.2 41.4 23.2
isopropyl
alcohol
DISCUSSION
Since electrolytes dissociate into ions
and glucose is a relatively large polar
compound, these compounds do not diffuse
into the cell membrane easily. Instead, these
compounds contribute to osmotic pressure.
Lower concentration of solute indicates a
higher concentration of water or a hypotonic
solution. When erythrocytes are subjected to a
hypotonic solution, water moves into the cell
by osmosis from outside to inside of the cell.
The movement of water causes the swelling of
cells to the point of cell lysis or hemolysis. [2]
Electrolytes dissociate into ions when
dissolved in water. For example, one mole of
sodium chloride dissociates into one mole of
sodium ions and one mole of chloride ions, it
is logical that the hemolytic point of sodium
chloride is about half of glucose, a
nonelectrolyte. The experimental isotonic
coefficient of sodium chloride is 1.818. This
indicates that 100 molecules of sodium
chloride exert the same osmotic pressure as
about 182 molecules of glucose. [7]
As seen in Figure 1, The molecular
weight of the compound shows a direct
relationship to the time of hemolysis. This
shows that an increase in molecular weight
results in a longer time for hemolysis and a
much harder time for solutes to penetrate the
cell membrane. Theoretically, a smaller
molecular weight should have a high rate of
diffusion which results to a fast rate of influx.
Alternately, large molecules need to undergo
carrier-mediated transport which implies a
slower rate of influx. Urea, having a molecular
weight of 60.07g/mol has the fastest hemolysis
time while glucose, with 180.18 g/mol was the
last to be hemolyzed among the compounds.
Ethylene glycol and glycerol having 62.08
g/mol and 92.11 g/mol respectively have an
intermediate hemolysis time with respect to
urea and glucose. [3].
By definition, osmolarity describes the
number of particles in solution which can be
expressed in the number of particles (ions or
intact molecules) per liter of solution. [4]. It is
thus defined on the basis of an ideal
osmometer in which the osmotic membrane
allows water to pass. [3]. On the other hand,
tonicity is a physiological term used to
describe a solution and how that solution
affects cell volume. Hence, a solution is called
isosmotic when two solutions contain the same
number of solute particles per unit volume
while a cell that does not change size is
considered as a isotonic solution. [4]
In the experiment, solutions that are
isosmotic with respect to intracellular fluid
was used, and underwent hemolysis.
Erythrocytes rupture or swell because when a
cell is exposed to such hypotonic conditions,
there is net water movement into the cell. Cells
without walls like animal cells will swell and
may burst (lyse) if excess water is not
removed from the cell. [5] Hence, solutions
could be isosmotic but not isotonic as depicted
in the experiment.
Partition coefficient is defined as a
ratio of concentrations of un-ionized
compound in organic and aqueous phases at
equilibrium. It can be viewed as an indicator
of intrinsic lipophilicity in the absence of
ionization or dissociation of the compound.
[5]. It is the primary predictor of the diffusion
of a nonelectrolyte across the lipid bilayer. [3]
The equation of the partition coefficient is
shown below:
4

Figure 3. Partition coefficient equation
(Adapted from Eckert, 2002)
Hence, from the equation above it can
be implied that the higher organic
concentration would give a higher partition
coefficient. As mentioned before, the heavier
the molecular size would give a slower
hemolysis time. This would mean that the
longer the carbon chain length would also give
a slower hemolysis time implying a lower rate
of influx as shown in Figure 2, and it has a
higher partition coefficient. [3] The sequential
elongation of an alkyl chain increases the
partition coefficient by a factor of about 3 per
methylene group. The partition coefficient of
butanol is about 9 times higher than that of
ethanol.
In the experiment, methanol, ethanol,
isopropanol and n-butanol were examined in
increasing molecular weights and chain
lengths, respectively. These are also in
increasing partition coefficient, whereas
methanol would exhibit the fastest hemolysis
time for its lowest partition coefficient than the
other organic compounds. On the other hand,
n-butanol, the highest partition coefficient,
exhibited the slowest hemolysis time.
*OVERTON’S RULE OF
PERMEABILITY
Table 3 illustrates the relationship
between polarity and hemolysis time and
therefore cell permeability. Nonpolar
molecules can easily pass through the
membrane because of its lipid-solubility. Polar
and charged molecules will have difficulty in
crossing the membrane.
Polar molecules can easily interact
with the outer face of the membrane while the
charged molecules have difficulty to pass the
hydrophobic core. Since charged molecules
such as Na+, K+, Ca2+, and C1- cannot diffuse
through lipid bilayers, special protein
molecules act as pores for these molecules to
pass through the membrane. [3]
The chemical nature of the cell
membrane can be inferred with the data from
the different parameters. The cell membrane is
not an ideal membrane where it only allows
water to pass through; solutes can freely enter
and exit the cell under different conditions. In
Figure 1, it can be said that as the molecular
weight of the solute increases, the harder it is
for it to the enter the cell. It states that some
molecules undergo simple diffusion across the
cell membrane, where it is easier for small
molecules to pass through compared to bigger
ones.
The inversely proportional
relationship of partitioning coefficient to
hemolysis time shown in Figure 2 states that
molecules that have a higher solubility in
lipids would have easier access to the inside of
the cell. This is explained by the phospholipid
bilayer of the membrane, wherein non-polar
compounds would be transported faster
through the hydrophobic interior than polar
ones. This is also true for the factor of chain
length as shown in Figure 3, wherein longer
carbon chains would diffuse faster because
*MEASURE OF POLARITY ->
DIELECTRIC CONSTANT
may burst (lyse) if excess water is not
removed from the cell. [5] Hence, solutions
could be isosmotic but not isotonic as depicted
in the experiment.
Partition coefficient is defined as a
ratio of concentrations of un-ionized
compound in organic and aqueous phases at
equilibrium. It can be viewed as an indicator
of intrinsic lipophilicity in the absence of
ionization or dissociation of the compound.
[5]. It is the primary predictor of the diffusion
of a nonelectrolyte across the lipid bilayer. [3]
The equation of the partition coefficient is
shown below:
5

Figure 3. Partition coefficient equation
(Adapted from Eckert, 2002)
Hence, from the equation above it can
be implied that the higher organic
concentration would give a higher partition
coefficient. As mentioned before, the heavier
the molecular size would give a slower
hemolysis time. However, it would be
different when it comes to the carbon chain
length where a longer carbon chain would give
a shorter hemolysis time implying a higher
rate of influx as shown in Figure 2, and it has a
higher partition coefficient. [3] The sequential
elongation of an alkyl chain increases the
partition coefficient by a factor of about 3 per
methylene group. The partition coefficient of
butanol is about 9 times higher than that of
ethanol.
In the experiment, methanol, ethanol,
isopropanol and n-butanol were examined in
increasing molecular weights and chain
lengths, respectively. These are also in
increasing partition coefficient, whereas
methanol would exhibit the slowest hemolysis
time for its lowest partition coefficient than the
other organic compounds. On the other hand,
n-butanol, the highest partition coefficient,
exhibited the fastest hemolysis time.
Table 3 illustrates the relationship
between polarity and hemolysis time and
therefore cell permeability. Nonpolar
molecules can easily pass through the
membrane because of its lipid-solubility. Polar
and charged molecules will have difficulty in
crossing the membrane.
Polar molecules can easily interact
with the outer face of the membrane while the
charged molecules have difficulty to pass the
hydrophobic core. Since charged molecules
such as Na+, K+, Ca2+, and C1- cannot diffuse
through lipid bilayers, special protein
molecules act as pores for these molecules to
pass through the membrane. [3]
The chemical nature of the cell
membrane can be inferred with the data from
the different parameters. The cell membrane is
not an ideal membrane where it only allows
water to pass through; solutes can freely enter
and exit the cell under different conditions. In
Figure 1, it can be said that as the molecular
weight of the solute increases, the harder it is
for it to the enter the cell. It states that some
molecules undergo simple diffusion across the
cell membrane, where it is easier for small
molecules to pass through compared to bigger
ones.
The inversely proportional
relationship of partitioning coefficient to
hemolysis time shown in Figure 2 states that
molecules that have a higher solubility in
lipids would have easier access to the inside of
the cell. This is explained by the phospholipid
bilayer of the membrane, wherein non-polar
compounds would be transported faster
through the hydrophobic interior than polar
ones. This is also true for the factor of chain
length as shown in Figure 3, wherein longer
carbon chains would diffuse faster because
they are more non-polar than their
shorter counterparts. It can be said that the
polarity of a substance would have a larger
effect on the permeability of the cell compare
to its molecular size.
CONCLUSION
The experimental results in measuring
the effect of the different factors affecting
membrane permeability indicated that
hemolysis time varied with respect to
molecular size, presence of polar and nonpolar
groups, partition coefficient, the length of the
carbon chains being considered and also
whether the substance is an electrolyte or not.
Results for the plot of the molecular
weight and hemolysis time is directly linear
6
indicating that the reagents with increasing
molecular weight which are urea, ethylene
glycol, glycerol and glucose were respectively
hemolyzed from the fastest to slowest time.
Likewise, electrolyte solutions behave
similarly with respect to hemolysis since they
dissociate and they require less time to
hemolyze the red blood cells compared to non-
electrolyte substance like glucose.
The presence of polar atoms and the
relative chain length also affected the
hemolysis time. Compounds with a polar
group stronger than that of the other
compounds exhibited a shorter time for
hemolysis such as chloroform that is made up
of three strong electronegative chloride ions.
On the other hand, hexane which is made up
of entirely nonpolar alkane compound
exhibited the slowest hemolysis time. It can be
concluded that polar groups makes penetration
easier for molecules to the cell membrane
given that the compound is relatively small.
The partition coefficient also showed
varying times for the hemolysis of the red
blood cells. N-butanol exhibited the fastest
hemolysis time even though it is expected to
have the slowest hemolysis time because of
the presence of polar groups which overcame
its partition coefficient. Methanol was the
compound that exhibited the slowest
hemolysis time because of its relatively high
partition coefficient.
REFERENCES
[1] Cooper, G. (2000). Cell Membranes.
Retrieved from:
https://www.ncbi.nlm.nih.gov/books/NBK992
8/
[2] Sherwood, L., Klandorf, H., Yancey,
P.H. (2011). Animal Physiology: From Genes
to Organism. 2nd ed. Belmont, CA:
Brooks/Cole Cengage Learning.
[3] Randall, D. J., Burggren, W. W., French,
K., & Eckert, R. (2002). Eckert animal
physiology: Mechanisms and adaptations. 5th
ed. New York: W.H. Freeman and Co. 93-124
[4] Silverthorn, D. Johnson, B. Ober, W. Ober,
C. & Silverthorn, A. (2016). Human
physiology: An integrated approach. San
Francisco: Pearson/Benjamin Cummings. 159-
162
[5] n.a. n.d. Cells in Hypotonic Solution
Pearson.com. Prentice Hall. Pearson
Education, Inc. Retrieved from
http://www.phschool.com/science/biology_pla
ce/biocoach/biomembrane1/hypotonic.html
[6] Kwon Y (2001). Handbook of Essential
Pharmacokinetics, Pharmacodynamics and
Drug Metabolism for Industrial Scientists.
New York: Kluwer Academic/Plenum
Publishers. 44.
[7] Scott, L.A. (1993). Diffusion Across a
Sheep Red Blood Cell Membrane. Retrieved
from http://www.ableweb.org/volumes/vol-
14/7-scott.pdf
they are more non-polar than their
shorter counterparts. It can be said that the
polarity of a substance would have a larger
effect on the permeability of the cell compare
to its molecular size.
CONCLUSION
The experimental results in measuring
the effect of the different factors affecting
membrane permeability indicated that
hemolysis time varied with respect to
molecular size, presence of polar and nonpolar
groups, partition coefficient, the length of the
carbon chains being considered and also
whether the substance is an electrolyte or not.
Results for the plot of the molecular
weight and hemolysis time is directly linear
indicating that the reagents with increasing
molecular weight which are urea, ethylene
glycol, glycerol and glucose were respectively
hemolyzed from the fastest to slowest time.
7
Likewise, electrolyte solutions behave
similarly with respect to hemolysis since they
dissociate and they require less time to
hemolyze the red blood cells compared to non-
electrolyte substance like glucose.
The presence of polar atoms and the
relative chain length also affected the
hemolysis time. Compounds with a polar
group stronger than that of the other
compounds exhibited a shorter time for
hemolysis such as chloroform that is made up
of three strong electronegative chloride ions.
On the other hand, hexane which is made up
of entirely nonpolar alkane compound
exhibited the slowest hemolysis time. It can be
concluded that polar groups makes penetration
easier for molecules to the cell membrane
given that the compound is relatively small.
The partition coefficient also showed
varying times for the hemolysis of the red
blood cells. N-butanol exhibited the fastest
hemolysis time because it is the most lipid-
soluble of the group which yields to the fastest
hemolysis time Methanol was the compound
that exhibited the slowest hemolysis time
because of its relatively low partition
coefficient which states that it is the least
lipid-soluble among the other alcohol reagents.
REFERENCES
[8] Lodish, H., Berk, A., & Zipursky, S. L.
(2000). Molecular Cell Biology (4th ed.).
New York: W.H. Freeman. Retrieved
August 22, 2017, from
https://www.ncbi.nlm.nih.gov/books/NBK21626/.
[1] Cooper, G. (2000). Cell Membranes.
Retrieved from:
https://www.ncbi.nlm.nih.gov/books/NBK992
8/
[2] Sherwood, L., Klandorf, H., Yancey,
P.H. (2011). Animal Physiology: From Genes
to Organism. 2nd ed. Belmont, CA:
Brooks/Cole Cengage Learning.
[3] Randall, D. J., Burggren, W. W., French,
K., & Eckert, R. (2002). Eckert animal
physiology: Mechanisms and adaptations. 5th
ed. New York: W.H. Freeman and Co. 93-124
[4] Silverthorn, D. Johnson, B. Ober, W. Ober,
C. & Silverthorn, A. (2016). Human
physiology: An integrated approach. San
Francisco: Pearson/Benjamin Cummings. 159-
162
[5] n.a. n.d. Cells in Hypotonic Solution
Pearson.com. Prentice Hall. Pearson
Education, Inc. Retrieved from
http://www.phschool.com/science/biology_pla
ce/biocoach/biomembrane1/hypotonic.html
[6] Kwon Y (2001). Handbook of Essential
Pharmacokinetics, Pharmacodynamics and
Drug Metabolism for Industrial Scientists.
New York: Kluwer Academic/Plenum
Publishers. 44.
[7] Scott, L.A. (1993). Diffusion Across a
Sheep Red Blood Cell Membrane. Retrieved
fromhttp://www.ableweb.org/volumes/vol-
14/7-scott.pdf