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

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
 Alternately, large molecules need to undergo
0.3 M 11 32.6 21.6
hexane carrier-mediated transport which implies a
slower rate of influx. Urea, having a molecular
0.3 M 9.5 26.2 16.7 weight of 60.07g/mol has the fastest hemolysis
ethylene time while glucose, with 180.18 g/mol was the
glycol last to be hemolyzed among the compounds.
0.3 M 18.2 41.4 23.2 Ethylene glycol and glycerol having 62.08
isopropyl g/mol and 92.11 g/mol respectively have an
alcohol intermediate hemolysis time with respect to
urea and glucose. [3].

DISCUSSION By definition, osmolarity describes the

number of particles in solution which can be
Since electrolytes dissociate into ions expressed in the number of particles (ions or
and glucose is a relatively large polar intact molecules) per liter of solution. [4]. It is
compound, these compounds do not diffuse thus defined on the basis of an ideal
into the cell membrane easily. Instead, these osmometer in which the osmotic membrane
compounds contribute to osmotic pressure. allows water to pass. [3]. On the other hand,
Lower concentration of solute indicates a tonicity is a physiological term used to
higher concentration of water or a hypotonic describe a solution and how that solution
solution. When erythrocytes are subjected to a affects cell volume. Hence, a solution is called
hypotonic solution, water moves into the cell isosmotic when two solutions contain the same
by osmosis from outside to inside of the cell. number of solute particles per unit volume
The movement of water causes the swelling of while a cell that does not change size is
cells to the point of cell lysis or hemolysis. [2] considered as a isotonic solution. [4]

Electrolytes dissociate into ions when In the experiment, solutions that are
dissolved in water. For example, one mole of isosmotic with respect to intracellular fluid
sodium chloride dissociates into one mole of was used, and underwent hemolysis.
sodium ions and one mole of chloride ions, it Erythrocytes rupture or swell because when a
is logical that the hemolytic point of sodium cell is exposed to such hypotonic conditions,
chloride is about half of glucose, a there is net water movement into the cell. Cells
nonelectrolyte. The experimental isotonic without walls like animal cells will swell and
coefficient of sodium chloride is 1.818. This may burst (lyse) if excess water is not
indicates that 100 molecules of sodium removed from the cell. [5] Hence, solutions
chloride exert the same osmotic pressure as could be isosmotic but not isotonic as depicted
about 182 molecules of glucose. [7] in the experiment.

As seen in Figure 1, The molecular Partition coefficient is defined as a

weight of the compound shows a direct ratio of concentrations of un-ionized
relationship to the time of hemolysis. This compound in organic and aqueous phases at
shows that an increase in molecular weight equilibrium. It can be viewed as an indicator
results in a longer time for hemolysis and a of intrinsic lipophilicity in the absence of
much harder time for solutes to penetrate the ionization or dissociation of the compound.
cell membrane. Theoretically, a smaller [5]. It is the primary predictor of the diffusion
molecular weight should have a high rate of of a nonelectrolyte across the lipid bilayer. [3]
diffusion which results to a fast rate of influx. The equation of the partition coefficient is
shown below:

4
 through lipid bilayers, special protein
molecules act as pores for these molecules to
pass through the membrane. [3]
Figure 3. Partition coefficient equation
(Adapted from Eckert, 2002) The chemical nature of the cell
membrane can be inferred with the data from
Hence, from the equation above it can the different parameters. The cell membrane is
be implied that the higher organic not an ideal membrane where it only allows
concentration would give a higher partition water to pass through; solutes can freely enter
coefficient. As mentioned before, the heavier and exit the cell under different conditions. In
the molecular size would give a slower Figure 1, it can be said that as the molecular
hemolysis time. This would mean that the weight of the solute increases, the harder it is
longer the carbon chain length would also give for it to the enter the cell. It states that some
a slower hemolysis time implying a lower rate molecules undergo simple diffusion across the
of influx as shown in Figure 2, and it has a cell membrane, where it is easier for small
higher partition coefficient. [3] The sequential molecules to pass through compared to bigger
elongation of an alkyl chain increases the ones.
partition coefficient by a factor of about 3 per
methylene group. The partition coefficient of The inversely proportional
butanol is about 9 times higher than that of relationship of partitioning coefficient to
ethanol. hemolysis time shown in Figure 2 states that
molecules that have a higher solubility in
In the experiment, methanol, ethanol, lipids would have easier access to the inside of
isopropanol and n-butanol were examined in the cell. This is explained by the phospholipid
increasing molecular weights and chain bilayer of the membrane, wherein non-polar
lengths, respectively. These are also in compounds would be transported faster
increasing partition coefficient, whereas through the hydrophobic interior than polar
methanol would exhibit the fastest hemolysis ones. This is also true for the factor of chain
time for its lowest partition coefficient than the length as shown in Figure 3, wherein longer
other organic compounds. On the other hand, carbon chains would diffuse faster because
n-butanol, the highest partition coefficient, *MEASURE OF POLARITY ->
exhibited the slowest hemolysis time. DIELECTRIC CONSTANT
*OVERTON’S RULE OF may burst (lyse) if excess water is not
PERMEABILITY removed from the cell. [5] Hence, solutions
could be isosmotic but not isotonic as depicted
Table 3 illustrates the relationship in the experiment.
between polarity and hemolysis time and
therefore cell permeability. Nonpolar Partition coefficient is defined as a
molecules can easily pass through the ratio of concentrations of un-ionized
membrane because of its lipid-solubility. Polar compound in organic and aqueous phases at
and charged molecules will have difficulty in equilibrium. It can be viewed as an indicator
crossing the membrane. of intrinsic lipophilicity in the absence of
ionization or dissociation of the compound.
Polar molecules can easily interact [5]. It is the primary predictor of the diffusion
with the outer face of the membrane while the of a nonelectrolyte across the lipid bilayer. [3]
charged molecules have difficulty to pass the The equation of the partition coefficient is
hydrophobic core. Since charged molecules shown below:
such as Na+, K+, Ca2+, and C1- cannot diffuse

5
 molecules act as pores for these molecules to
pass through the membrane. [3]

Figure 3. Partition coefficient equation The chemical nature of the cell

(Adapted from Eckert, 2002) membrane can be inferred with the data from
the different parameters. The cell membrane is
Hence, from the equation above it can not an ideal membrane where it only allows
be implied that the higher organic water to pass through; solutes can freely enter
concentration would give a higher partition and exit the cell under different conditions. In
coefficient. As mentioned before, the heavier Figure 1, it can be said that as the molecular
the molecular size would give a slower weight of the solute increases, the harder it is
hemolysis time. However, it would be for it to the enter the cell. It states that some
different when it comes to the carbon chain molecules undergo simple diffusion across the
length where a longer carbon chain would give cell membrane, where it is easier for small
a shorter hemolysis time implying a higher molecules to pass through compared to bigger
rate of influx as shown in Figure 2, and it has a ones.
higher partition coefficient. [3] The sequential
elongation of an alkyl chain increases the The inversely proportional
partition coefficient by a factor of about 3 per relationship of partitioning coefficient to
methylene group. The partition coefficient of hemolysis time shown in Figure 2 states that
butanol is about 9 times higher than that of molecules that have a higher solubility in
ethanol. lipids would have easier access to the inside of
the cell. This is explained by the phospholipid
In the experiment, methanol, ethanol, bilayer of the membrane, wherein non-polar
isopropanol and n-butanol were examined in compounds would be transported faster
increasing molecular weights and chain through the hydrophobic interior than polar
lengths, respectively. These are also in ones. This is also true for the factor of chain
increasing partition coefficient, whereas length as shown in Figure 3, wherein longer
methanol would exhibit the slowest hemolysis carbon chains would diffuse faster because
time for its lowest partition coefficient than the they are more non-polar than their
other organic compounds. On the other hand, shorter counterparts. It can be said that the
n-butanol, the highest partition coefficient, polarity of a substance would have a larger
exhibited the fastest hemolysis time. effect on the permeability of the cell compare
Table 3 illustrates the relationship to its molecular size.
between polarity and hemolysis time and
therefore cell permeability. Nonpolar CONCLUSION
molecules can easily pass through the
membrane because of its lipid-solubility. Polar The experimental results in measuring
and charged molecules will have difficulty in the effect of the different factors affecting
crossing the membrane. membrane permeability indicated that
hemolysis time varied with respect to
Polar molecules can easily interact molecular size, presence of polar and nonpolar
with the outer face of the membrane while the groups, partition coefficient, the length of the
charged molecules have difficulty to pass the carbon chains being considered and also
hydrophobic core. Since charged molecules whether the substance is an electrolyte or not.
such as Na+, K+, Ca2+, and C1- cannot diffuse
through lipid bilayers, special protein Results for the plot of the molecular
weight and hemolysis time is directly linear

6
indicating that the reagents with increasing physiology: Mechanisms and adaptations. 5th
molecular weight which are urea, ethylene ed. New York: W.H. Freeman and Co. 93-124
glycol, glycerol and glucose were respectively [4] Silverthorn, D. Johnson, B. Ober, W. Ober,
hemolyzed from the fastest to slowest time. C. & Silverthorn, A. (2016). Human
Likewise, electrolyte solutions behave physiology: An integrated approach. San
similarly with respect to hemolysis since they Francisco: Pearson/Benjamin Cummings. 159-
dissociate and they require less time to 162
hemolyze the red blood cells compared to non- [5] n.a. n.d. Cells in Hypotonic Solution
electrolyte substance like glucose. Pearson.com. Prentice Hall. Pearson
Education, Inc. Retrieved from
The presence of polar atoms and the http://www.phschool.com/science/biology_pla
relative chain length also affected the ce/biocoach/biomembrane1/hypotonic.html
hemolysis time. Compounds with a polar
group stronger than that of the other [6] Kwon Y (2001). Handbook of Essential
compounds exhibited a shorter time for Pharmacokinetics, Pharmacodynamics and
hemolysis such as chloroform that is made up Drug Metabolism for Industrial Scientists.
of three strong electronegative chloride ions. New York: Kluwer Academic/Plenum
On the other hand, hexane which is made up Publishers. 44.
of entirely nonpolar alkane compound
exhibited the slowest hemolysis time. It can be [7] Scott, L.A. (1993). Diffusion Across a
concluded that polar groups makes penetration Sheep Red Blood Cell Membrane. Retrieved
easier for molecules to the cell membrane from http://www.ableweb.org/volumes/vol-
given that the compound is relatively small. 14/7-scott.pdf

The partition coefficient also showed

varying times for the hemolysis of the red they are more non-polar than their
blood cells. N-butanol exhibited the fastest shorter counterparts. It can be said that the
hemolysis time even though it is expected to polarity of a substance would have a larger
have the slowest hemolysis time because of effect on the permeability of the cell compare
the presence of polar groups which overcame to its molecular size.
its partition coefficient. Methanol was the
compound that exhibited the slowest CONCLUSION
hemolysis time because of its relatively high
partition coefficient. The experimental results in measuring
the effect of the different factors affecting
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