Abstract lipids with proteins inserted into it (Alberts

Abstract

            The cell membrane is the most important part of a cell and
selected organelles as it keeps everything intact, therefore it is important to
take into account its permeability. This experiment was conducted to determine
if the addition of methanol would increase the permeability of a cell membrane
and if increasing the concentration would more so increase the permeability. It
was specifically evaluating if the cell membrane of a beet would experience
this affect. A beet was cut into individual uniform pieces and placed into test
tubes with different methanol concentrations. The absorbance of the dye in the methanol
and water mixture was determined by using a spectrophotometer. The experimental
data demonstrated that there was a positive correlation between the methanol
concentration and the absorbance. Therefore, as the methanol concentration
increased, the absorbance increased as well. It was concluded that the results
were consistent with the hypothesis that an increased methanol concentration
will affect permeability proportionately.

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Introduction

            The most crucial organelle present on earth that is vital
for the survival of all cells, is the cell membrane. Not only does it surround
the cell, but it also surrounds specific organelles and their contents. About
70% of a cell is water and therefore movement of water and materials across the
cell membrane is crucially important (Alberts et al 2014). A cell’s membrane is very flexible has a very simple
structure, it consists of a thick double layer sheet of lipids with proteins
inserted into it (Alberts et al 2014).
The function of a cell membrane involves many different things. It is used to
prevent the contents that are inside the cell from escaping and combining with
the medium outside of the cell, as well as it must pass nutrients across the
membrane and into the cell and pass waste out of the cell (Alberts et al 2014). To expedite this transport,
the cell includes many selective channels and transporter proteins. Many
molecules can pass across the cell membrane without any help by simple
diffusion, but the more complex molecules such as inorganic ions, sugars, amino
acids, nucleotides, and other cell metabolites need assistance (Alberts et al 2014). These cells move by
facilitated transport. Some proteins provide exclusive transport for a specific
water-soluble molecule (Alberts et al
2014). It allows them to evade the hydrophobic core of the cell membrane to
enter the cell. While transport across membranes is very important, its
important to note that other factors can interrupt cell membranes.  Certain substances have the possibility of
binding to the membrane near the surface, this will displace the proteins in
the membrane causing it to become disrupted (Alberts et al 2014). Disruptions in the cell membrane will allow materials
that are not necessarily able to cross the membrane through facilitated
transport to cross the membrane. This works in both directions, materials can
leave or enter the cell as a factor of this.

            Different cells have different characteristics, such as the
beet cells used in this experiment. Beets have a significant pigment called
betalains that can indicate the permeability of the cell membrane present on
the beet. When the membrane is damaged, the pigment will be released into the
cell. In this experiment, it was hypothesized that methanol at different
concentrations affects the permeability of the cell membrane of a beet. It was
predicted that the permeability of the cell membrane of a beet will increase
with an increased methanol concentration. This was done by placing cut beets
into different concentrations of methanol and measuring the absorbance of the
pigment found in the solution.

 

 

Methods

            The flow of fluids throughout a cell was investigated
thoroughly through first hand analysis. The experiment was conducted in a room
at a temperature of 21.5°C.  The
investigation was initiated by obtaining a beet root. The beet root was then
cut into pieces of a uniform surface area (BIOL 2070H Lab Manual, 2018). Test
tubes were then prepared with 30 mL solutions containing different methanol
concentrations. Specific dilutions were calculated for the methanol
concentrations that were not provided (BIOL 2070H Lab Manual, 2018). Three sets
of three test tubes were created. Three containing a 1.5% methanol solution
containing 22.5 mL of methanol and 7.5 mL of distilled water. Another three
containing, a 1% methanol solution containing 15 mL of methanol and 15 mL of
distilled water and lastly another three containing a solution that was a 2%
methanol solution that was 30 mL of methanol. A control was created in a test
tube using room temperature water. A piece of beet was then placed in each test
tube, including the control (BIOL 2070H Lab Manual, 2018). The test tubes were
observed for 58 minutes and inverted frequently during the observation. They
were inverted every 5 minutes during the experiment (BIOL 2070H Lab Manual, 2018).
They were first observed visually to determine that a suitable amount of dye
had been released.

            The beet was then removed from the test tube and the
liquid was placed in a new test tube. The solutions were one at a time
transferred into a cuvette and the absorbance was measured using a
spectrophotometer (BIOL 2070H Lab Manual, 2018). The spectrophotometer used had
a model number of 333142. The spectrophotometer was first set to zero using the
control. The control was then replaced with each sample, one at a time, and the
absorbance was measured (BIOL 2070H Lab Manual, 2018). A graph was then created
to plot the mean absorbance of each set of test tubes against the concentration
that was used. The standard deviation was used to detect any significant error.

Results

            The permeability of the beet membrane was seen visually
before the spectrophotometer was used to determine the absorbance
quantitatively. The three test tubes that had the 2% methanol concentration had
pink dye flowing out almost immediately. There was one test tube that was a significantly
darker pink than the other two, which had a significantly different absorbance
of 0.61. The three test tubes that had the 1.5% methanol concentration, had the
pink dye present after a short period of time. There was also an individual
test tube that was significantly darker than the other two for this
concentration as well. The other set of test tubes at 1% had a significantly
less amount of dye present after the same amount of time. There were also
bubbles present rising infrequently when the beet was initially dropped into
the methanol.

            The results demonstrated in figure 1 suggest that there
is a relationship between the concentration of methanol and the permeability of
the beet cell membrane. There is a positive correlation indicated between the
absorbance and the methanol concentration where as the methanol concentration
increases, the absorbance also increases. It is also suggested that as the
concentration increases, the difference in the absorbance increases. Between 1%
and 1.5% there is a 0.097 difference and between 1.5% and 2% there is a 0.29
difference. Further, figure 1 also demonstrates that with an increased methanol
concentration, there is significantly more standard error. At 1% there is a
standard deviation of 0, at 1.5% there is a standard deviation of 0.055 and at
2% there is a standard deviation of 0.17. The standard error at 2% methanol
concentration is substantially higher than at lower concentrations.

 

 

 

 

 

 

 

 

 

Figure 1:
A plot of absorbance obtained using a spectrophotometer versus the associated methanol concentration.

Discussion

            Since cell membranes can easily damaged or impaired, they
are susceptible to many different factors. They respond to many different
factors in their environment and therefore it would be assumed that by changing
these conditions, it would be visible to see the impact of specific conditions.
Alcohol can interrupt the proteins found in a cell membrane, therefore the
addition of methanol should affect the permeability of a membrane. It was
expected that by submersing a beet in methanol and increasing the
concentration, the permeability would increase with a higher concentration of
methanol. This hypothesis was supported by the data that was obtained in this
investigation.

            Taking into consideration that the hypothesis was
correct, it is important to realize that depending on a cell’s environment, a
cell’s membrane will change accordingly. The methanol that the cell was
immersed in, impacted the composition of the proteins in the cell membrane.
Thus, since there was a positive correlation, an increased amount of alcohol
will increase the damage to the membrane. By increasing the damage imposed upon
the membrane, it will become more permeable and allow intracellular components
to exit the cell. It will also work in reverse and allow fluids outside of the
cell to enter the cell. In the case of this experiment, the beet’s red pigment,
called betalain, is stored inside intracellular components such as the vacuole
to keep it contained. The membrane on the vacuole is like the outer cell
membrane and when it is impaired, it releases the red pigment, indicating the
permeability of the membrane. Since the absorbance increased with the concentration
of methanol, it means that there was a larger amount of red dye found in the
solution. Thus, the permeability of the membrane was influenced by the amount
of alcohol that was found in the solution.

            Cell membranes are not only influenced by different
concentrations of alcohol, such as methanol and ethanol, the temperature can
affect the permeability, with a range of temperatures from boiling to freezing,
or different detergents can affect the permeability. In an experiment conducted
by Stephen Adam and his associates, they were studying biochemical events in
nucleocytoplasmic transport (Adam, Marr and Gerace, 1990). They used a
non-ionic detergent called digitonin to permeabilize the cell membrane. At low
concentrations, the detergent selectively perforates the plasma membrane which
in turn releases the cytosolic components of the cell (Adam, Marr and Gerace,
1990). It interrupts the protein and cholesterol content of the cell, thus
causing the membrane to become permeabilized (Adam, Marr and Gerace, 1990).
Therefore, this indicates that different treatments can alter cell membranes,
besides the alcohol content of the surrounding cell.

            It is important to note that there is also a key
relationship between these cells and the cells found in the body. Cells in the
body are apoptotic and therefore will usually die before their membrane becomes
permeable. Cells that have an apoptotic microtubule network are more likely to
be impermeable to selected conditions (Oropesa-Avila et al 2013). Where there was a
damaged apoptotic microtubule network that was disorganized, the cell became more
susceptible to membrane permeability (Oropesa-Avila et al 2013). The
permeability indicates that one cell is more likely to affect other cells
around it, if the cell does not die (Oropesa-Avila et al 2013). Other than the apoptotic microtubule network, similar
factors like alcohol can also affect the body, like the beet cells. Consuming
alcohol in large quantities can induce the production of reactive oxygen
species, lower cellular antioxidant levels and enhances oxidative stress in the
liver (Wang, 2014). Reactive oxygen species damage complex cellular molecules
like lipids, proteins, and nucleotides (Wang, 2014). Thus, cell membranes in
the body will be damaged by these reactive species (Wang, 2014). Therefore, the
cells in the body are very similar to the beet cells used in this experiment.

            In this investigation, there were certain errors that
occurred that could have been avoided under different circumstances. During the
experiment, there was a large time constraint. There was very little time for the
beet to be immersed in the alcohol, if more time was used, more dye could have
been observed. In an experiment also using alcohol treated red beet tissue, the
beet was left for 10-15 hours, therefore it had much more effective results
(Grunwald, 1968). Further, the methanol used in this experiment was of very low
concentration, 2%. If the concentration was much higher, the cell membrane
would have become more permeable at a much faster rate, therefore creating more
effective results. If this investigation were to be conducted again, it would
be important to focus on these errors as well as possible additions to the
experiment. It could be investigated how to reverse the permeability effect of
the alcohol on the beet. In the investigation previously mentioned, calcium
ions were able to reverse the effect of methanol on the cell membrane
(Grunwald, 1968). Finally, in future investigation with a larger timeframe, it
would be effective to investigate each of the different factors that affect
permeability to get a more widespread explanation of the permeability of a
membrane.

            In conclusion, while this was investigation was not
perfect, it conducted what it was proposed and confirmed the hypothesis and
prediction. With the increasing methanol concentration, the permeability of the
beet membrane increased proportionately. Therefore, cell membranes, a vitally
important structure, can be easily influenced by different factors.

 

 

 

 

 

 

 

 

 

 

 

 

 

References

Adam, S. A., R.S. Marr
and L. Gerace. 1990. Nuclear Protein Import in Permeabilized

Mammalian
Cells Requires Soluble Cytoplasmic Factors. The Journal of Cell Biology.

111:
807-816.

Alberts, B.,
D. Bray, K. Hopkin, A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter. 2014.
 

Essential Cell Biology (4th e.d.).  Garland Science: New York, NY. P. 4-12.

BIOL 2070H Lab
Manual, 2018, pp. 1-3.

Grunwald, C. 1968. Effect
of Sterols on the Permeability of Alcohol-Treated Red Bet Tissue. 

Plant
Physiol. 43: 481-488.

Oropesa-Avila, M., A.
Fernandez-Vega, M. de la Mata, J. G. Maraver, M.D. Cordero, D. Cotan,

M.
de Miguel, C.P. Calero, M.V. Paz, A.D. Pavon, M.A. Sanchez, A.P. Zaderenko, P.

Ybot-Gonzalez
and J. A. Sanchez-Alcazar. 2013. Apoptotic microtubules delimit an

active
caspase free area in the cellular cortex during the execution phase of
apoptosis.

Cell
Death and Disease. 4: 1-13.

Wang, K. 2014. Molecular
mechanisms of hepatic apoptosis. Cell Death and Disease. 5: 1-10.

 

 

 

 

Appendix A

Dilution Calculations

C1V1 = C2V2

1.5 % Methanol

V1 = 0.15 x 30 mL / 0.2

     = 22.5 mL methanol + 7.5 mL distilled water

 

1 % Methanol

V1 = 0.1 x 30 mL / 0.2

     = 15 mL
methanol + 15 mL distilled water

 

Table 1: Absorbance
correlated with amount of beet dye and its associated methanol

concentration.

Methanol
Concentration

Test
Tube 1 Absorbance

Test
Tube 2 Absorbance

Test
Tube 3
Absorbance

Average
Absorbance

Control
0%

0.04

 

 

 

2%

0.35

0.61

0.3

0.42

1.5
%

0.1

0.09

0.19

0.13

1%

0.03

0.03

0.03

0.03

 

Appendix B

 

Table 2: A
description of cow blood cells in an unknown solution viewed under 40X

 magnification with an Ernst Leitz Wetzlar
phase-contrast microscope, model 2112.

Solution

Description

0.9% NaCl (Isotonic)

-Uniform
circles
-Dark
membrane
-Relatively
small (60 across)

A (Hypertonic 1.5%)

-Uniform
circles
-Slightly
smaller than isotonic
-Dark
membrane
-Slightly
pinched

B (Hypotonic 0%)

-Cells
no longer visible
-Bits
and pieces present
-Cell
had already lysed

C (Isotonic 0.6%)

-Uniform
circles
-Dark
membrane
-Relatively
small

 

Three
4 different solutions were used to identify isotonic, hypertonic and hypotonic
solutions. By viewing the cow blood cells under the microscope, different
characteristics could be seen that helped identify which solution was which. An
isotonic solution is where the concentration of the solute inside of the cell
is equal or very similar to that of the concentration outside of the cell,
therefore, the cell has no significant changes (BIOL 2070H Lab Manual, 2018).
Thus, solution C could be identified as the isotonic solution of 0.6% NaCl as
it experienced little change. A hypotonic solution has a low solute
concentration outside of the cell compared to the inside, therefore water will
move into the cell (BIOL 2070H Lab Manual, 2018). Since the cells for solution
B were present in small pieces, this indicates that the cells took in to much
water and burst before they could be observed and is therefore hypotonic.
Finally, a hypertonic solution has a higher concentration of solute outside of
the cell and therefore water will move out of the cell (BIOL 2070H Lab Manual, 2018).
The cells that were present in solution A were slightly smaller and appeared to
be pinched, therefore since the cell shrunk, it is in a hypertonic solution.