Abstract
In this experiment the integrity of beet root cell membranes exposed to harsh environmental conditions was assessed. These harsh environmental conditions include varying pH, varying temperature, and the introduction of solvents into the environment. Upon the degradation of the cell membranes the molecule responsible for the red pigment inside beets known as betanin was released. This release of pigment allowed for solutions to be tested through the use of a spectrophotometer. A greater degradation of the cell membrane led to a greater release in pigment and a higher absorbance reading from the spectrophotometer. From this lab it has been concluded that the integrity of cell membranes is severely reduced in acidic environments, extreme temperatures, and in the presence of highly concentrated solutions of acetone and methanol. All of these factors have a detrimental effect on the effectiveness and structural sanctity of the fluid mosaic cell membrane which surrounds cells.
Introduction
The basis of this lab is formulated on the degradation of the cell membranes found in the beta vulagaris root samples. The fluid mosaic model of a cell membrane explains the many defining characteristics demonstrated by cell membranes and its various components each play an integral role in the overall function of the cell. The "fluid" description of the membrane is due to the ability of the many components of the membrane to move laterally. This motion throughout the membrane means that the membrane itself is not entirely solid. Although it has a defined structure its movement is more closely related to a fluid[1]. The second component of the naming process is the "mosaic" description. The word mosaic is used because the cell membrane is a collection of many different parts which all work to allow the membrane to properly function. A cell membrane is formulated of many different components however the phospholipid bilayer is the defining structural piece. The phospholipid bilayer is a collection of phospholipids which each have a hydrophobic tail and a hydrophilic head. The hydrophobic regions become attracted towards one another and eventually form tight bundles. The density of these bundles is dependent on the type of lipid which makes up the tail. As saturated fats are more tightly bundled the density of the membrane is greater than that formed by phospholipids with unsaturated fat tails. Unlike the tails, the phosphate heads are polar in nature and love water [2]. The two contrasting characteristics form a layer which keeps the movement of substances in and out of the cell controlled and ultimately defines the boundary of the cell. Other components of the cell membrane include the globular protein, carbohydrate chains and protein chains. The various chains which are attached to the cell membrane are used primarily for communication between cells however the globular protein has a variety of different functions [3]. The globular protein can act as a transport channel which allows substances from the outside environment to enter the cell, it can act as a cell identity marker by displaying a small oligosaccharide "flag", and it can aid in the structural support of the cell by attaching to other globular proteins or cytoskeletons. The fluid mosaic model of the cell membrane works to define the many characteristics that cell membranes adopt [4]. They control which substances enter the cell environment and provide a defining shape to the cell. In this lab these membranes must be destroyed in order to extract the betanin which is found inside the cell. Please refer to the appendix, Figure 1, for a visual representation of the fluid mosaic model.
One of the major threats to the health and structure of cell membranes in the body is the presence of free radicals. One of the purposes of the cell membrane is the definition of a cell's shape and the containment of its many components. Free radicals are atoms and molecules which are left with unpaired electrons when weak molecular bonds are broken. These atoms desperately work to find electrons in order to stabilize themselves. As a result these atoms take electrons from stable atoms which in turn become free radicals themselves[5]. This process can form a chain reaction which destroys many cell components including DNA and cell membranes. Free radicals can form as a result of regular bodily functions such as metabolism or immune reactions however there are many environmental stresses present in the world which can create free radicals. Some of these environmental stresses include pollution, radiation, harsh chemical and smoke inhalation[6]. Antioxidants are compounds which counteract the negative effects of free radicals in the body. Certain compounds such as vitamin C and vitamin E are considered antioxidants because they donate electrons to free radicals without becoming free radicals themselves. As antioxidants remain stable with or without their donated electron they travel through the body collecting and donating electrons in an effort to maintain total body health. By ending the chain reaction caused by the presence of free radicals antioxidants effectively save the body from the degradation that the free radicals induce. If left unchecked free radicals can cause a variety of ailments in the body including the possibility of cancer, heart disease and general aging. By incorporating antioxidant rich foods into your diet many of the negative effects of free radicals can be repressed[7]. It has been proven that vitamin E can help in the treatment and prevention of both cancer and heart disease. Through its ability to inhibit the conversion of nitrites in the stomach into nitrosamines vitamin E helps prevent cancer. Nitrosamines are cancer promoters found in the body and with large doses of vitamin E the risks of oral cancer developing are cut in half. Vitamin E has also been linked to cases of heart disease, eye disorders, neurological disorders and aging. Through the presence of vitamin E the detrimental effects associated with these ailments can be delayed and minimized [8]. Please refer to the appendix, Figure 2, for a diagram demonstrating the actions of free radicals and antioxidants.
Betanin is another antioxidant which contributes greatly to the overall health of the body. Found in the vacuoles of red beets, betanin is a water soluble compound which is responsible for the red colouration of the beets. With a molecular formula of C24H26N2O13 and a molecular mass of 550.46884 g/mol betanin is considered to be a fairly large molecule found in the betalain family [9]. Betalain compounds are classified by the combination of a sugar and a coloured compound present in their synthesis as well as the presence of nitrogen in their chemical formulas. Please refer to the appendix, Figures 3 & 4, for two and three dimensional structural diagrams of betanin respectively. The synthesis of betanin is an extremely complex process which begins with betalamate, also known as betalamic acid, and the amino acid tyrosine. Tyrosine reacts with oxygen and dihydroxyphenylanine to form dopaquinone. From there, dopaquinone as a tendency to release a hydrogen ion and become leucodopachrome. The release of the hydrogen ion is spontaneous in nature. Now that there is leucodopachrome present in the environment it can react with betalamate to form betanidin. Betanidin is the coloured compound which binds with a sugar to form a betalain, specifically betanin. The betanidin reacts with a specialized form of glucose known as UDP-D-glucose. With the assistance of an enzyme called betanidin 5-0-glucosyltransferase betanin is formed[10]. The biosynthesis of betanin is a long and complex process which relies heavily on the assistance of enzymatic activity and the spontaneous release of hydrogen ions. Despite the complex chemical nature of betanin there have been many useful applications found for it. Betanin is used regularly in food dyes as it is naturally occurring and is known to provide health benefits due to its antioxidant nature. Betanin is also used in more complex processes which are on the cutting edge of science. By creating sensitized gelatin microlenses with the addition of betanin a variety of different applications have been identified. These new lenses can be placed in cell phone cameras, medical devices, and optical storage devices. The sensitized gelatin that is created from the betanin is an effective tool that has the potential to revolutionize the technological world, or at the very least make complex technologies more affordable. The lens that is created is extremely clear and can be bent without the traditional "fish-eye" effect common to concave lenses[11].
The results of this lab are quantified entirely by values provided by a spectrophotometer. Spectrophotometry is the measure of the amount of light that is absorbed by a sample. By setting the spectrophotometer to a certain wavelength within the spectrum of visible light an absorbance amount (Abs) can be identified[12]. As the colour of the betanin which was being released into the solutions was known an absorbance spectrum could be preset. An increased presence in betanin would result in a higher Abs value due to the solutions new found ability to absorb light of a variety of different colours[13]. In this lab the process of spectrophotometry was used to measure the presence of betanin in each of the samples. Please refer to the appendix, Figure 5, for an image of a spectrophotometer. The other component of spectrophotometry is Beer's Law. Beer's Law states that, "the absorbance is directly proportional to the concentration of a solution. If you plot absorbance versus concentration, the resulting graph yields a straight line. The equation for the straight line can be used to determine the concentration of an unknown solution[14]." The idea presented by Beer's Law is that the concentration of an unknown solution can be derived through the use of spectrophotometric data and a mathematic equation. By identifying the concentration of a solution something as precise as the number of molecules could be derived given simple mathematical equations and the help of Avagadro's constant. Please refer to the appendix, Figure 6, for a table including Beer's Law and a molecule calculation.
The objective of this lab is to identify trends in cell membrane integrity upon the introduction of harsh environmental stresses. The ability for a cell membrane to remain intact upon the introduction of varying pH, temperature and solvent concentrations will all be tested and quantified through the use of a spectrophotometer. By recording absorbance values the amount of betanin released into the varying solutions can be recorded in a unified and comparable way across all three tests. The major control present in the lab was a blank burette used in the spectrophotometer. Through its use the apparatus could be properly calibrated and a true negative result could be identified. There were also individual controls present for the pH and temperature stress tests. The pH test contains a sample with a pH of 7. The presence of water will provide a good baseline with which to compare the other results to. The temperature test has one sample being incubated at room temperature. This provides a good baseline to measure and compare the other results gathered in the lab to. By having samples in which no changes have been made the results that are recorded are more reliable. The data from multiple labs was also compiled and averaged to remove individual human error from the experiment. The anticipated results of the lab vary between each test. During the pH it is expected that the most extreme of pHs, two and twelve, will yield the largest absorbance values. In the temperature test the coldest and hottest temperature are expected to yield the largest Abs values. In the solvent test the highest concentrations are expected to result in the highest Abs values. The 50% acetone and 50% methanol should provide the highest absorbance values in their respective tests. As the stresses on the cell membrane become more extreme it is hypothesised that the cell membrane will degrade and release more betanin than a cell membrane in near normal conditions. The increased release in betanin will result in an increased Abs reading from the spectrophotometer.
Methodology
Sample Preparation
The experiment began by splitting up the class into five separate groups. Each group was assigned to one of three variables to research. Upon the assignment of each variable the groups began to prepare the samples and required apparatus. A test tube rack containing six large test tubes and six cuvettes was distributed to each group for sample storage. These test tubes were subsequently labelled to correspond with the values represented in the previously provided data chart. The cuvettes were not taped as the tape would interfere with the spectrophotometric analysis. Using a cork borer a cylindrical sample was extracted from a beet. A probe was then inserted into the cork borer to remove the beet cylinder. This process was repeated numerous times to allow for a sufficient number of test samples. Once the cylinders were prepared a razor blade was used to cut the cylinders into disks. Each disk was approximately three millimetres in thickness. The pH and solvent test groups prepared a total of thirty beet disks while the temperature test groups prepared only fifteen. These values correspond with the number of samples necessary for each test. Once the disks were cut they were placed into a beaker and washed. The beaker was filled with water and swirled to immerse the beet disks. The washing of the beets was repeated a minimum of three times. The washing process stopped once the water used to clean the beet disks stopped adopting the red colour of the beets. Once the washing liquid had been removed from the beaker tweezers were used to distribute five disks into each test tube. For the temperature test groups the -18oC and 4oC samples were prepared prior to the day of the experiment and stored in refrigeration units. The samples were prepared and ready for the cell membrane stress studies.
Cell Membrane Stress Studies
The pH groups used transfer pipettes with graduations to a maximum of 2mm to transfer 10mm of each stock pH sample into respective graduated cylinders. The meniscus of each graduated cylinder was observed to ensure that 10mm of solution was placed into each test tube. Once the contents of each graduated cylinder were checked the contents were deposited into the test tubes containing the beet samples. Each graduated cylinder solution had a corresponding test tube which was previously labelled. The different pH solutions that were used were of pH 2, 4, 6, 7, 10, and 12. The samples were then incubated at room temperature for ten minutes. Upon the conclusion of the ten minute incubation period each sample was agitated in the test tube through a series of flicking and shaking manoeuvres. This was done until the solution appeared uniform. Once a uniform appearance was achieved a portion of each test tube's sample was placed in the previously identified cuvette in the test tube rack. The samples were then ready for spectrophotometric analysis.
The temperature groups filled five graduated cylinders with exactly 10mm of tap water. The meniscus of each graduated cylinder was observed to ensure that precisely 10mm of water was present in each cylinder. Once the contents of each graduated cylinder were checked 10mm of water was deposited in each test tube. The test tubes containing the frozen (-18oC), refrigerated (4oC), and room temperature (21oC) samples were incubated at room temperature for ten minutes. The 40oC and 80oC samples were placed in respective water baths of the desired heat using tongs. These samples were also incubated for a duration of ten minutes. At the conclusion of the ten minutes the test tube racks in the water baths were removed using tongs and placed back into the test tube racks. Each sample was agitated in the test tube through a series of flicking and shaking manoeuvres. This was done until the solution appeared uniform. Once a uniform appearance was achieved a portion of each test tube's sample was placed in the previously identified cuvette in the test tube rack. The samples were then ready for spectrophotometric analysis.
The solvent groups used transfer pipettes with graduations to a maximum of 2mm to transfer 10mm of each stock solvent sample into respective graduated cylinders. The meniscus of each graduated cylinder was observed to ensure that 10mm of solution was placed into each test tube. Once the contents of each graduated cylinder were checked the contents were deposited into the test tubes containing the beet samples. Each graduated cylinder solution had a corresponding test tube which was previously labelled. The different solvent samples that were used included 1% acetone, 25% acetone, 50% acetone, 1% methanol, 25% methanol, and 50% methanol. The samples were then incubated at room temperature for ten minutes. Upon the conclusion of the ten minute incubation period each sample was agitated in the test tube through a series of flicking and shaking manoeuvres. This was done until the solution appeared uniform. Once a uniform appearance was achieved a portion of each test tube's sample was placed in the previously identified cuvette in the test tube rack. The samples were then ready for spectrophotometric analysis.
Spectrophotometric Analysis
The spectrophotometer was prepared at a wavelength of 560nm for use by all of the groups. One at a time each group utilized the spectrophotometer. The first step that was taken was the calibration of the apparatus. By using a cuvette containing a control the spectrophotometer was tuned to demonstrate optimal results. Once the machine was properly calibrated the samples were inserted one at a time into the machine. The cuvette was inserted into the slot and the lid was closed. Once the lid is closed a reading appeared on the digital display. This value was recorded in the data table. The original sample was removed and another one was inserted into the machine. This entire process was repeated until each sample was properly analyzed by the spectrophotometer and all of the data had been recorded. Once all of the data had been recorded it was submitted to Mr. Melegos for the purpose of creating a larger sample size. By pooling the results gathered in both SBI4U1 courses running this semester the possibility for human error is removed and the results become more reliable. Mr. Melegos went on to determine average values for all the spectrophotometric analysis. These values were used in the analytical portions of the lab.
Results
By introducing cell membrane samples to a variety of different environmental stresses a base of data was created which demonstrates very specific trends. When the beet cells were exposed to varying pH the greatest cell membrane degradation occurred at a pH of 2 with a value of 0.52Abs. The second greatest value was recorded at a pH of 4 with a value of 0.16Abs. The lowest value which represents the least amount of cell membrane disturbance was recorded at a pH of 10. The value was 0.01Abs. The graph which was formulated using the recorded results demonstrates a parabolic nature. It begins trending upwards at the ends of the graph with two defined x-intercepts and a vertex. Please refer to the appendix, Figure 7, for a graph of Absorbance vs. pH. The second test included changing temperature as its variable. In this test there was a definitive parabolic shape in the graph. The highest absorbance occurred at -18oC and the second highest absorbance occurred at 80oC with Abs values of 1.61Abs and 1.05Abs respectively. The lowest absorbance value was recorded at 40oC with a value of 0.02. Please refer to the appendix, Figure 8, for a graph of Absorbance vs. Temperature. The final test involved the introduction of varying solvents to the cell environment. Both solvents demonstrated similar effects as both trend lines on the graph demonstrate exponential growth. The absorbance values were lowest when the solvent concentration was at 1%. Both samples yielded an absorbance value of 0.02Abs. Acetone in its 50% concentration yielded the greatest absorbance value with a value of 0.46Abs followed closely by methanol in its 50% concentration at 0.32Abs. Please refer to the appendix, Figure 9, for a graph of Absorbance vs. Solvent Concentration, and Figure 10, for the data table in which all the results are contained.
Discussion
All of the recorded results followed defined trends which varied from expected results to unexpected outcomes. In the pH test the high level of absorbance demonstrated by the low pH sample indicates that cell membranes tend to denature in highly acidic environments. Highly acidic solutions contain an excess of H+ ions. These ions look to bond with other substances in order to stabilize themselves. In essence the H+ ions act just like free radicals. In an effort to bond with the different components of the phospholipid bilayer the H+ ions dissociate the different bonds that have been formed. By breaking apart the phosphate groups in the hydrophilic heads of the phospholipid bilayer the H+ ions disrupt the cohesive nature of the bilayer that was formed. This action happens whenever there are H+ ions however the extremely high level of acidity at a pH of two means that there is an incredibly large amount of the ions. The number of ions tearing at the different bonds present in the components of the phospholipid bilayer becomes too much and the membrane breaks apart or separates. This opening allows for betanin to leave the cell and removes a cell's ability to function effectively. The information presented in the graph also began to show an upward trend as the pH approached an excessively high value. Although not wholly demonstrated in the lab it is reasonable to infer that the same process of bonds being destroyed due to rogue ions looking for bonding partners could be present in extremely basic solutions. The OH- ions which are released in basic solutions maintain the same role as the H+ ions in an extremely acidic solution however the OH- ions are fat soluble. They act on the lipid component of the phospholipid bilayer. In pH values which approach neutral the effect of the rogue ions is minimal. As a result the destruction of the bonds in the phospholipid bilayer is also minimal. The absorbance values for pH support the theory that strong acids and bases cause disassociation of bonds in the cellular membrane which leads to a release of the cells inner contents, namely betanin. These ions also have the ability to diffuse through fats. As the ions have the ability to permeate lipids they can pass through the lipid bilayer however due to their charge they are constantly attracting other molecules or atoms. As a result there is possibility for spheres of water to form around the ion. If one of these ions were too attempt and form water in the hydrophobic region of the bilayer dissociation of the membrane may occur[16].
In the second test the data collected demonstrated that both extreme heat and intense cold could cause damage to the cell membrane. When substances experience a reduction in heat they begin to solidify. During the solidification process many substances, water for example, form crystalline structures. As there were liquids present in the cell environment upon introduction to the cold ice crystals began to form. Water expands when it changes state from liquid to solid. As a result any water that was present in the cell would crystallize and expand. The expansion of the water would take up space and put pressure on the cell membrane. As the water continues to expand the cell membrane would eventually break. The cell membrane is not a solid mass but simply a bonded connection of individual pieces. As a result if a great force were to act against it the bonds would dissociate and the cell membrane would be broken down. The nature of water and its expansive characteristics explain why the frozen samples demonstrated the highest recorded absorbance values in the lab. Heat also caused the degradation of the cell membrane but for an entirely different reason. Heat is one factor which is responsible for denaturing the peptide bonds in protein structures. When extreme heat is applied to proteins the peptide bonds which hold the amino acids together are broken. These bonds cannot be re-established and as a result the protein is destroyed. One component of the fluid mosaic membrane is the globular proteins. These proteins act as pathways in and out of the cell and also work to provide structural support. When exposed to high heat the peptide bonds which compose these proteins are destroyed and as a result so too is the protein. Without the globular proteins or protein chains the cell membrane loses much of its rigidity and shape[15]. A lack of shape and effective bonds leads to the destruction of the membrane and the release of betanin. As a result it seems that both hot and cold temperatures can destroy different components of the membrane and lead to the release of betanin into the surrounding environment. At less stressful conditions the cell membrane works to effectively separate the cell from the outside environment. Its fluid structure allows it to move and avoid rupture under normal circumstances.
The final test involved the introduction of acetone and methanol in varying concentrations into the cell environment. It was demonstrated that high concentrations of both solvent caused the cell membrane to degrade and break down. Both acetone and methanol have the ability to break down the phospholipid bilayer present in cell membranes. As both solvents are hydrophilic or polar in nature they can easily destroy the phospholipid bilayer which makes up the cell membrane. Both solvents contain active groups which foster a polarity in their structure due to the molecular shape which they adopt. Polar substance have the ability to break down other polar substances and bonds. The polar nature of the two solvents coupled with the polar nature of the phosphate heads at the top of the phospholipid bilayer result in a destructive interaction. As a result they destroy the phosphate heads of the layer and destroy the cell membrane[17]. Recent research has also proven that acetone and methanol have a tendency to attach to certain proteins and denature them. By fixing themselves onto the proteins found in the cellular membrane acetone and methanol both break down peptide bonds and demonstrate the ability to denature proteins[18].The results seen in the lab support the theory that cell membranes are extremely soluble in the presence of both acetone and methanol. More specifically, the phospholipid components of the cell membrane are soluble in the presence of the two tested solvents. As the concentration of the two substances increased the absorbance value recorded in the solution increased at an alarming rate.
As a whole the lab demonstrated that cell membranes are doomed to fail under extreme circumstances. They are designed to work in an optimized environment and can exist outside of it however when situations become too extreme the structure fails. The phospholipid heads present in the bilayer are the main targets of many of the extreme conditions and have proven that under stress the bonds that are formed can be broken. As the cell membrane is a composition of moving parts its inability to stay intact at all times is understandable. This lab has proven that acidic, basic, hot, cold, and solvent rich environments can all destroy cell membranes. As many of the compounds in the environment of the cell demonstrate lipid solubility the bilayer of the cell becomes exposed. When substances can dissolve in the integral component of a structure the possibility for it to change and fail is magnified.
Despite the wealth of results and insight that this lab has provided there were multiple sources of error throughout the process. The first source of error was the inability to properly add the different liquid components to the beet disk samples in one motion. As the test tubes were all filled at slightly different times throughout the lab the ten minute incubation period fluctuated. Some samples were left for more than ten minute while others were not left long enough. With the proper apparatus to simultaneously load the six tubes at one time this lab could have been much more efficient and effective. Another source of error present in the lab was the sizing of the beet disks. As the actual size was simply estimated at around 3mm the sample sizes between groups could have been wildly different. An increase in beet disks leads to an increase in the amount of material the different solutions have an effect on and as a result the amount of betanin released varies. By using a mandolin set to a 3mm setting a consistent size could be guaranteed every time and each group would be working with equivalent beet product in each test tube. Another source of error was the temperature of the water bath. As the only means of measuring its temperature was a thermometer the readings had the possibility for change. In the sample that was conducted in the first class the water bath temperature was at 41oC however the recorded temperature for the lab was 40oC. Slight variations in temperature between different samples could affect the results and cause discrepancies with the data. By having a water bath with a precise temperature setting the need for human involvement and "guesswork" could be removed. The final source of error present in this lab was the methodology used in the temperature case study. The samples which were prepared prior to the lab day were left out on the workbench while the other samples were being prepared. This exposure to room temperature air began to warm up the samples. Compounding the warming effect was the addition of room temperature water to the samples. As the samples were warmed up the data collected may have skewed too far towards that of the room temperature test. By keeping the samples in refrigeration until just prior to the addition of the water this source of error could be mostly eliminated. All in all there are flaws with this lab however by pooling data from a large contributing group the effect of individual error is all but removed.
This lab was an effective tool in the understanding of the formulation of the cell membrane and which environmental stresses degrade its structure. Despite the many great lessons learned there are extensions which could be added to the lab which make it more exciting and allow for a deeper understanding of the different factors effecting cellular membrane structure. One factor that could be changed would be the subject being used for the experiments. By using a meat like chicken which has absorbed a dye the experiments can be repeated in an effort to prove a relation between the structure of both plant and animal cells. Another possibility for an extension of the lab would be to increase the number of increments between each measurement. By doing so in the temperature variable the parabolic shape of the trend line could be proven with true data. A final possible extension to the lab would be the completion of the lab with a new variable. By submerging the beet root disks in samples of varying concentration the effects of other substances could be examined. Some different substances include sucrose solutions of varying concentration or oil suspension of varying oil contents. This lab allows for a better understanding of the different characteristics that make up a cell membrane. It develops an understanding in the variety of stresses that cells can and cannot cope with in their environment. Overall it is an effective laboratory endeavour which also teaches components of biostatistics and spectrophotometric analysis. Through continued studies involving cell membranes breakthroughs can be made which benefit whole body health. By understanding the miniature components which make up the body the entire entity can be better maintained.
References
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3. Cell Theory, Form, and Function: Fluid Mosaic Model of Membrane Structure and Function — Infoplease.com. (n.d.). Infoplease: Encyclopedia, Almanac, Atlas, Biographies, Dictionary, Thesaurus. Free online reference, research & homework help. — Infoplease.com. Retrieved March 4, 2012, from http://www.infoplease.com/cig/biology/fluid-mosaic-model-membrane-structure-function.html
4. Melegos, D. N. (Director) (2012, February 13). The Fluid Mosaic Model of Cell Membranes. SBI4U1 - 70. Lecture conducted from Dunbarton H.S., Pickering.
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