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Permeability Beet Lab

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Brianna Bader

on 19 June 2012

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Transcript of Permeability Beet Lab

Determining the Degradation of a Root Cell Membrane for the Beta Vulgaris through the Absorbance while placed in Various Temperatures
Introduction
S.J. Singer and Garth Nicolson in 1972, described the structural features of a biological membrane, discovering the fluid mosaic model. [1] The fluid mosaic model or plasma membrane, is a biological membrane that separates’ the interior of all cells from the outside environment. The cell membrane contains a monolayer arrangement, of a hydrophobic fatty acid tail, and hydrophilic phosphate head. The head of the phospholipids is polar, but the tail is non-polar.
This mono-layer makes up a phospholipid bilayer arrangement. This is formed spontaneously when placed in water. The bilayer does not accept large charges polar molecules to pass through [2]. Proteins in the bilayer also help with the passageways. The organization in the fluid mosaic model, with the phospholipids, proteins, carbohydrates, and glycolipids help allow molecules needed to pass through and absorb nutrients. They also help protect the membrane, and keep it warm with the fat carbohydrates.
Free radicals are unstable and incomplete atoms, which steal electrons from other molecules to remain stables. A product of oxidation, helping with our metabolism, digestion, and energy, also in taking more oxygen would produce more free radicals. Although they exist naturally within our body, having to many can cause harm to our bodies (eg. cancer) [4]. Antioxidants is a molecule in which can possibly help to prevent cancer. From the damaged caused by the free radicals, antioxidants can help repair the cell before it becomes damaged producing cancerous cells [3].
Abstract
This lab was performed to test the permeability of the cell membrane of a beet using various and extreme temperatures (high and low). The prediction of this lab is testing how much stress the cell membrane can withstand under different polarities and strengths of bonds using our understanding of the fluid-mosaic model. A spectrophotometer was used to calculate the absorbance of each variable after the beet was placed in each environment. When the lab was completed, the data stated that under each different reactant and condition the cell membrane showed multiple changes in tolerability of the beets.

Betanin, a red food dye and antioxidant, is water soluble and comes from the red beet (more specifically betacyanin). When subjected to different conditions the betacyanin can leak from the beet cell. Betanin is sensitive to heat, light and oxygen and deteriorates when in the presence of them [5]. Therefore, betanin is commonly used in non-perisable food items, powders and frozen foods. It is also known to be an anti-oxidant when absorbed in the stomach [5]. Its’ molecular formula is C24H27N2O13 and has a molecular mass of 550.46884 g/mol and is considered to be quite large in the betalain family [7]. 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[8]. 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.
A spectrophotometer is a machine that is used to measure the amount of light that a sample absorbs. The machine beams a light through the test tube (where the sample is held) and measures the amount of light that passes through the other side. Beer’s Law relates the amount of light absorbed by a solution to the concentration of a specific substance dissolved in the solution. [6]
The objective is to test the tolerability of the cell membrane in a beet. Under what pH, temperature and solvent is the cell membrane the weakest and strongest? With high temperature and very low temperature we anticipate that there will also be more red dye shown in the sample do to the shock it is placed in.
Methodology
Discussion
References
[1]"Fluid Mosaic Model." Biology-Online.org. Web. 03 Mar. 2012. <http://www.biology-online.org/dictionary/Fluid_mosaic_model>.

[2]"Fluid Mosaic Model." Wikipedia. Wikimedia Foundation, 03 Apr. 2012. Web. 04 Mar. 2012. <http://en.wikipedia.org/wiki/Fluid_mosaic_model>.

[4] Mithra, S., and L. S. Wynn. "What Are Free Radicals?" WiseGeek. Conjecture, 22 Dec. 2011. Web. 03 Mar. 2012. <http://www.wisegeek.com/what-are-free-radicals.htm>.

[3]"Antioxidant." Wikipedia. Wikimedia Foundation, 18 Mar. 2012. Web. 03 Mar. 2012. <http://en.wikipedia.org/wiki/Antioxidant>.

[5] "Betanin." Wikipedia. Wikimedia Foundation, 18 Mar. 2012. Web. 04 Mar. 2012. <http://en.wikipedia.org/wiki/Betanin>.

[6] http://www.chm.davidson.edu/vce/spectrophotometry/index.html

[7] Betanine - PubChem. (n.d.). The PubChem Project. Retrieved March 5, 2012, from http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=91559#x291

[8] Pathway: betacyanin biosynthesis. (n.d.). MetaCyc Database. Retrieved March 5, 10. Pathway: betacyanin biosynthesis. (n.d.). MetaCyc Database. Retrieved March 5, 2012, from ecocyc.org/META/NEW-IMAGE?type=PATHWAY&object=PWY-5399&detail-level=2

Images:

http://homepage.smc.edu/wissmann_paul/anatomy2textbook/phospholipids.html

http://apbrwww5.apsu.edu/thompsonj/Anatomy%20&%20Physiology/2010/2010%20Exam%20Reviews/Exam%201%20Review/Ch03%20The%20Cell%20and%20Membrane%20Structure.htm
By Stephanie Gertsakis
and Brianna Bader
First we took the beet and cut off the top and bottom section and placed it flat on the cutting board. We then took a cylindrical tool and punched it through the beet, creating a circular piece of the vegetable. Taking that piece, we cut thirty small slices of the beet, approximately half a centimeter thick. We then placed those slices into a large beaker and rinsed the sample thoroughly three times until the water was clear with no red dye. Five slices were each placed into five test tubes with tweezers (no water included).
We tested the beets with temperatures ranging at -18°, 4°, 21°, 40°, and 80°. Each test tube was set into their designated temperatures for ten minutes. After the time, 10 mL of distilled water was added to the test tubes. They were then agitated before 5 mL of each sample’s fluid was placed into a smaller test tube.
This smaller test tube was placed, one by one, into the spectrophotometer and results were recorded. The machine was set at 560 nm and at 0% light absorbance (recorded with just water).
Results
Red dye was best extracted under absolute high (80°) and absolute low (-18°) temperatures.
The results were found by how much absorbance each sample detected; the higher the absorbance means the more red dye was leaked. At about room temperature the absorbance was lowest.
Temperature Absorbance
-18 0.66
4 0.21
21 0.01
40 0.07
80 0.38
The predictions we made were right based on what we understood about the cell membrane and fluid-mosaic model. Knowing that cell membranes are being a part of our living system, you can guess that they are susceptible to changing when put under different environmental conditions. Membranes are most often used in osmosis and functions that are related to it are affected. Like most parts of the biological system, it can only stand so much change, and has a small range of function when subjected to temperatures, solvents and pH’s.
The different levels of pH affect the pigment in the beet root because the proteins are denatured depending on if they are subjected to the extremes of pH. The more acidic the levels get, the more the proteins are damaged, and meaning more of the red dye is excreted. When proteins become denatured they become useless and do not take up their usual activity. Even if subjected to the extreme level of alkalinity, the structure will still be compromised because it will have become damaged too, but not as much as the extreme acid. That is why in the experiment pH 12 is a higher absorbance than pH 10.
When a cell membrane is put under different temperatures, it becomes compromised in the extremes. The phospholipid bilayer is able to move and adapt. When put under high temperatures, the phospholipids move apart and behave like a fluid, and when this happens there is no barrier. This results in cytoplasm and other cell components being able to escape, thus being able to see more betacyanin in the sample. When put under very low temperatures, -18°, just like a human system, phospholipids become closer together and instead of being able to move they become stiff. When this happens, they are in danger of shattering or becoming damaged. Thus again why you are able to see more red dye.
Acetone and methanol are two organic solvents that are able to dissolve hydrophobic properties like phospholipids because they are hydrophilic. If phospholipids are broken down, the cell membrane has changed and it is more easily manipulated. More molecules are able to get in and out of the cell, thus the leakage of betacyanin. Acetone is less hydrophilic than methanol, and because cell membranes are composed primarily of lipid (phospholipids), the acetone is able to interact with and disrupt the lipid core of the phopholipid bi-layer more easily. In the experiment this is shown due to the higher absorbance with 50% Acetone than 50% Methanol.
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 minutes while others were not left long enough. With the proper machine to load all six tubes at once, this lab could have been timed better.
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. Slight variations in temperature between different samples could affect the results.
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 much 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 machine to cut the perfect size of beet it could be guaranteed that each group would be working with equivalent beet product.
Spectophotometer
Heat Incubator
Refridgerated
Room Temp.
80 degrees
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