Beta Vulgaris Root Tonoplast and Cell Membrane Permeability Increase Through Immersion in Solutions of; Low pH, Extreme Temperature, and of High Solvent Concentration.

Beta Vulgaris Root Tonoplast and Cell Membrane Permeability Increase Through Immersion in Solutions of; Low pH, Extreme Temperature, and of High Solvent Concentration

Eric Horton

March 5^th 2012

SBI 4U1 – 70

Prepared for D. Melegos

Abstract

            This manuscript explores the affects of various stresses on the permeability of cell membranes and tonoplasts. Some background information will be provided covering various topics including the fluid mosaic model, free radicals, antioxidants, betanin, spectrophotometry and Beer’s law. The experimental approach for the lab will be highlighted along with the objectives and anticipated results. A clear outline of the methodology followed will be covered followed by the results obtained through the lab. The results will be explained along with sources of error present during the lab and future lab prospects.      

 Introduction

In order to understand the logic behind the methodology of this lab, and understand the results, it is important to have a firm grasp of a few preliminary concepts. For example, the fluid mosaic model, seen as figure 1 in the appendix section, is the most widely accepted model used to describe the composition of biological membranes. The term mosaic implies the nature of the membrane, showing how it is composed of many different structures that work side by side without creating an intermediate substance ^[1]. In general, the structure of biological membranes consists of a lipid bilayer, with impeded proteins ^[1]. The term fluid mosaic implies the presence of lateral motion within the membrane. The proteins float in the lipid bilayer, free to move along the plane of the membrane ^[1]. Other components of the membrane include oligosaccharide chains, membrane proteins and cholesterol ^[1]. Understanding the composition of the cell membrane is key to understanding what stresses will be the most successful in altering the permeability of the Beta vulgaris’ cell membranes.

Free radicals are very unstable molecules that possess an odd unpaired electron. This is a result of a bond splitting, leaving the odd unpaired electron as depicted in figure 2. As a result of their unstable nature, free radicals react quickly with other compounds attempting to capture the needed electron, regaining stability. This means that free radicals tend to attack the nearest stable molecule in order to “steal” an electron, leaving that molecule as a free radical. This creates a chain reaction that eventually can disrupt the living cell. Free radicals can be created by your body naturally during metabolism. Sometimes the body’s immune systems cells purposely create free radicals in order to neutralize viruses and bacteria present in the body. On the other hand, environmental factors the body is exposed to such as pollution, radiation, cigarette smoke and herbicides seen in figure 3 can cause free radical formation. Interestingly enough, Free radical damage tends to accumulate with age. ^[2]

 Antioxidants are substances such as vitamin C or E that remove potentially damaging oxidizing agents from a living organism. Vitamins C and E are thought to protect the body against the damaging effects of free radicals. These antioxidants neutralize free radicals by donating one of their own electrons. This puts an end to the “electron stealing” chain reaction created by free radicals. Antioxidant nutrients don’t become free radicals themselves after donating an electron because they are stable in both forms. Their unique properties help prevent cell and tissue damage that could lead to disease. Vitamin E tends to protect against cardiovascular disease by defending against LDL oxidation and artery-clogging plaque formation. Many studies have linked high vitamin C intakes with lower rates of cancer, particularly cancers of the mouth, larynx and esophogus. ^[2]

Betanin is a food additive used to give a red colour to many food products including meat, ice cream and various beverages. Betanin is found in red beats or Beta vulgaris and is a type of antioxidant. Being an antioxidant, the consumption of betanin provides many benefits against oxidative and stress related disorders. Betanin is also known to increase the red blood cell count in the human body and maintain a proper metabolism. The flow chart labelled figure 5 in the appendix section shows the complex series of steps followed during the biosynthesis of betanin. Betanin has a structure as seen in figure 4, which consists of a great deal of nitrate, which can also be hazardous to human health in high doses. ^[3]

Spectrophotometry is the measurement of a solutions colour by determining the amount of light absorbed in the ultraviolet, infared, or invisible spectrum, used to calculate the concentration of a substance in solution ^[4]. When a beam of incident light passes through a solution, part of the light is reflected, part is absorbed and the rest is transmitted. The relationship that exists between the concentration of a compound and the extent of absorption of light is based on Lambert’s Law and Beer’s Law ^[5].  According to Lambert’s law, the light absorbed by a solution is directly proportional to the length of the light path through the solution ^[5]. According to Beer’s law, the amount of light absorbed is directly proportional to the concentration of absorbing solute in the solution ^[5]. During this lab, spectrophotometry will be a key tool used to determine the relative effectiveness of each stress on the Beta vulgaris’ cellular membrane.

The objective of this lab was to investigate the extracellular concentrations of betanin, and correlate it with the cell membrane/tonoplast permeability. Factors used to test the viability of these cell parts will include the determination of the optimal pH, temperature, and solvent. These results will be quantified using a spectrophotometer.

The approach taken with this experiment involves various controls coupled with isolation of the various factors. In order to test for the optimal pH value for cell membrane permeability, five beet root disks were immersed for 10 minutes in a test tube containing one of the six solutions, with pH’s of 2,4,6,7,10 and 12. Within this portion of the experiment, water (pH of 7) acts as a control of sorts. This “neutral” pH should have little to no effect on the membrane.

For the second portion of the experiment, temperature was isolated as a factor affecting the membrane permeability. Five different tests were run, one with five disks inserted into a test tube with water at a temperature of -18⁰ C, the second set in a test tube with water at 4⁰ C, the third set was placed in water at 21⁰ C, the fourth set was put in water at 40⁰ C and the final five disks were put in water at 80⁰ C. All of these solutions were left to incubate for 10 minutes. The room temperature water in this case (21⁰ C) acted as a control.

For the final variable, five beet root disks were put into each of the six test tubes containing various solvents of varying concentrations for 10 minutes. The solutions used included; 1% acetone, 25% acetone, 50% acetone, 1% methanol, 25% methanol, 50% methanol.

After the incubation time for each of the samples was complete, the extracellular fluid from each test tube was poured into a small cuvette and then inserted into the spectrophotometer. The resulting absorbance values were examined and a conclusion was drawn.

It is anticipated that the greatest absorbance will occur when the cells have been submitted to the harsher stresses. Various high and very low temperatures should return a high absorbance value, along with the higher concentrations of solvents. High and low pH’s should also cause greater cell membrane and tonoplast permeability,

resulting in higher absorbance values. The more moderate conditions aren’t as likely to increase the cells permeability and in turn should have low absorbance values.

Methodology

The methodology for this lab was broken down into several steps. The first step involved the preparation of the Beta vulgaris root for testing. The beet was prepared by slicing the top and bottom off of a fresh beet. Using a cork borer, a core sample of the root was extracted. After driving the borer into the beet, the tweezers were used to remove the core sample. The cylindrical root section was then sliced into small 3mm thick disks using a scalpel. For our section of the experiment, fifteen disks were prepared. Once all the cutting was complete, the disks were put into a beaker and then immersed in tap water. The beaker was then agitated, swirled and then emptied. This process was repeated three times in order to adequately rinse the contents of the burst cells from the samples.

In order to test the permeability of the cells under various temperature conditions, five pre cut and rinsed disks were placed in the freezer at a temperature of eighteen degrees below zero Celsius. Five disks were also placed in the fridge at a temperature of four degrees Celsius. Five disks from the freezer were then put into a large test tube, five disks from the fridge were placed in another large test tube, and then the five of freshly cut disks were placed into each of the three remaining test tubes. Using a graduated cylinder, 10ml of tap water was measured out and added to each of the five test tubes. Each of the samples was incubated for 10 minutes. The samples containing the disks from the fridge and freezer were incubated at room temperature along with the 21⁰ C sample. The 40⁰C and 80⁰C samples were incubated in water baths at their assigned temperatures.

In order to investigate the relationship between membrane and tonoplast permeability and varying pH, a series of steps were followed. As for the previous test, 25 3mm beet root disks were prepared using the cork borer and scalpel. 5 disks were placed into each of the six clean test tubes prepared. Each test tube was labelled using masking tape and a permanent marker with the pH value of either 2,4,6,7,10 or 12. Then 10mL of each solution of a varying pH was measured using a graduated cylinder, and then added to the corresponding test tube.

As for the test involving pH, a key sequence of steps was followed in order to test the effect of various solvents of numerous concentrations on membrane and tonoplast permeability. 25 3mm beat root disks were prepared using a cork borer and a scalpel. 5 disks were placed in each of the 6 test tubes. Each test tube was labelled with one of the following solvents and concentration; 1% acetone, 25% acetone, 50% acetone 1% methanol, 25% methanol, 50% methanol. Then 10ml of each solvent was measured using a graduated cylinder and added to the corresponding test tube.

All of the samples were then allowed to incubate for 10 minutes, some at specific temperatures, and the rest at room temperature. After the 10 minute incubation period, the samples were gently agitated and then the liquid portion was poured into small test tubes. The spectrophotometer was then set to 560nm and calibrated using a test tube containing tap water. Once calibrated, each of the five small test tubes were placed in the spectrophotometer and their absorbance readings were recorded in table 1 in the appendix section.

Results

            After conducting the above experiment, a clear and relatively definitive set or results were obtained. As seen in table 1, it was clearly observed that as the different stresses were applied, varying absorbance values resulted. With regards to pH, the lower acidic pH values returned higher absorbance values than the moderate to basic pH solutions. At a pH of 2, the highest absorbance value of 0.52 was observed compared to the 0.02 value at the pH of water and 0.05 at a pH of 12. This clearly indicates that the cell membranes and tonoplasts are significantly more permeable in acidic solutions. As indicated by chart 1 found in the appendix section, a clear logarithmic trend associating increasing pH with reduced absorbance values can be observed.

            The temperature related stress results were rather interesting as well. Table 1 indicates that the cell membrane and tonoplast are the most permeable at extreme temperatures. At -18⁰ C an absorbance value of 1.61 is observed and at 80⁰ C an absorbance value of 1.05 results. These values contrast the absorbencies indicated at a more moderate temperature of 21⁰ C where absorbance values of 0.04 were observed. Through plotting this data on chart 2, a parabolic trend is observed, this indicates that extreme temperatures provide the most stress to a cells membrane and tonoplasts.

            In the final test, it was noted that with increasing the concentration of solvent used in the test, greater membrane and tonoplast permeability was achieved. The absorbencies for this test ranged from 0.02 for the 1% acetone solution, to 0.46 for the 50% acetone solution. For the methanol, 0.02 absorbance values was observed at a 1% concentration, and at a 50% concentration a 0.32 absorbance value was found to exist. Through the exponential trends illustrated on chart 3 it became clear that not only as the concentration increased the absorbency increased, but the acetone was more effective than methanol at creating permeability.

Discussion     

            The results obtained through the series of test conducted can be justified using some basic knowledge of cell membranes. The results of the temperature portion of the lab can be explained using the properties of cell membranes. When the cell is submitted to high temperatures, the phospholipids become increasingly fluid, increasing the permeability of the membrane. This allows materials previously unable to travel in and out of the cell to do so. The extremely high temperatures can also break the hydrogen bonds found in the protein structure which could cause protein de-naturation. When the cell is frozen, the membrane bursts because of the ice crystals and extreme temperatures make the cell brittle, causing it to fracture. This allows an increase in permeability as well.

When subjected to a solution consisting of a solvent the membrane also becomes more permeable. Acetone and methanol are both polar solvents. The phospholipids bilayer is also polar, meaning they will tend to combine easily. Solvents denature the proteins found in the phospholipids bilayer, meaning the sulphide bridges between cysteine amino acids found in the quaternary and tertiary may be disrupted. The alteration to the proteins structure stops them from being able to function correctly. This protein alteration creates gaps in the cell membrane that allow for increased permeability. As the concentration of the solvents increased, this effect is further magnified. Acetone has a greater effect on the cell membrane because acetone has a greater molecular dipole (2.91) than that of methanol (1.69). The difference in polarity makes acetone a stronger solvent, resulting in a more significant effect on the cell membrane. 

pH also has a similar affect on the cell membrane. With the increase in acidity during the pH stress test, protein denaturation occurs as well. The change in pH tends to disrupt the ionic bonds found in the proteins tertiary and quaternary structures. These disruption cause changes to the shape of the protein and as a result of this the protein is denatured. Changes to the proteins composition can cause an increase in the membrane permeability.

 Along with the increase in permeability through each of the stresses, comes an increase in the absorbency. As the cell membrane becomes increasingly permeable, the more betanin is released into the extracellular fluid. With the increase in betanin release, there is an increase in the absorbency measured using the spectrophotometer.  

Through conducting this lab several sources of error became evident. During the temperature stress test, the incubation time was supposed to be 10 minutes. However, by the time the water was added to each of the test tubes, and each test tube made it to its designated temperature environment, the times were skewed. With the present lab set up it was impossible to incubate each of the samples for the same period of time. The difference in incubation time could alter the absorbency values significantly. This could be remedied by setting separate timers for each of the test tubes incubation. Another source of error that became evident was the insertion of the frozen or chilled beet disks into room temperature water. This would moderate the temperature during incubation, rather than having a consistent temperature. This problem however is somewhat unavoidable, unless that water was made the same temp as the disk. This would pose a problem for the frozen disk test however. The final source of error that was evident during the lab was the consistency in the size of beet disks used. There was no step taken to ensure absolute accuracy with respect to disk size. Different disk sizes could contribute to large or fewer available cells and therefore lesser or greater absorbency values. This problem could be fixed using a tool that cuts consistent disk sizes. An example of this may include a cheese slicer. A consistent disk size would make for far more accurate results.  

A potential lab proposal may be one that incorporates a comparison between polar and non polar solvents. Despite its carcinogenic nature, benzene would be a good non polar solvent that could be compared to acetone and methanol. 3 test tubes, one with 1% concentration the others 25% and 50% respectively. The results of this test could be graphed with the results of the polar solvent test. This would give insight into the effects of the disruption of the London forces between non polar amino acids. Another potential lab proposal might involve the isolation of betanin in order to explore its abilities as an antioxidant. One might consider introducing free radicals into a group of plant or animals and observing the effects. Then a comparison might be made with the results after antioxidants are added to the samples. This would allow us to verify and explore the importance of antioxidants in our diet.

References

[1] Campbell, M. K., & Farrell, S. O. (2011). Lipids and Proteins are Associated in Biological Membranes. Biochemistry (7th ed., pp. 207,208). Belmont: Brooks/Cole.

[5] Chauhan, B. (2008). Principles of Biochemistry and Biophysics. Bangalore, New Delhi: University Science Press.

[2] Understanding Free Radicals and Antioxidants. (n.d.). HealthCheck Systems. Retrieved March 3, 2012, from http://www.healthchecksystems.com/antioxid.htm

[3] Walker, M. (2011, February 25). What is Betanin Used For? Health Benefits and Side Effects of Betanin. Kay Circle | Everyday Reference. Retrieved March 3, 2012, from http://www.kaycircle.com/What-is-Betanin-Used-For-Health-Benefits-and-Side-Effects-of-Betanin

[4] spectrophotometry - definition of spectrophotometry in the Medical dictionary - by the Free Online Medical Dictionary, Thesaurus and Encyclopedia.. (n.d.). Medical Dictionary. Retrieved March 3, 2012, from http://medical-dictionary.thefreedictionary.com/spectrophotometry