To Determine the Effects of Temperature on the Permeability of the Beetroot Cell Membrane

Research Question: To investigate how different temperatures affect the beetroot pigment and finding out the absorbance and transmittance % using a visible spectrophotometer.

Background Information: Beetroots contain betalains which are the red pigments present in the cell vacuole. Betalains are soluble in water and they contain nitrogen. Betalains extracted from beetroot is commonly used as food dye because it is not known to cause any allergic reactions.

Cell membrane is the barrier that separates the inner environment of the cell from the outer environment. The membrane is selectively permeable. The cell membrane is made up of mainly lipids, carbohydrates and proteins. The lipids are the phospholipid bilayer which consists of a hydrophilic polar head and a hydrophobic tail. Among the proteins present in the cell membranes are integral proteins and peripheral proteins. {1}

Lipids increase in fluidity as temperature increases. Once denatured, proteins start to unravel and are unable to carry out their function. Diffusion is the movement of molecules from high concentration to a low concentration down a concentration gradient. Facilitated diffusion and active transport requires assistance from proteins imbedded in the cell membrane to transport substances in and out of the cell. Any damage to the cell membrane will cause the cytoplasm to leak out of the cell. Energy for the movement of molecules comes from the kinetic energy of molecules. Cell membranes are composed of phospholipids and proteins, which contribute to the selectively permeable nature of the membrane. Small, uncharged molecules can diffuse across the membrane, while certain other substances must be transported across the membrane by proteins. Since the membrane is impervious to some substances except through channel proteins, important solutes can be retained. Beet cells, which are plant cells, contain a water-soluble pigment called betacyanin that is stored in the vacuole. {2}

A spectrophotometer can measure the absorbance of a solution. As more betacyanin leaks out into the extracellular luid, the solution will absorb more light. Since the spectrophotometer reading is proportional to the leakage of pigments from the beet cells, the absorbance of the solution can be used to evaluate the level of permeability of the membrane in beet cells. Students will be expected to incorporate the use of the spectrophotometer into their experimental design to measure the intensity of color in the surrounding environment. The intensity of color should be proportional to the amount of damage sustained by the beet cell membrane. {3}

With increasing temperature, the structural integrity of the cellular membrane is compromised because the proteins that make up the plasma membrane are denatured and lose their shape. Secondly, the effects of temperature extremes like extreme cold and heat were tested on the permeability of the beet root membrane.

It has to do with the fluidity of the membrane. If the temperature is warmer, the membrane will be more fluid and so more permeable (also a reason why bacteria can be killed by high heat, the membrane gets TOO fluid and ruptures). The lower the temperature, the less fluid the membrane becomes and so the less permeable the membrane is. Other things, such as saturated or unsaturated fatty acids in the lipid membrane part also affect fluidity. Beetroot tissue will be used as the model to investigate the types of environmental stresses that affect membrane integrity. {4}

Reference:

{1} http://s3.frederickding.com/shared/school/beet-lab.pdf

{2} http://www.studymode.com/essays/The-Effect-Of-Temperature-On-Membrane-447197.html

{3} http://lawrencekok.blogspot.sg/2011/02/absorbance-of-beetroot-pigment-using.html

{4} http://rebekah-lee.wikispaces.com/02a-Beetroot+Experiment

Literature Graphs:

Hypothesis:

1. If the temperature is increased, then the membrane will become more permeable and the absorbance will be greater because the extra energy of higher temperatures disrupts the structure of proteins in the membrane; the absorbance reading is directly proportional to the greater concentration of leaked pigments in the solution.

2. Our hypothesis of the study was that as the amount of damage increased, the more betacyanin would be released. The betacyanin would absorb light of a specific wavelength and we would be able to measure absorbance maximally.

3. If the temperature is increased, then the membrane will become more permeable and the absorbance will be greater, because the extra energy of higher temperatures disrupts the structure of proteins in the membrane.

4. The absorbance reading is directly proportional to the greater concentration of leaked pigments in the solution.

5. The extreme temperatures that the beet cell membranes were put through should have caused denaturation of the proteins that make up the membrane and allow the purple-red pigment bound within the plasma membrane to leak out of the cell.

1. Temp. ranging 20° C to 30° C- The rate of diffusion would speed up due to the low heat, thus increasing the cell permeability.

2. Temp. ranging 30° C to 40° C – Vital proteins such as betacyanin and phospholipids will start to break down, hence making the membrane more permeable than before.

3. Temp. ranging 40° C to 50° C – Integrity of the cell membrane becomes compromised, as betacyanin and other proteins become liquids, increasing the cell permeability.

4. Temp. ranging 50° C to 60° C – Betacyanin and other proteins start leaking out, causing gaps in the membrane. Cell permeability continues increasing.

Variables:

Variables

Description

How to control it

Independent

Temperature

Experiment should be conducted in the lab at a constant room temperature, which is approximately 25° C.

We will be changing the temperature ranges 4 times.

Dependent

1. Absorbance rate

2.% of Transmittance ray

1. Maximum wavelength should be fixed, because the absorbance is measured relatively to the maximum wavelength. The maximum absorbance is determined by the value of the maximum wavelength.

2. Results will be converted from qualitative to quantitative depending on the independent variable, using a spectrophotometer, which will measure the % light transmission through the different temperatures.

Controlled

1.Time

2. Wavelength maximum

3. Size, colour and type of beetroot

4. Mass of beetroot

5.Temperature

1. Specific timings should for all trials. Trials should be simultaneously stopped by taking out beetroot pieces at the same time.

2. Absorbance will be measured relatively to the same at the maximum wavelength. That is how the maximum absorbance is determined.

3. Beetroot pieces of identical shape and size should be prepared. They should have the same colour so that there is no difference in the colour of the dye. Only the middle part of the beetroot should be used. Same beetroot is used for all trials.

4. The mass should always remain constant. Because as size of beetroot decreases, surface area is more and thus permeability is more. We should take the beetroot cores which have approximately the same masses.

5. During the experiments, the lids on the water baths will be removed and replaced. This may have an effect on the actual temperatures inside them and could mean a deviation from the originally planned temperatures.

Apparatus:

1. Spectrophotometer

2. 5 test tubes

3. 5 water baths (20oC, 30oC, 40oC, 50oC, 60oC)

4. Hot water

5. Ice

6. Labels

7. Stopwatch

8. Glass stirring rod

1. Mounting needle/forceps to handle beetroot cores

2. Syringe

1. Thermometers for each water bath

2. 5 cuvettes

Procedure:

1. Obtain the beetroot cores from the lab technician.

2. We put each of the beetroot cores in each of test tubes.

3. Using a syringe, we fill up 10 ml of water and pour it into the test tubes.

4. Meanwhile prepare the water baths (20oC, 30oC, 40oC, 50oC, 60oC).

5. When the water baths are ready, place each test tube in each of the water baths. Have your stopwatches ready.

6. Keep the test tubes for exactly 10 minutes in the water baths.

7. After 10 minutes, take out the test tubes, discard the beetroot cores and pour out its contents, the pigmented water, into the cuvette.

8. Ask the lab technician to help you calibrate and warm up the spectrophotometer to 480.5nm

9. Using one cuvette for each trial, measure out the absorbance and transmittance %.

10. Repeat steps 1-9 for all trials.

11. Record your observations in a table.

Precautions:

1. Make sure you don’t spill out anything.

2. Beware of the hot water baths; use a test tube holder to take out the test tube from the hot water baths.

3. Be cautious when using the knife to cut out the beetroot cores.

4. Report any spills so they may be cleaned up.

5. After the experiment, all the apparatus should be cleaned and wiped properly. Equipments must be stored back to their respective places.

6. Before starting the experiment, we should just wipe the apparatus to ensure zero error.

7. Do not overtime the experiment.

8. When the beetroot was cut some of the membrane was broken causing the pigment to leak out this must be thoroughly cleaned and washed off to secure the accuracy of results.

Data Presentation:

Conclusion: Higher temperatures also lead to increases in absorbance at which the cell loses a large amount of its betacyanin. The hypothesis that increases in temperature lead to increases in membrane permeability has been shown to be valid. As the size of beetroot gets smaller, surface area is more, therefore permeability is more. Denser the colour, more pigment is present. If absorbance is higher, transmittance ray is lower and vice versa. The effect of temperature on membrane permeability is generally that increased temperatures cause more leakage through the membrane, with the most notable exception being the temperatures close to the freezing point of water. For most of the temperature range, higher temperatures appeared to effect greater permeability, hence higher betacyanin concentration in the solution, and thus greater absorbance.

The membrane becomes more permeable at higher temperatures. This was explained by the fact that proteins denature with high temperatures and the phospholipids structure changes and becomes less stable. When the temperature rises, the molecules have increased kinetic energy which means that the phospholipids and other substances move around more, making the membrane more ‘leaky’ which means that substances that wouldn’t usually do so can enter and leave the cell. As long as the temperature does not go beyond what the membrane is supposed to withstand, the permeability of the plasma membrane should not be affected. Water expands, putting pressure on the membranes from within. The lipid part of the membrane liquefies, making it more prone to leakage. The proteins that span the membrane fall apart, creating holes in the fabric. All this combined will allow compounds to exit the cell.

Higher temperature makes all molecules shake and vibrate more. The faster movement disrupts any ordered structure there might have been, eventually destroying the structure altogether. The absorbance increases with a raise in temperature while the transmittance is quite the opposite it decreases with an increase in temperature.

Evaluation:

Error

How will it affect?

Rectification

1. Different beetroots used for taking out cores

We might have obtained cores of different beetroots, and since different beetroots have different amount of pigments, it might lead to variation in the colour of pigmented water.

We obtain cores of the same beetroot, or the same colour, size and type. If the colour is same then it’s still acceptable, as the pigmented water will have the respective colours for the respective temperatures.

2. Inability to maintain temperature of water baths

Many times we were unable to follow the temperature trail, which would result in a varied membrane absorbance, with the temperature drop when all test tubes were put in the water baths, hence giving us inaccurate results.

3. Spilling of liquids

Most if the coloured pigment water may spill out, hence not giving us the respective colours, and will result in inaccurate spectrophotometer readings which acts as major error instigator.

Be more careful in handling the liquids to prevent spilling of anything. If anything does spill, redo the entire experiment again.

4.Timekeeping

The test tubes of beetroot cores were places a little too early or a little too late. As the amount of time increases, the amount of diffused pigment will also increase. Overshooting of time for the test tubes in the water baths will have resulted in accumulated errors.

Coordinate efforts and place the test tubes in the water baths at the same time to reduce errors as mentioned above. Stay to your time limit, and never overshoot the time.

5. Confusion to procedure

Many times confusion in procedure will contribute lots of errors to the readings.

Come prepared and read the procedure and precautions before you start the lab. If there is any confusion, do not hesitate to ask the lab assistant.

6. Large margins between different trials

7. The pH of the water

Most of the times tap water is used, and tap water contains a lot of impurities, which affect the permeability of the membrane.

Use of distilled water to prevent impurities that might alter the pH.