An investigation to determine the effects that various temperatures have on beetroot membrane using colorimetry
Abstract/Aim
The aim of this experiment is to look at how temperature has an effect on the movement of pigments through a beetroot membrane, this will involve investigating how the membranes in the beetroot will split and leak the red pigment, anthocyanin.
The experiment conducted was to test the effects of various temperatures on a beetroot membrane. Previous studies have shown that the higher the temperature the more permeable the membrane will become. In plant cells proteins in the membrane act as a molecular signal that lets cells communicate with each other, the substances outside the cell, and within the cell wall.
In the experiment I will be paying particular attention to the roots of the beetroot. Beetroot is widely produced and consumed, healthy vegetable with many different health benefits. There is a vast array and many different varieties of beetroots the beetroot we mostly consume is known as ‘beta vulgaris’.
Hypothesis
Do various different temperatures affect a cell membranes permeability? It is thought that if the temperature increases then so does the beetroots cell membrane permeability, which can be seen by the optical density of the solution dipped with the beetroot sample.
The null hypothesis for this experiment is that even as the temperature increases, the permeability of the plasma membrane remains the same and so the optical density of light of all solutions is the same.
Prediction
My prediction for this experiment after researching this particular area and using what I’ve learnt in this topic is that as I increase the temperature of the water not a lot will actually happen below 40áµ’c, but once the temperature increases to 60áµ’ or higher the beetroot membrane will start to become unstable and the fluidity will change, as the lipids that make up the membranes will begin to lose their structure and become weak. This will then lead to the membrane edge becoming perforated, which will then eventually lead to the red pigment anthocyanin by diffusion, as the high concentration of the pigment inside the cell looks for a balance to the system the low concentration of the pigment outside. Whilst the temperature increases the membrane will breakdown at a rapid pace.

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Introduction
The dark red/purple pigment is a family of chemical compounds called ‘betalains’. The specific betalian found in the common beetroot is ‘betanin’, this is also found in some types of fungi. Also it is used as red food colouring, which has been found to have an anti-oxidant quality. The colour of betanin depends on its pH level. A yellow/brown colour indicates an alkaline pH level, whilst a deep red/purple colour indicates an acidic pH level.
Betalins can be found within the cells the ‘vacuole’ acts as a general storage area containing nutrients, ions and waste products. It also exerts ‘hydrostatic pressure’ which gives the cell structure. The vacuole is surrounded by a semi permeable membrane called the ‘tonoplast’ its main function is to separate the fluid filled vacuole from the cytoplasm surrounding it. Plant cell vacuoles are bigger than that of animal cells. There is a second membrane which surrounds the whole cell this is known as the ‘plasma membrane’. This again is semi permeable which is selectively permeable as to what comes in and out of the cell. It is made up of a bi-layer of ‘phospholipids’. The extrinsic layer have hydrophilic heads which attract water whilst the intrinsic layer have hydrophobic tails which deters water. In-between the bi-layer is fatty acid chains. Proteins are embedded in both extrinsic and intrinsic layers but can also cover through both layers exposed to both sides. These proteins exchange nutrients as well as communicating with other cells. The plasma membranes separates the contents within the cell from its outside environment.
Previous studies taken from “How Temperature Effects the Movement of Pigment through Cell Membranes.” 123HelpMe.com on temperature protein interactions have been recorded and results have shown that at normal temperatures proteins allow ions and nutrients into the cell, and lipids within the plasma membrane are ordered and organised. At increased raised temperatures lipids in the fatty acid chains begins to liquefy which increases the fluidity of the cell, the membrane then becomes more permeable, letting things in and out of the cell that it normally wouldn’t which will then denature (breakdown) the proteins within the cell. Also the speed increases within the cell. With low, decreased temperatures lipids in the fatty acid chains become more rigid, this decreases the fluidity o the cell and makes the membrane less permeable, which leads to vital nutrients and waste not being able to enter or leave the cell. Additionally cellular reactions decrease and become slower.
This experiment I will conduct will look at the different temperatures and how they affect the membrane. In general the effect temperature has on protein is that by being heated it denatures the tertiary structure of the proteins. The structure gives protein its function. When the beetroot is heated the pigment leaks out of the cell, indicating how permeable the cell is.
In the experiment I will be using a technique called ‘colorimetry’ which is used to determine the concentration of coloured compounds within a solution. In terms of temperature this is measured by a colorimeter. By using various different temperatures we can see what effects it has on the beetroot membrane. For this experiment I will be looking at temperatures of 20áµ’c 40áµ’c and 60áµ’ c. At increased temperature the phospholipid bi-layer greatly increases the permeability of the membrane, this should show the proteins staring to breakdown (denature) and lose their structure, this would mean they could no longer function effectively.
Once this part of the experiment has been done I will then look at the relevant beetroot membrane with a colorimeter. The main principles of a colorimeter are to measure the absorbance of light, this is vital in determining the concentration of a substance. For this I will use a Colorimeter these measure the amount of colour and light that’s is being transmitted. All colorimeters have a ‘light’ source i.e. a bulb, adjustable hole, coloured filters, cuvette which holds the relevant solution, a detector to measure light absorbed and a meter to display the reading. Colorimeters are quick and simple to set up and use and are very effective. Whilst researching this experiment I came across “principles of colorimetry” written by Rachel Predeepa (June 2011) she explains on the Ecology website that ‘beers law’ is a monochromatic light that passes through the coloured solution, the amount of light transmitted decreases exponentially with an increase in concentration of the coloured substance.” She goes onto explain that colorimetry is a technique used often in these types of biochemical experiments and investigations, and that it involves the quantitive estimation of colours.
Research
After looking at various sources such there are many experiments carried out within this area. The beetroot is probably the most popular one this is probably because beetroot contains the red pigment and they are soluble in water, and it’s the betanin in the beetroot that’s responsible for the red pigment. Also the beetroot is from the plant kingdom. The plant cell walls are much thicker than its plasma membrane, and ranges from 0.1µm to several micrometres. This could provide a challenge with the experiment and various temperatures.
The protein structure of the beetroot is what dictates its specificity and the tertiary structure (3 dimensional) is very important, The majority of the proteins are quite specific about the various tasks and the way they perform, if this structure is disturbed the protein would lose its functionality and would of then undergone denaturation. The proteins molecules are extremely important and carry out many important tasks.
When the beetroot experiments or any experiments carried out around temperature and permeability it seems that if a solution containing a protein is heated, it will reach a temperature that properties such as the absorption of ultraviolet light will change abruptly. This temperature is called the melting temperature of the protein. The melting temperature varies for different proteins, but temperatures above 41°C will break the interactions in many proteins and denature them. This temperature is not that much higher than normal body temperature (37°C), so this fact demonstrates how dangerous a high fever can be. Another example of heat-caused denaturation are the changes observed in the albumin protein of egg whites when they are cooked. When an egg is first cracked open, the “whites” are translucent and runny, but upon heating they harden and turn white. The change in viscosity and colour is an indication that the proteins have been denatured.
Planning
Before carrying out the experiments a full risk assessment was needed to ensure full safety was adhered to beforehand, during and after the experiment, this involved checking all equipment was in a safe place. There were no harmful chemicals being used so the care needed to be taken would be with some of the equipment such as scalpel and borer and then the temperature of the hot water. I will make sure that there is plenty of space around the area and safety goggles, gloves and a lab coat would be worn.
Whilst conducting this experiment it was important that I controlled the variables. On this occasion the independent variable is the part that you can control. In this case it was the temperature, volume of liquid being used the beetroot disc i.e. how many will I use, what size will they be etc., and the time taken,
The dependent variable was the estimated concentration of betalain in the tube. If these were not controlled at the right time it could of produce anomalies and the experiment probably wouldn’t have worked effectively.
For the experiment I will use a colorimeter as this measures the absorbance of different wavelengths of light in a solution. It can be used to measure the concentration of a known solute. I will also use beetroot discs which will be washed to remove and dirt and any leaked pigment and will also be cut to the same length to give an average result, the beetroot is being used because of the red pigment that is produced. I will need three test tubes per experiment as I will be testing three different temperatures, I will need a sharp piece of apparatus so I will use a scalpel and a borer, forceps will be used so I can gently place the beetroot into the test tubes. Finally I will need a sous vide cooking bath which is used to reheat prepared food which are contained in baths, this allows me to keep the relevant temperatures constant for the 30 minutes needed. I will be doing this 11 times to get a good average of results
Method

Cut pieces of beetroot using a cork borer and a scalpel
Place discs in running water for roughly 5 minutes to remove dirt and leaked pigment.
Prepare heated water baths (Sous vide) at 3 relevant temperatures i.e. 20áµ’c, 40áµ’c and 60áµ’c
Using forceps, place washed beetroot discs in the water baths for minimum of 30 minutes
Using three different test tubes each with 10cm of distilled water and leave for set time
Repeat for all three temperatures
After the discs have been distilled in water for 30 minutes transfer liquid into clean tubes leaving the discs behind
Before using colorimeter machine blank/zero it first to remove impurities of the machine
After looking at all 3 compare the colour of the liquids with the machine.
Repeat above measures ten times to get comparable results.

SAFETY: Take care carrying scalpels or knives around the laboratory. Always carry in a tray1
Results
First experiment

Temperature áµ’c

Test 10

20áµ’c

0.03

40áµ’c

0.15

60áµ’c

1.73

The next 10 experiments

Temperature

1

2

3

4

5

6

7

8

9

10

11

Mean

Range

20áµ’c

0.07

0.15

0.20

0.08

0.17

0.04

0.05

0.02

0.05

0.25

0.11

0.02-0.25

40áµ’c

0.44

0.88

0.82

0.27

1.34

0.24

0.26

0.13

0.22

0.59

0.73

0.13-1.34

60áµ’c

1.25

2.00

2.00

0.77

2.00

1.17

1.81

1.40

1.55

2.00

1.60

0.77-2.00

Evaluation
Looking at the results the numbers aren’t that far out compared to each other which shows that the variables were controlled well. The lower temperature of 20áµ’ c doesn’t really show any significant changes this s because the proteins are still present in the membrane, the integral part of the tonoplast is still intact and the pigment has no leakage.
When the temperature is increased to 40áµ’c the absorbance reading on the first result shows 0.15 whereas the average result came out as 0.73 again there is a slight difference. But again the temperature it still doesn’t affect the beetroot membrane. Once we hit 60áµ’ c the absorbance reading in the first experiment was 1.73 and the average result was 1.60 this however is slightly lower than the average result but again the variables were consistent and was still relatively close. At this temperature however a significant change occurs. Looking at these results it clearly shows that the membrane begins to break down (denature). As the water temperature increases the absorbency level increases. As said in the introduction by heating the beetroot membrane the pigment clearly starts to leak which makes it more permeable, the proteins start to ‘denature’ and they can no longer function effectively. Each temperature has a range of absorbance and so the higher temperature causes the membranes completely disappear. There is a limit to this though and even if you increased the temperature and used hotter water the colorimeter would only show up to a certain point and would hit its maximum reading. If we look at the overall results there are similarities and differences between the results even though the exact same experiment was carried out. As much as they had a similar range at each different temperature the range in results at 20áµ’ c was 0.02-0.25 at 40 c it ranged from 0.13-1.34 and then at 60áµ’ c ranged from 0.77-2.00. You would expect that all the values were the same if the same steps were carried out, but this wasn’t the case as each experiment produced a different absorbency level. If investigated further this could be because the beetroot was cut slightly bigger/smaller, larger discs would inevitably give more pigment volume, the beetroot may not have been washed as thoroughly the first time as the second or third time etc. or for a longer period as other. All of the above reasons could quite easily explain the difference but ultimately the range in the three different temperatures would produce similar results anyway.
All the independent variable were controlled, as said previously so when it came to the three different temperatures it was imperative that the baths i.e. sous vide were kept at the necessary temperatures for the 30 minutes needed I did a practice run beforehand and no problems occurred.
The temperature was measured with a thermometer to make sure the temperatures were exactly as they should be. The volume of liquids also had to be accurate as again any errors could make a crucial difference with the beetroot discs. I measured 10cm of water with a measuring jug, this could also cause problems if the water was from different sources i.e. if tap water was used or bottled water tap water may contain impurities and have different acidity levels so distilled water would be used. The beetroots used were from the same batch as if they were bought from different places or from different batches this could of produced difference results as they could have been grown in different temperatures, grown in different countries size could have been different this could have a minor effect on their anthocyanin concentration or the strength of the cell membranes was cut into separate discs and measured accurately, they will be cut with a scalpel and then a borer is used to remove the disc. The beetroot discs will be submerged in the test tube and in the heated water aids for 30 minutes, the time will be measured by a stopwatch.
The dependent variable is what is measured, in this case it will be the temperature, and this is going to be the main point of my analysis. The experiment will be repeated 10 times and by using a range of temperatures, 20áµ’c, 40áµ’c, and 60áµ’c I will then be able to find out which temperature the membranes begin to denature.
The above are all control variables however there is one variablethat, although slightly controlled we do not have total control over,this is the constancy of the temperature. Although it is the dependantvariable it is also the one which we have 100% control of, thisis because as we heat the distilled water to the desired temperaturethe water automatically begins to cool therefore my own judgement hasto be reached as to when to put the beetroot cylinder into the heated water. If the beetroot is put into the water a few degrees above thedesired temperature then as it cools the membranes will begin tofracture at the right temperature or as close to there as possible.Water baths would have been preferred for the experiment however I didn’t have access to these so I sous vide was used. The room temperature mustalso be considered as an independent variable.
In future when carrying out this experiment I could make sure the beetroot discs are measured more accurately and the comparable data could be made from a wider sample, also a range of temperature may show more evidence, but as said previously the lower temperature i.e. less than 40 c will not affect the beetroot membrane and will be intact and anything over 40 c will inevitably break down the membrane and prevent it from functioning effectively. In comparison with other studies such as beers law that was mentioned in the introduction the experiment clearly sets out a clear relationship between temperature and protein interactions.
Conclusion
The conclusion is that the beetroot membrane does become denatured and fails to function effectively when a temperature above 40áµ’c degrees is applied
References
Predeepa, Rachel (2011) Principles of colorimetry Available at: https://Ecoplants.wordpress.com (Accessed 1 February 2015).
Ursula, Hinz How temperature effects the movement of pigment through cell membranes. Available at: http://www.123helpme.com/view. (Accessed 1 Mar 2015)
Ariel G. Loewy & Philip Siekevitz. – Cell Structure and function, 2nd Edition Ursula Hinz – University lecturer (web-site Accessed 15th Feb 2015)
Branden, C., and Tooze, J. (1998). Introduction to Protein Structures, 2nd edition. London: Taylor and Francis.
Campbell, Neil A. (1990) Biology, 2nd edn. The Benjamin/Cummings publishing company, inc. Redwood City CA
Creighton, T. E. (1993). Proteins, 2nd edition. New York: Freeman.
Fagain, C. O. (1997). Protein Stability and Stabilization of Protein Function. Georgetown, TX: Landes Bioscience.