2.3 Core Practical 3: Investigating Membrane Structure & Permeability

• The permeability of cell membranes is affected by several factors such as
  • Temperature
  • pH
• The effect of such factors on membrane structure and permeability can be investigated using beetroot
  • Beetroot cells contain a dark purple-red pigment
  • The higher the permeability of the beetroot cell membrane, the more of this pigment leaks out of the cell

• The amount of pigment in a sample can be measured using a piece of equipment known as a colorimeter
  
  • A colorimeter is a machine that passes light through a coloured liquid sample and measures how much of that light is absorbed by or transmitted through the sample
    • This gives an indication of how much of the colour is present in the solution, e.g. more absorbance or less transmission both indicate a darker coloured solution
  • A colour filter is used in the light path to ensure that the correct wavelength of light is used to measure the optical density of the specific pigment in the solution, e.g. the beetroot pigment called betalain
  • The colorimeter must be zeroed before each colorimeter is used; this can be done using distilled water in a small container called a cuvette
    • This process is known as calibrating the colorimeter

Investigating the effect of temperature on membrane permeability

Apparatus

• Scalpel
• Cork borer 
• Cutting board
• Ruler
• Digital balance
• Test tubes
• Measuring cylinder
• Water baths
• Digital stopwatch
• Pipettes
• Colorimeter and cuvettes

Method

1. Using a cork borer and scalpel, cut five equal-sized sections of beetroot
   • The pieces must have the same dimensions so that they all have equal surface areas and volumes, as these factors could affect the rate at which the pigment leaks out
   • A cork borer enables cores of equal diameter to be cut, which can then be cut to the same length
2. Rinse the beetroot pieces
   • To remove any pigment released during cutting
3. Add the beetroot pieces to five different test tubes, each containing the same volume of water, e.g. 5 cm³
4. Put each test tube in a water bath at a different temperature, e.g. 10 ℃, 20 ℃, 30 ℃, 40 ℃, and 50 ℃, for the same length of time
   • The time should be long enough to allow the pigment to diffuse into the water, e.g. around 30 minutes
5. Remove the beetroot pieces, leaving just the coloured liquid in the five test tubes
6. Use pipettes to transfer samples of the coloured liquid to colorimeter cuvettes
   • Cuvettes are small cuboid containers that hold the liquid to be measured in a colorimeter
   • A different pipette should be used for each sample to avoid the transfer of pigment molecules between samples
7. Use a colorimeter to measure how much light is absorbed as it passes through each of the five samples of coloured liquid
   • The higher the absorbance, the more pigment must have been released due to a greater membrane permeability

Results

• The general pattern you would expect to see is that as temperature increases, membrane permeability also increases
  • As temperature increases, the phospholipids within the cell membrane move more because they have more kinetic energy; increased movement means the phospholipids are not as tightly packed together, increasing the permeability of the membrane
  • Temperature affects the 3D shape of proteins; at high temperatures membrane proteins denature, increasing the permeability of the membrane
  • The volume of water inside the cells expands, putting pressure on the membrane and damaging membrane components; this can increase membrane permeability
• If experimenting with temperatures below 0 ℃, membrane permeability may be increased once the cells have thawed again
  • Ice crystals that form can pierce the cell membrane, making it highly permeable

Both high and very low temperatures can increase membrane permeability

Limitations

• Cuvettes may differ in thickness; a thicker cuvette will absorb slightly more light than a thinner cuvette
  • Solution: use the same cuvette for every reading, or repeat the investigation many times and find a mean
  • Note that scratched cuvettes can have the same impact on absorbance as thicker cuvettes
• The beetroot pieces may not be identical in size and shape, meaning some test tubes could contain slightly more beetroot tissue than others
  • Solution: cut the discs as accurately as possible using a scalpel and ruler, and repeat each investigation several times to find a mean
• Some parts of beetroot tissue have more pigment in their cells than others
  • Solution: conduct several repeats, using different parts of the beetroot and find a mean

Variations

• The impact of alcohol on membrane permeability could also be investigated
• The expected trend is
  • As alcohol concentration increases the permeability of the membrane increases
  • This is because alcohol dissolves lipids in the cell surface membrane so the membrane loses its structure and the beetroot pigment can leak out