PAG 8 - Transport In and Out of Cells

• State 2 factors that affect the permeability of cell membranes.

  • Temperature
  • Concentration of solvents (ethanol)
• How is beetroot used to measure the permeability of cell membranes?

  The higher the permeability, the more red pigment that leaks out into the surrounding solution within a given time. A colorimeter can be used to determine the absorbance, hence concentration of pigment
• Outline the procedure to investigate the effect of temperature on permeability of cell membrane.

  1. Cut beetroot into 6 identical cubes with a scalpel
  2. Place each cube in a different test tube with equal volumes of distilled water
  3. Place each tube into water baths ranging from 30 - 80 °C. Leave for 20 minutes.
  4. Filter each solution out into a cuvette and measure the absorbance.
• What are the safety hazards involved in testing the effect of ethanol concentration on membrane permeability?

  • Ethanol is an irritant and is flammable, keep away from naked flames, wear eye protection
  • Keep sharp scalpel away from fingers
  • Handle hot liquid with care
• What is the effect of temperature on membrane permeability?

  Increasing temperature results in increase membrane permeability
• What is the effect of ethanol concentration on membrane permability?

  Increasing ethanol concentration leads to increased membrane permeability, as ethanol causes gaps to form in the membrane.
• Outline the materials used to measure the rate of diffusion.

  • Make agar jelly mixed with phenolphthalein
  • Place in HCl and measure the time taken for the agar jelly to turn colourless
  • Change the factor and repeat e.g. concentration of HCL, surface area of agar jelly
• What is the effect of concentration on the rate of diffusion?

  Increasing concentration increases the steepness of the concentration gradient, hence increasing the rate of diffusion (decreasing the time taken for the agar jelly to decolourise)
• What is the effect of surface area on the rate of diffusion?

  Increased surface area to volume ratio increases the rate of diffusion
• • You can investigate how these different factors affect membrane structure and permeability 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
• • A colorimeter is a machine that passes light through a coloured liquid sample and measures how much of that light is absorbed (and therefore gives an indication of how much of the colour is present in the 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 tube (called a cuvette) is inserted. This can be done using distilled water in a cuvette
• Apparatus

  • Scalpel
  • Cork borer (optional)
  • Cutting board
  • Ruler
  • Digital balance
  • Test tubes
  • Measuring cylinder
  • Water baths
  • Digital stopwatch
  • Colorimeter
• Method (1)

  • Using a scalpel, cut five equal-sized cubes 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 can also be used, as long as the cores are cut to the same length
  • You should also use a digital balance to check that all pieces have the same mass
  • Rinse the beetroot pieces
  • To remove any pigment released during cutting
  • Add the beetroot pieces to five different test tubes, each containing the same volume of water (e.g. 5cm3)
• Method (2)

  • Put each test tube in a water bath at a different temperature (e.g. 10℃, 20℃, 30℃, 40℃, 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)
  • Remove the beetroot pieces, leaving just the coloured liquid in the five test tubes
  • 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 (1)

  • 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 energy
  • Increased movement means the phospholipids are not as tightly packed together, increasing the permeability of the membrane
  • At high temperatures, the phospholipid bilayer may even start to melt and breakdown, further increasing the permeability of the membrane
• Results (2)

  • In addition, the volume of water inside the cells expands, putting pressure on the membrane, causing channel and carrier proteins to deform so they can no longer control what enters and leaves the cell. These factors also increase the permeability of the membrane
  • Temperature also affects the conformation (3D shape) of proteins as at high temperatures the intermolecular forces between amino acids are broken which affects the protein’s specificity and function
• Results (3)

  • If experimenting with temperatures below 0C, membrane permeability may also be increased (once the cells have thawed again)
  • Increased permeability can be caused by channel or carrier proteins deforming at these low temperatures
  • Ice crystals that form can also pierce the cell membrane, making it highly permeable
• Limitations (1)

  • Cuvettes are the small cuboid containers that hold the liquid to be measured in a colorimeter
  • Cuvettes may differ in thickness (very slightly). A thicker (or scratched) cuvette will absorb slightly more light than a thinner unscratched cuvette
  • Solution: use the same cuvette for every reading, or repeat the investigation many times and find a mean
• Limitations (2)

  • 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