Membranes

The phospholipids form a bilayer: two layers of phospholipid molecules with their hydrophilic phosphate heads facing outwards towards the watery cytoplasm and extracellular fluid, and their hydrophobic fatty-acid tails pointing inwards, away from the water.

The proteins are scattered throughout this bilayer in an irregular, mosaic-like pattern. Some proteins (intrinsic/integral) span the whole bilayer, while others (extrinsic/peripheral) sit on one surface only.

Because the phospholipids and many of the proteins can move sideways within the layer, the whole structure is described as 'fluid'.

A glycoprotein is a membrane protein with a short, branched carbohydrate chain attached, and a glycolipid is a phospholipid with a carbohydrate chain attached. In both cases the carbohydrate chain projects from the outer surface of the membrane.

One role is cell recognition: the carbohydrate chains act as markers/antigens that allow cells to recognise each other as 'self', which is important in the immune system (and explains transplant rejection when the markers are 'non-self').

A second role is acting as receptors: specific glycoproteins bind specific molecules such as hormones (for example the insulin receptor) or neurotransmitters, triggering a response inside the cell. They can also help cells adhere to one another to form tissues.

Beetroot cells store a red/purple pigment (betalain) in the vacuole, retained by the cell membranes. The amount of pigment that escapes depends on how permeable these membranes are.

As temperature rises, the phospholipid and protein molecules gain kinetic energy and move more, so small gaps begin to open up in the bilayer and it becomes more permeable.

Above about 45 °C, the membrane proteins denature: the heat breaks the hydrogen bonds and other weak interactions holding their tertiary structure, so the proteins (including channel and carrier proteins) change shape and the bilayer is increasingly disrupted.

The membrane therefore becomes much more permeable and can no longer retain the contents of the cell and vacuole.

As a result, more betalain pigment leaks out of the cells into the surrounding water, which is detected as a darker solution — a higher absorbance (or lower percentage transmission) on the colorimeter.

Ethanol is an organic solvent that is less polar than water and can dissolve the lipids of the cell surface membrane and tonoplast.

As the ethanol concentration increases, more of the phospholipid bilayer is dissolved/disrupted, so the membrane loses its ordered structure and larger gaps form in it.

This makes the membrane increasingly permeable, so it can no longer keep the pigment inside the vacuole.

Therefore more red betalain pigment leaks out of the cells into the surrounding solution as the ethanol concentration rises.

This appears as a progressively darker solution, measured as a higher absorbance (lower transmission) on a colorimeter, showing a positive correlation between ethanol concentration and membrane permeability.

The cell surface membrane must remain fluid enough for proteins to move and for processes such as endocytosis and exocytosis to occur, while still acting as a partially permeable barrier that controls what enters and leaves the cell. Both cholesterol and the type of fatty-acid tails help to maintain this balance.

Cholesterol is positioned between the phospholipid molecules in the bilayer. At high temperatures the phospholipids have more kinetic energy and would move more, making the membrane too fluid and leaky; cholesterol restricts their movement, increasing stability and reducing permeability. At low temperatures the phospholipids would pack tightly and the membrane could become rigid; cholesterol prevents them packing too closely, so the membrane stays fluid and does not crack. In this way cholesterol acts as a 'buffer', keeping fluidity within a suitable range across a range of temperatures, and it also helps reduce the leakage of water-soluble ions.

The proportion of unsaturated fatty acids in the phospholipid tails also affects fluidity. Unsaturated fatty acids contain C=C double bonds that put a kink in the tail, so the phospholipids cannot pack closely together. A higher proportion of unsaturated tails therefore keeps the membrane more fluid, which is especially important at low temperatures to stop the membrane becoming rigid. Organisms in cold environments often have more unsaturated fatty acids for this reason, whereas more saturated (straight) tails pack tightly and make the membrane less fluid.

Together, cholesterol and the balance of saturated/unsaturated fatty acids allow the membrane to stay fluid yet stable, maintaining its function as a controlled, partially permeable barrier.

Preparing the beetroot. Use a cork borer to cut several beetroot pieces of equal diameter, then cut them to the same length with a tile and scalpel so each disc has the same surface area and volume. Rinse the pieces in distilled water until no more pigment comes off, to remove betalain released from cells damaged during cutting; otherwise the starting readings would be falsely high.

The independent variable is temperature. Set up water baths across a range of temperatures (for example 10, 20, 30, 40, 50, 60 and 70 °C). Place one equal-sized piece into a test tube containing the same volume of distilled water at each temperature, and leave each for the same length of time (e.g. 30 minutes).

Controlled variables include the size/surface area of the beetroot, the volume of water, the time submerged, the variety and age of the beetroot, and using the same colorimeter and filter — so that any change in pigment release is due to temperature alone.

Measuring the dependent variable. After the set time, remove the beetroot and shake each tube to mix. Measure the colour of each solution with a colorimeter fitted with a blue/green filter (the colour the red pigment absorbs most). First zero the colorimeter using distilled water as a blank. Record the absorbance (which increases) or percentage transmission (which decreases) of each sample.

Reliability and processing. Repeat the procedure several times at each temperature and calculate a mean absorbance, discarding clear anomalies. Plot a graph of mean absorbance against temperature. The results should show that absorbance stays low at lower temperatures and rises sharply above about 45 °C, because higher temperatures denature the membrane proteins and disrupt the bilayer, making the membrane more permeable so more pigment leaks out.

A molecule is amphipathic when it has both a hydrophilic region and a hydrophobic region — in a phospholipid, the hydrophilic phosphate head and the two hydrophobic fatty-acid tails.

Because there is water on both sides of the membrane, the molecules orient with the hydrophilic heads facing the water and the hydrophobic tails turned away from the water towards each other, producing two layers — a bilayer.

The interior of the bilayer is formed by the hydrophobic fatty-acid tails, creating a non-polar core.

Oxygen and carbon dioxide are small, non-polar (lipid-soluble) molecules, so they can dissolve in and diffuse directly through the hydrophobic core of the bilayer, down their concentration gradients, by simple diffusion.

Sodium ions are charged and glucose is a large, polar (water-soluble) molecule; both are repelled by the hydrophobic tails and cannot pass freely through the bilayer.

Instead they must cross through intrinsic transport proteins — $Na^+$ through channel proteins (or carrier proteins by active transport), and glucose through specific carrier proteins by facilitated diffusion (or co-transport).

This selectivity is exactly why the membrane is a partially permeable barrier: lipid-soluble substances pass through the bilayer, while ions and large polar solutes depend on specific proteins.

The fatty-acid tails of the phospholipids can be saturated (no C=C double bonds, straight tails) or unsaturated (one or more C=C double bonds).

A C=C double bond puts a 'kink' in the tail, so unsaturated phospholipids cannot pack closely together. The more unsaturated tails present, the greater the spacing between phospholipids and the more fluid the membrane. Saturated, straight tails pack tightly and make the membrane less fluid.

In cold Arctic water there is low kinetic energy, so the phospholipids move slowly and the membrane would tend to become too rigid / gel-like, which would stop transport proteins functioning and could cause the membrane to crack.

The higher proportion of unsaturated fatty acids counteracts this: the kinked tails keep the phospholipids apart so the membrane remains fluid even at low temperature.

This is an advantage because a fluid membrane keeps the cell membrane functional — allowing carrier and channel proteins to work, vesicles to form, and the membrane to remain a controlled partially permeable barrier.

Tropical fish do not need this; in warmer water their membranes have enough kinetic energy to stay fluid, so more saturated tails maintain an appropriate (not excessive) fluidity.

Describe the trend. As temperature increases, the mean absorbance of the solution increases, showing that more betalain pigment leaks out and the membrane becomes more permeable. The rise is small and gradual from 20 °C to 40 °C (absorbance only rises from 0.12 to 0.22, a change of 0.10), but then increases sharply between 40 °C and 60 °C (from 0.22 to 0.95, a change of 0.73). Above 60 °C the increase levels off / plateaus (60 °C to 70 °C rises only from 0.95 to 1.04).

Explain the gradual region (20–40 °C). At these temperatures the phospholipids and proteins have relatively low kinetic energy; the bilayer stays largely intact and stable, so the membrane remains partially permeable and little pigment escapes.

Explain the sharp rise (40–60 °C). As temperature rises further the molecules gain more kinetic energy and move more, opening gaps; crucially, above about 45 °C the membrane proteins denature — the hydrogen bonds and other interactions holding their tertiary structure break, so the proteins change shape — and the phospholipid bilayer becomes increasingly disrupted. The membrane is therefore far more permeable, so a much larger amount of betalain leaks from the vacuole into the water, giving the steep rise in absorbance.

Explain the plateau (above 60 °C). Above 60 °C the membrane is already maximally disrupted and most of the available pigment has already leaked out, so absorbance cannot rise much further.

Evaluation. The data do support the conclusion that membrane disruption is greatest between 40 °C and 60 °C, because that is where absorbance rises most steeply (the largest gradient), consistent with protein denaturation occurring over this range. However, the support is limited: the temperature intervals are wide (10 °C), so the exact threshold between 40 °C and 50 °C is not resolved — readings at, say, 42, 44, 46 and 48 °C would pinpoint it. There is also no indication of repeats or error bars, so the reliability and spread of the means cannot be judged, and a single anomalous tube could distort a point. To strengthen the conclusion the student should repeat each temperature and calculate means with a measure of spread, and take smaller temperature intervals around 40–60 °C.