Effect of Temperature on Membrane Permeability

Spec Mapping: This lesson is mapped to OCR H420 Module 2.1.5 — Biological membranes (refer to the official OCR H420 specification document for exact wording). It develops the factors that affect membrane structure and permeability and the related Practical Activity Group investigation using beetroot pigment release as a proxy for membrane damage.

The structural features of biological membranes described in Lesson 1 have direct consequences for how they behave when environmental conditions change. Temperature is one of the most important variables, and it is also the focus of a required practical. This lesson develops the OCR H420 Module 2.1.5 content on factors that affect membrane structure and permeability, with the beetroot practical as the worked example.

---

1. Why Temperature Affects Membrane Permeability

Membrane permeability depends on the tight packing of phospholipids and the functional integrity of membrane proteins. Temperature affects both.

1.1 Low temperatures (below about 0 °C)

• Phospholipids have less kinetic energy and pack tightly together.
• Fatty acid tails are relatively rigid — the membrane becomes gel-like and less fluid.
• Channel and carrier proteins may become deformed; they are held in a rigid matrix and cannot change conformation easily.
• Ice crystals can form inside the cell, physically piercing membranes and causing large holes.
• Paradoxically, permeability to some molecules increases at very low temperatures because the membrane cracks or proteins no longer seal properly.

1.2 Moderate temperatures (0–45 °C)

• Membranes are fluid and selectively permeable.
• Phospholipids move laterally; proteins can carry out their functions (transport, signalling, catalysis).
• Permeability increases gently with temperature — more kinetic energy means faster diffusion.

1.3 High temperatures (above about 45 °C)

• Phospholipids have high kinetic energy and move apart — gaps open in the bilayer.
• Water and solutes can leak through these gaps.
• Membrane proteins denature: the hydrogen bonds and ionic interactions maintaining their tertiary structure break, the proteins unfold and their shape changes irreversibly.
• Channels may become permanently open (or closed); receptors stop functioning; enzymes are inactivated.
• Permeability rises sharply.

graph LR
  A[Temperature] -->|Low| B["Reduced fluidity<br/>Protein deformation<br/>Ice crystal damage"]
  A -->|Moderate| C["Normal fluidity<br/>Functional proteins<br/>Controlled permeability"]
  A -->|High| D["Gaps in bilayer<br/>Protein denaturation<br/>Increased permeability"]

---

2. Other Factors Affecting Permeability

Before we look at the practical, two other factors are examinable:

2.1 Solvents

Non-polar solvents such as ethanol, propanone (acetone) and methanol dissolve the hydrophobic core of the membrane:

• At low concentrations they disrupt phospholipid packing, increasing fluidity and permeability.
• At high concentrations they dissolve membranes entirely, causing catastrophic leakage and cell death.
• This is why ethanol above ~70% kills bacterial cells (and why it is used in hand sanitiser).

2.2 pH

Extremes of pH denature membrane proteins and alter the ionisation of phospholipid heads. Both effects increase permeability.

---

3. Required Practical — Investigating Membrane Permeability Using Beetroot

This is a classic OCR required practical. Beetroot is an ideal material because its vacuoles contain a red-purple water-soluble pigment called betacyanin (also written betalain). Betacyanin is normally trapped inside the tonoplast (the vacuolar membrane) and plasma membrane. When these membranes are damaged, betacyanin leaks out into the surrounding water and can be quantified using a colorimeter.

3.1 Hypothesis

As temperature increases, membrane permeability increases, so more betacyanin will leak out and the colour intensity of the surrounding water will increase.

3.2 Method

1. Use a cork borer to cut several beetroot cylinders of equal diameter from a single beetroot.
2. Cut the cylinders into equal-length discs (e.g. 1 cm) using a ruler and scalpel — a control for surface area.
3. Rinse the discs thoroughly in cold distilled water to remove betacyanin released from cells damaged during cutting. Blot dry.
4. Place one disc into a labelled test tube containing 5 cm³ of distilled water at a known temperature. Use a water bath to control temperature.
5. Test a range of temperatures, e.g. 10, 20, 30, 40, 50, 60, 70 °C.
6. Leave each tube for a fixed time (e.g. 30 minutes).
7. Remove the disc. Shake the tube gently to mix the contents.
8. Transfer the solution to a cuvette and measure absorbance at a suitable wavelength (around 480 nm or a green filter — because betacyanin is red, it absorbs strongly in the green/blue region).
9. Calibrate the colorimeter with distilled water first.
10. Repeat each temperature at least three times and calculate a mean absorbance.

3.3 Variables

Type	Variable	How controlled
Independent	Temperature	Water bath
Dependent	Absorbance (pigment release)	Colorimeter
Controlled	Disc size	Same cork borer; equal-length discs
Controlled	Volume of water	5 cm³ per tube
Controlled	Time in bath	30 min timer
Controlled	Beetroot	One beetroot cut on the same day
Controlled	Rinsing	Cold distilled water for set time

3.4 Risk Assessment

• Hot water baths and scalpels — handle with care; use tongs for hot tubes.
• Beetroot stains — wear eye protection and consider gloves.
• Distilled water spills — mop immediately.

3.5 Expected Results

Temperature (°C)	Absorbance	Membrane behaviour
10	Low	Membrane intact; slight passive leakage
20	Low	Membrane intact
30	Low–Moderate	Gentle increase with temperature
40	Moderate	Fluidity increasing; small increase
50	High	Proteins beginning to denature; gaps in bilayer
60	Very high	Proteins denatured; gaps open
70	Very high	Severe damage; near-maximum leakage

graph LR
  A[Low temp] --> B["Small increase<br/>gentle slope"]
  B --> C["Transition<br/>sharp rise around 45-55 C"]
  C --> D["Plateau<br/>maximum pigment released"]

The curve of absorbance against temperature is typically sigmoidal, with a slow rise at low temperatures and a sharp rise between about 45 and 55 °C as membrane proteins denature and the bilayer loses integrity.

3.6 Analysis

• Plot a line graph of mean absorbance against temperature. Use a line of best fit (not dot-to-dot).
• Identify the temperature at which the sharp increase begins — this corresponds to the point at which the membrane loses its integrity.
• Discuss any anomalies and calculate standard deviation or range to assess reliability.

3.7 Limitations and Improvements

• Cell damage during cutting — betacyanin from damaged cells can inflate readings. Rinse thoroughly and cut neatly with a sharp blade.
• Different beetroot cells have different vacuole sizes — use discs from the same beetroot.
• Evaporation at high temperatures can concentrate the solution — seal tubes with foil.
• Water bath temperature drift — use a thermometer and regulate carefully.
• Colorimeter cuvette fingerprints — wipe before reading.

---

4. Interpreting a Graph of Absorbance vs Temperature

A typical candidate graph shows:

• A gentle rise at low temperatures — small amounts of passive leakage increase as kinetic energy rises.
• A sharp increase around the denaturation temperature of membrane proteins (roughly 45–55 °C for beetroot).
• A plateau at high temperatures when all betacyanin has been released — the membrane can get no more permeable.

You may be asked to explain such a graph. Use the vocabulary of the fluid mosaic model:

"Below 45 °C the membrane is intact; small amounts of betacyanin leak out through gaps in the bilayer as kinetic energy increases phospholipid movement. Above about 50 °C the hydrogen and ionic bonds in membrane proteins break, causing denaturation, and the phospholipids have high kinetic energy, so the bilayer develops large gaps. Betacyanin leaks out rapidly, producing a sharp rise in absorbance. At very high temperatures the curve levels off because the vacuole has lost all its betacyanin."

---

5. Common Exam Mistakes

• Forgetting to rinse the discs — the resulting "baseline" leakage can mask the real pattern.
• Confusing absorbance with transmittance or "colour darkness". Higher pigment = higher absorbance (and lower transmittance).
• Saying proteins "melt" — they denature.
• Claiming the membrane "is destroyed" at 30 °C — at this temperature it is working normally.
• Failing to mention that betacyanin is in the vacuole, not the cytoplasm, so the tonoplast must also be damaged for it to escape.
• Writing that betacyanin is "in the cell membrane" — it is a vacuolar pigment.
• Not controlling surface area — larger discs release more pigment regardless of temperature.
• Reading "colour intensity" by eye — the colorimeter gives a quantitative measure.

---

6. Exam-Style Questions

1. Describe how you would investigate the effect of temperature on membrane permeability of beetroot cells. (6)
2. Explain, in terms of membrane structure, why permeability increases sharply above 50 °C. (4)
3. Suggest two variables, other than temperature, that must be controlled in this experiment. (2)
4. A student found high absorbance in their 10 °C tube. Suggest one reason. (2)

Model answer for (2): "Above 50 °C the kinetic energy of phospholipids increases, so they move apart and gaps open in the bilayer. The hydrogen and ionic bonds in the tertiary structure of membrane proteins break, so the proteins denature, changing shape and losing function. Channels may become permanently open. Together these changes allow the red pigment betacyanin to leak out of the vacuole and across the cell surface membrane rapidly."

---

Summary

• Membrane permeability increases with temperature because phospholipids move apart and proteins denature.
• The beetroot practical uses release of the vacuolar pigment betacyanin into surrounding water, measured by colorimeter absorbance, as a proxy for membrane damage.
• Key controls: disc size, volume of water, time, rinsing to remove pre-released pigment.
• The expected graph rises slowly at low temperatures, sharply between about 45 and 55 °C as proteins denature, and plateaus at high temperatures.
• Solvents and extremes of pH also increase permeability — ethanol dissolves phospholipids; pH denatures proteins.
• Use the language of the fluid mosaic model to explain experimental results in exam answers.

---

7. The Membrane-Damage Quantitative Framework

7.1 Why beetroot?

Beetroot is the model material because:

1. The pigment betacyanin is large and polar, so it does not normally cross intact membranes.
2. It is contained inside the vacuole, behind both the tonoplast and the plasma membrane. Two membranes must be damaged before significant release occurs — this gives a sharp threshold response.
3. It absorbs strongly in the visible spectrum (red wavelengths transmitted, green/blue absorbed) and is therefore quantifiable on a basic school colorimeter.
4. It is cheap, non-hazardous and reliably available year-round.

7.2 Calibrating absorbance

Absorbance $A$A is defined by the Beer–Lambert law:

$A = \varepsilon c \ell$A=εcℓ

where $\varepsilon$ε is the molar absorption coefficient (a constant for betacyanin at the chosen wavelength), $c$c is the concentration of pigment and $\ell$ℓ is the path length of the cuvette. Because $\varepsilon$ε and $\ell$ℓ are constant, absorbance is directly proportional to pigment concentration — and therefore to the fraction of pigment released from the vacuoles.

7.3 Anomalies and how to handle them

Anomalous observation	Likely cause	Remedy
High absorbance at 10 °C	Damage during cutting / inadequate rinsing	Rinse cold for 5–10 min before assay
Low absorbance at 70 °C	Pigment partly degraded by heat	Cap absorbance at saturation; degradation noted as a limitation
Step change at one specific temperature	Discontinuous water-bath control	Confirm with thermometer at start and end
Variability between repeats	Inconsistent disc size or beetroot heterogeneity	Use one beetroot; same cork borer; calliper-measured disc lengths

---

8. Quantitative SVG — A Schematic Absorbance vs Temperature Curve