The surface of a sheet of ice

Exploring antifreeze proteins

Dani Bancroft pulls on her thermals to learn about these molecules that help organisms survive in extremely low temperatures

Antifreeze proteins (AFPs) have evolved in many different species of plants, fungi, bacteria and fish as a natural survival response to the freezing cold. They help prevent the formation of ice crystals that damage cells. Organisms that have these proteins can live in extreme environments and are therefore known as ‘extremophiles’.

Why does freezing kill cells?

Cells are densely packed units containing cytoplasm and organelles. The majority of cytoplasm is water; in the average human cell, up to 70 per cent of cytoplasm is water.

At low temperatures, liquid water becomes ice when the water molecules join together with hydrogen bonds. They gradually recruit other nearby water molecules and the microcrystals join together to form larger ice crystals.

Ice is less dense than water, so it takes up more space. This increase in volume in the cytoplasm causes cells to burst, causing irreparable damage to their cell membranes and death.

Defrosting frozen tissues can also cause damage, because when ice thaws it undergoes recrystallisation (melting and refreezing to form different types of crystals), which can puncture cells from the outside.

A higher salt concentration lowers the freezing point of water to around –10˚C, while the melting point remains at 0˚C (an effect called thermal hysteresis). Therefore, in salt-water habitats, water can remain liquid even when it is below zero.

However, even though a salt-water habitat may be liquid, the tissues inside extremophiles such as Arctic notothenioid fish can still freeze when below 0˚C. This is because cytoplasm and tissue fluid in vertebrates (including humans) have a lower salt concentration than seawater, so the freezing point does not change. This is why antifreeze proteins are needed.

What structure do antifreeze proteins take?

Antifreeze proteins are compound proteins that have a sugar group attached to give them a charge. Often, antifreeze proteins are small and have one side that contains a high concentration of the amino acid threonine. Threonine provides a hydrophilic (water-loving) surface that water molecules attach to weakly (adsorption), which prevents ice microcrystals joining up to form larger ones and keeps the water liquid.

However, at low enough temperatures, water will solidify regardless. Antifreeze proteins work by causing thermal hysteresis even though the tissue fluid has too low a salt concentration for this effect to occur naturally.

Are they similar to car antifreezes?

Antifreeze proteins differ from the chemical antifreeze used in cars (ethylene glycol) as they act purely as ‘ice-restructuring’ proteins. Ethylene glycol disrupts hydrogen bonding to prevent water crystallising and forming ice altogether.

Ethylene glycol can be toxic when ingested by humans because it is converted by ethanol dehydrogenase to oxalic acid, a highly toxic substance with powerful effects on the central nervous system, heart, lungs and kidneys.

In some cases, people who have drunk ethylene glycol have survived by drinking ethanol (alcohol) afterwards. Ethanol competes with ethylene glycol molecules for the active site of the ethanol dehydrogenase enzyme, so the amount of oxalic acid produced is reduced. Ethylene glycol is regularly used in killing and preserving insect specimens.

How might we use antifreeze proteins in humans?

Antifreeze proteins have a range of biomedical applications in an emerging field called cryonics, which involves freezing body tissues for preservation and later use. This encompasses a range of different practices – some more futuristic than others. For many years, some people have arranged for their remains to be frozen after death (either their head or their full body) in ‘cryonic’ vaults. They do this in the hope that future advances in medicine will mean that their preserved bodies can one day be revived.

Cryonics can also be used to preserve single organs. Organs taken from humans for transplantation do not remain viable for very long on ice because the tissue deteriorates rapidly. At most, a human heart can only last 4–5 hours. Transporting the donor heart to a patient in another part of the country can be a very risky and expensive task.

Antifreeze proteins could help by enabling human organs to be frozen (and kept for longer in storage) without being damaged by ice crystals. This would mean that organs could be stored in hospital donor ‘banks’ for use.

An Israeli experiment in 2005 successfully preserved a rat heart with antifreeze proteins sourced from an Arctic fish. The heart was frozen for 21 hours before transplantation and managed to pump without any problems in the recipient mouse. This technology is being developed with rabbit kidneys, female egg cells (oocytes) and other tissues.

Lead image:

The surface of a sheet of ice.

Wellcome Library, London


Further reading

About this resource

This resource was first published in ‘Proteins’ in January 2014.

Cell biology, Biotechnology and engineering
Education levels:
16–19, Continuing professional development