By Tom McKeag
When Arthur DeVries arrived at McMurdo Station in 1961, he was fresh from Stanford University where he had signed up for a 13-month stint to study the respiratory metabolism of the endemic Notothenioid fishes found in McMurdo Sound, Antarctica. Notothenioids are Antarctic icefish, a suborder of the order of Perciformes. This order is the most numerous order of vertebrates in the world and includes perch, cichlids, and sea bass. Five families of Notothenioid fish dominate the Southern Ocean, comprising over 90 percent of the fish biomass of the region. They are a key part of an entire ecosystem, but that ecosystem would not exist in its robust form if they had not evolved a way to beat the extreme cold of these polar waters. DeVries would eventually find out how.
McMurdo station is at the southern tip of Ross Island, the largest of three U.S. science installations in Antarctica. Established in 1958, McMurdo had all the features of any work camp on the edge of raw nature, with few embellishments beyond generators, supply pallets and Quonset huts. The research community there existed in defiance of the climate, rather than because of it: recorded temperature extremes are as low as minus 50 degrees Celsius and average annual temperatures reside at minus 18 degrees Celsius.
Despite the conditions, De Vries thrived in the close-knit academic atmosphere and the rugged fieldwork of catching, stocking and analyzing fish. The challenges of his temporary job there, however, would lead him unexpectedly to a ground-breaking discovery and a lifetime of polar science. Some of the fish he was catching and holding in tanks were dying, while others were not. His zeal to solve his problem and his curiosity to find its causes would lead to an entire branch of research. As he told Scientia Publications,
“During these experiments I noticed that a deep water Notothenioid fish would freeze to death if any ice was present in our refrigerated salt water while those caught in the shallow water survived in the presence of ice. I decided to investigate why there was a difference in these species living in water of the same temperature (-1.9°C) for my PhD thesis research at Stanford. I investigated what compounds were responsible for their capability to avoid freezing in this environment while fishes in temperate waters would freeze to death at -0.8°C. My study culminated in the discovery of the antifreeze glycoproteins, the compounds responsible for their extreme freeze avoidance.”
The Antarctic icefish DeVries was studying are in a special club of organisms with the ability to live at low-temperature extremes. Some of these organisms, like the North American Wood Frog, are able to recover from freezing, and some, like the icefish, survive by avoiding being frozen. A great range of creatures from insects to diatoms to fungi and bacteria are also in this group that uses so-called ice-binding proteins (IBP) to survive. They use one of five general mechanisms for this: producing antifreeze; structuring ice where, for instance, an alga will create a more moderate liquid pocket within ice; adhering to ice, such as certain bacteria do; nucleating ice; and inhibiting ice recrystallization. Recrystallization is the consolidation of small ice crystals into bigger ones as they are attracted by hydrogen bonding in a cascade effect.
The icefish have evolved the first strategy of creating their own antifreeze. Anti-freeze proteins (AFP) can be defined as any ice-binding proteins that depress the hysteresis freezing point below the hysteresis melting point, thereby creating a “thermal hysteresis gap”. They are typically alpha helix glycoproteins also known as antifreeze glycoproteins (AFGP) or thermal hysteresis proteins (THP). Thermal hysteresis is the separation of freezing and melting temperatures. The fish are able to lower the point at which the water inside them freezes, while the point at which it melts remains the same (more on surprising developments on this later). To understand how this works requires a brief discussion of water itself.
Water is the universal medium on earth, with unique properties essential to a wide range of livable conditions and is a critical part of living things themselves. No other common material exists naturally on our planet in all three phases, liquid, solid and gas. Strong covalent bonds hold oxygen and hydrogen atoms together in a single molecule, but weaker hydrogen bonds connect water molecules to each other. The polar nature of the molecule, with oxygen negative and hydrogen positive, allows it to bind readily to other molecules, making for an excellent and universal solvent. Water has a high thermal capacity, which might be described as a reticence to change temperatures despite its surroundings. This creates an important moderating influence on climate at many scales. It has been estimated that our oceans can absorb one thousand times the heat as our atmosphere without significantly changing temperature. Most of the increased heat of global climate change, for example, has been absorbed by the earth’s oceans.
When water becomes colder, its density follows a predictable material trend, growing denser with each drop in temperature, until 4 degrees C. When water turns to ice it becomes lighter, less dense (approximately 9%) as the hydrogen atoms link to form a crystal lattice structure. This characteristic allows ice to float on top of its denser liquid phase, making overwintering aquatic life possible around the globe, including in the Antarctic Ocean. The expansion of water in the change from liquid to the solid phase can also be a powerful disruptive force; able to split granite.
This force can be equally straining at the intracellular and cellular level. Expansion of solid water inside of cells may cause them to burst, and the freezing of the intercellular spaces causes water loss and ion and metabolite buildup as ice forms. This water imbalance prompts a flow of liquid out of the cells and into the spaces between. This can lead to a toxic concentration of ions within the cell or a significant loss of pressure resistance and cell collapse.
A range of organisms across kingdoms has adapted to temperatures that freeze water: plants, yeasts, bacteria, and animals like fish and insects. They employ different stratagems, but all must live by the physical rules of their environments, especially the characteristics of water.
When salt is dissolved in water it lowers its freezing point. Seawater, therefore, has slightly different properties than fresh as the dissolved salts (3.5% for typical seawater) lower the freezing point to minus 1.9 degrees C. This is called freezing point depression and is a common evolved stratagem for many cold climate dwellers or psychrophiles. De Vries realized that the freezing point depression exhibited in his surviving shallow water fish could not be explained solely by common body salts in the serum of the fish. He devised a series of experiments to differentiate the chemical makeup of his two types of fish and isolated the glycoproteins that were key to his discovery. The proteins were attaching themselves to ice crystals within the blood of the fish and preventing them from growing. This, combined with body salts, allowed the fish to maintain liquid blood at minus 2.5 degrees C.
What he and his colleagues eventually found out was that these glycoproteins were binding to ice crystals irreversibly in a process they termed adsorption-inhibition (DeVries and Raymond, 1977). This is a so-called “step pinning” process in which crucial physical sequences necessary for freezing are interrupted or curtailed. In this case, the AFP’s were binding to small nascent ice crystals and forcing ice formation into smaller spaces between adsorption sites thereby bending the ice lattice’s growth front into a curve. This created a higher surface free energy and effectively lowered the freezing point in a phenomenon called the Gibbs-Thomson effect.
AFP’s are typically small compound proteins with an eccentric load of the amino acid threonine. Threonine has a hydrophilic surface that water molecules attach to weakly. This adsorption inhibits the microcrystals from coalescing into larger crystals and keeps the water in the liquid state.
It appears that these small ice crystals remain in the fish for their lifetimes, but this is still being studied. While there is no evidence that the fish are adversely affected by the year-round presence of the crystals, DeVries believes that they must have a mechanism to void them. One surprising recent discovery has been that the presence of the AFP’s make the crystals resist melting; higher temperatures are needed to melt them as well as lower temperatures needed to form them.
What is not known, according to DeVries, is just how these proteins are able to recognize solid phase water molecules within this liquid environment and preferentially bind to them. How they prevent growth is also still be investigated, with the adsorption inhibition model still open to debate and refinement. Nonetheless, there is no refuting this as a successful survival strategy. Indeed, it is an example of convergence, often an indicator, if not a guarantee, of effective and durable solutions in nature. Two genetically distinct populations of fish, one in the Arctic (the Arctic Cod) and one in the Antarctic (Notothenioids), have developed these techniques.
The discovery of these anti-freeze proteins may have touched off an entire research industry into their abilities, but do they perform as well as their commercial namesake? It seems that they do, as a matter of fact much better by an order of magnitude. The reason is the selectivity that they exhibit in attaching to the small ice crystals. Ethylene glycol, the green liquid typically used in car radiators, works by mass action effect, disrupting hydrogen bonding by the chemical equivalent of carpet bombing. Although it is not persistent, the chemical is a moderately toxic poison. When swallowed it is converted into oxalic acid by ethanol hydrogenase. Oxalic acid is highly toxic, affecting the central nervous system, heart, lungs and kidneys. It is responsible for tens of thousands of animal poisonings and thousands of human poisonings each year. Ethylene glycol has been demonstrated as a developmental toxicant in higher doses in rats.
Propylene glycol with metal nanoparticles has been developed as a safer alternative to ethylene glycol, but lacks the efficiency of the AFP’s. It is cheaper, however, readily available and uses a material already employed in the food industry and approved by the FDA.
Despite decades of research into the mechanism of these proteins, industry applications remain few, with proteins from the Arctic pout fish used in ice cream to prevent recrystallization, and AFP’s and growth hormones introduced to transgenic farmed salmon for cold-weather hardiness and increased growth. It is in the biomedical field, however, where the use of these proteins promises the most rewards and challenges.
Transporting and transplanting organs, preserving human bodies for the future miracles of medicine (cryonics), and performing surgery are all endeavors where AFP’s could play a revolutionary role. Single cells, like sperm and eggs, are routinely frozen and stored, but larger tissue is more difficult to preserve. AFP’s have been employed successfully to preserve rat and pig hearts in below freezing temperatures. In one experiment, researchers removed a rat heart, preserved it in sterile water and AFP’s at minus 1.3 degrees C for 24 hours, then transplanted the warmed up (non-pumping) heart into a new rat.
Notwithstanding these early successes and the great promise of AFP’s, the technology of preserving human organs still lags far behind the medical demand. The US Department of Health and Human Services estimates that approximately 21 patients a day die waiting for an organ that is not available. Lungs remain usable for only twelve hours and hearts only four or five, using the current techniques. The toxicity of cryoprotectants and the disruptive effects of thawing are two of the most challenging problems. While vitrification is an effective technique of quick freezing of organs to a glass state, most techniques rely on pumping the cells full of toxic chemicals, and it is in the thawing where damage is most severe. Differential warming causes splintering and fracturing of material subjected to opposing forces. One University of Minnesota team, however, is working on a method of using nanoparticles to gently and uniformly heat organs back to living temperatures. The magnetic nanoparticles are excited to activity (and heat) by radio waves in a process the team calls “nanowarming”, and the technique has been used successfully on clusters of cells.
Other research teams are looking elsewhere in nature for even more effective anti-freeze compounds. One is a glycolipid found in a freeze-tolerant Alaskan beetle, Upis ceramboides which allows the insect to endure temperatures of minus 60 degrees C and still recover. Cell and Tissue Systems of South Carolina is employing it successfully in the preservation of tissues for days at below zero temperatures without deterioration, according to the company. The glycolipid appears to coat the membrane of the cell, armoring it against external ice and sealing it against the osmotic draw of liquid from the cell.
Whether using a protein or a glycolipid, lowering freezing temperatures or enduring being frozen, pumping themselves full of cryoprotectants, sealing themselves up or drying themselves out, nature’s organisms of all domains have come to live with the uncommon cold. It is still up to human researchers to fully unlock these secrets and put them to use in the better preservation of life.