Antifreeze proteins (AFPs) or ice structuring proteins (ISPs) refer to a class of polypeptides produced by certain vertebrates, plants, fungi and bacteria that permit their survival in subzero environments. AFPs bind to small ice crystals to inhibit growth and recrystallization of ice that would otherwise be fatal (Madura, 2001). There is also increasing evidence that AFPs interact with mammalian cell membranes to protect from cold damage. This work suggests the involvement of AFPs in cold acclimatization (Fletcher et al, 2001).
Non Colligative Properties
Unlike the widely used automotive antifreeze, ethylene glycol, AFPs do not lower freezing point in proportion to concentration. Rather, they work in a non colligative manner. This allows them to act as an antifreeze at concentrations 300-500 times lower than other dissolved solutes. This minimizes their effect on osmotic pressure (Fletcher et al, 2001). The unusual capabilities of AFPs are attributed to their binding ability at specific ice crystal surfaces. (Jorov et al, 200
AFPs create a difference between the melting point and freezing point known as thermal hysteresis. The addition of AFPs at the solid ice and liquid water interface inhibits the thermodynamically favored growth of the ice crystal. Ice growth is kinetically inhibited by the AFPs covering the water-accessible surfaces of ice. (Jorov et al, 2004).
Thermal hysteresis is easily measured in the lab with a nanolitre osmometer. Different organisms have different values of thermal hysteresis. The maximum level of thermal hysteresis shown by fish AFP is approximately 1.5ºC. However, insect antifreeze proteins are 10-30 times more active than any known fish protein. This is probably because insects encounter lower temperatures on land than the -1ºC or -2ºC that fish face in freezing waters. During the extreme winter months, the spruce budworm can battle temperatures approaching -30ºC and resist freezing (Fletcher et al, 2001).
The rate of cooling can influence the thermal hysteresis value of AFPs. Rapid cooling can substantially decrease the non-equilibrium freezing point and hence the thermal hysteresis value. This means that organisms may not be able to adapt to their subzero environment if the temperature drops abruptly (Fletcher et al, 2001).
Freeze Tolerance Versus Freeze Avoidance
Species containing AFPs may be classified as:
Freeze Avoidant: These species are able to prevent their body fluids from freezing altogether. Generally, the AFP function may be overcome at extremely cold temperatures, leading to rapid ice growth and death.
Freeze Tolerant: These species are able to survive body fluid freezing. Some freeze tolerant species are thought to use AFPs as cryoprotectants to prevent the damages of freezing but not freezing altogether. The exact mechanism is still unknown. However, it is thought that AFPs may inhibit recrystallization and stabilize cell membranes to prevent damage by ice (Duman, 2001). They may work in conjunction with protein ice nucleators (PINs) to control the rate of ice propagation following freezing (Duman, 2001).
There are many known non homologous types of AFP.
Antifreeze glycoproteins or AFGPs are found in Antarctic notothenioids and northern cod. They are 2.6-3.3 kD (Crevel et al, 2002).
Type I AFPs are found in winter flounder and shorthorn sculpin. They are the best documented AFP because it was the first to have its 3D structure determined (Duman and DeVries, 1976). Type I AFPs consist of a single, long, amphipathic alpha helix. They are approximately 3.3-4.5 kD in size. There are three faces to the 3D structure: the hydrophobic, hydrophilic, and Thr-Asx face (Duman and DeVries, 1976).
Type I-hyp AFP (where hyp stands for hyperactive) are found in several righteye flounders. It is approximately 32 kD (two 17 kD dimeric molecule). The protein was isolated from the blood plasma of winter flounder. It is considerably better at depressing freezing temperature than most fish AFPs (Scotter et al, 2006).
Type III AFPs are found in Antarctic eelpout. They exhibit similar overall hydrophobicity at ice binding surfaces to type I AFPs. They are approximately 6kD in size (Crevel et al, 2002).
Type IV AFPs are found in longhorn sculpins. They are alpha helical proteins rich in glutamate and glutamine (Deng et al, 1997).
Type V AFPs are the hyperactive (i.e. greater thermal hysteresis value) AFPs found in insects (Graham et al, 1997).
Tenebrio and Dendroides AFPs are both found in different insect families. They are very similar to one another. These AFPs consist of varying numbers of 12- or 13-mer repeats with approximately 8.3 to 12.5 kD. Throughout the length of the amino acid, at least every sixth is a cysteine residue (Duman, 2001). They are also hyperactive.
The classification becomes more complicated when antifreeze proteins from plant were discovered (Griffith et al, 1992). Plant AFPs are rather different from the other AFPs in the following aspects:
1. they have much weaker thermal hysteresis activity when compared to other AFPs (Griffith and Yaish, 2004)
2. their physiological function is likely in inhibiting the recrystallization of ice rather than in the preventing ice formation (Griffith and Yaish, 2004)
The remarkable diversity and distribution of AFPs suggests that the different types evolved recently in response to sea level glaciation occurring 1-2 million years ago in the Northern hemisphere and 10-30 million years ago in Antarctica. This independent development of similar adaptations is referred to as convergent evolution (Fletcher et al, 2001). There are two reasons why many types of AFPs are able to carry out the same function despite their diversity:
1. Although ice is uniformly composed of oxygen and hydrogen, it has many different surfaces exposed for binding. Different types of AFPs may interact with different surfaces.
2. Although the five types of AFPs differ in their primary sequence of amino acids, when each folds into a functioning protein they may share similarities in their 3D or tertiary structure that facilitates the same interactions with ice. (Fletcher et al, 2001)
Mechanisms of Action
AFPs are thought to inhibit growth via an adsorption–inhibition mechanism (Raymond and DeVries, 1977). They adsorb to non-basal planes of ice, inhibiting thermodynamically favored ice growth (Raymond et al, 1989). The presence of a flat, rigid surface in AFPs seems to facilitate its interaction with ice via Van der Waals force surface complementarity (Yang et al, 1998).
Binding to ice
Normally ice crystals grown in solution only exhibit the basal (0001) and prism faces (1010) and appear as round and flat discs (Jorov et al, 2004). However, it appears the presence of AFPs exposes other faces. It now appears that the ice surface 2021 is the preferred binding surface, at least for AFP type I (Knight et al, 1991). Through studies on type I AFP, it was initially thought that ice and AFP interacted through hydrogen bonding (Raymond and DeVries, 1977). However, when parts of the protein that were thought to facilitate this hydrogen bonding were mutated, the hypothesized decrease in antifreeze activity was not observed. Recent data suggests that hydrophobic interactions could be the main contributor (Haymet et al, 1998). It is difficult to discern the exact mechanism of binding because of the complex water-ice interface. Currently, attempts to uncover the precise mechanism are being made through use of molecular modelling programs (molecular dynamics or Monte Carlo method) (Madura, 2001; Jorov et al, 2004).
In the 1950s, Canadian scientist Scholander set out to explain how Arctic fish can survive in water colder than the freezing point of their blood. His experiments led him to the belief that there was “antifreeze” in the blood of Arctic fish (Madura, 2001). Then in the late 1960s, animal biologist Arthur DeVries was able to isolate the antifreeze protein through his investigation of Antarctic fish (De Vries and Wohlschlag, 1969). AFGPs were the first AFPs to be discovered. At the time they were called "glycoproteins as biological antifreeze agents" (De Veries and Wohlschlag, 1969). These proteins were later called antifreeze glycoproteins (AFGPs) or antifreeze glycopeptides to distinguish them from newly discovered non-glycoprotein biological antifreeze agents (AFPs). DeVries worked with Robert Feeney (1970) to characterize the chemical and physical properties of antifreeze proteins. In 1992, Griffith et al. documented their discovery of AFP in winter rye leaves. Around the same time, Urrutia, Duman and Knight (1992) documented thermal hysteresis protein in angiosperms. In 1993, Duman and Olsen noted that AFPs had also been discovered in over 23 species of angiosperms, including ones we eat. As well, they reported their presence in fungi and bacteria.
Recent attempts have been made to relabel antifreeze proteins as ice structuring proteins in order to more accurately represents their function and to rid of any assumed negative relation between AFPs and automotive antifreeze, ethylene glycol. These two things are completely separate entities bearing loose similarity only in their function.
Commercially, there appears to be infinite uses for antifreeze proteins. Numerous fields would be able to benefit from the protection of tissue damage by freezing. Businesses are currently investigating the use of these proteins in:
- increasing freeze tolerance of crop plants and extending the harvest season in cooler climates
- improving farm fish production in cooler climates
- lengthening shelf life of frozen foods
- improving cryosurgery
- a therapy for hypothermia
One recent, successful business endeavor has been the introduction of AFPs into ice cream and yogurt products. This ingredient, labelled ice-structuring protein, has been approved by the Food and Drug Administration. The proteins are isolated from fish and replicated, on a larger scale, in yeast.
There is concern from anti-genetically modified (GM) organizations, stating that modified antifreeze proteins may cause inflammation (Dortch, 2006). However, as stated, ISPs have been approved for human consumption following diligent tests. Intake of AFPs in diet is likely substantial in most northerly and temperate regions already (Crevel et al, 2002). Given the known historic consumption of AFPs, it is safe to conclude that their functional properties do not impart any toxicologic or allergenic effects in humans (Crevel et al, 2002).
As well, the transgenic process of ISP production is widely used in society already. This is how mass amounts of insulin are made to treat people with type I diabetes each year. The process does not impact the product, it merely makes production more efficient and prevents the death of many fish who would, otherwise, be killed for the extraction of such protein.
Currently Unilever incorporates AFPs into some of its products including some popsicles and a new line of Breyers Light Double Churned ice cream bars. In ice cream, AFPs allow the production of very creamy, dense, reduced fat ice cream with fewer additives. They control ice crystal growth brought on by thawing on the loading dock or kitchen table which drastically reduces texture quality (Regand et al, 2006).
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