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). Insulin crystals Recrystallization is an essentially physical process that has meanings in chemistry, metallurgy and geology. ... Two alpinists Acclimatization, uh kly muh tuh ZAY shuhn. ...

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, 2004) Ethylene glycol (monoethylene glycol (MEG), IUPAC name: ethane-1,2-diol) is an alcohol with two -OH groups (a diol), a chemical compound widely used as an automotive antifreeze. ... In chemistry, colligative properties are factors that determine how the properties of a bulk liquid solution change depending on the concentration of the solute in the bulk solution. ... Osmotic pressure or turgor (also called turgor pressure) is the pressure produced by a solution in a space that is enclosed by a differentially permeable membrane. ...

Thermal Hysteresis

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. ...

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). Clifton nanolitre osmometer setup A Clifton nanolitre osmometer is a device that allows the determination of the melting and freezing point of an aqueous solution. ... Spruce Budworm is a group of closely related insects in the genus Choristoneura. ...

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). Insulin crystals Recrystallization is an essentially physical process that has meanings in chemistry, metallurgy and geology. ...

Diversity

There are many known non homologous types of AFP.

FISH AFPs

Figure 1. The three faces of Type I AFP

Antifreeze glycoproteins or AFGPs are found in Antarctic notothenioids and northern cod. They are 2.6-3.3 kD (Crevel et al, 2002). Image File history File links AFPnew. ... Image File history File links AFPnew. ... A glycoprotein is a macromolecule composed of a protein and a carbohydrate (a sugar). ...

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). Binomial name Pseudopleuronectes americanus (Walbaum, 1792) The winter flounder, Pseudopleuronectes americanus, is a flatfish of the family Pleuronectidae. ...

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 II AFPs are found in sea raven, smelt and herring. They are cysteine rich globular proteins containing five disulfide bonds (Ng and Hew, 1992). Genera Blepsias Hemitripterus Nautichthys See text for species. ... Genera Allosmerus Hypomesus Mallotus Osmerus Spirinchus Thaleichthys Smelts are a family, Osmeridae, of small anadromous fish. ... Species Clupea alba Clupea bentincki Clupea caspiopontica Clupea chrysotaenia Clupea elongata Clupea halec Clupea harengus Clupea inermis Clupea leachii Clupea lineolata Clupea minima Clupea mirabilis Clupea pallasii Clupea sardinacaroli Clupea sulcata Herrings are small oily fish of the genus Clupea found in the temperate, shallow waters of the North Atlantic...

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). The eelpouts are a family (Zoarcidae) of perciform ray-finned fish. ...

Type IV AFPs are found in longhorn sculpins. They are alpha helical proteins rich in glutamate and glutamine (Deng et al, 1997).

INSECT AFPs

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.

PLANT AFPs

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)

3. most of them are evolved pathogenesis-related proteins, sometimes retaining antifungal activities (Griffith and Yaish, 2004). Something antifungal kills or inhibits the growth of fungus. ...

Evolution

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: In evolutionary biology, convergent evolution is the process whereby organisms not closely related, independently evolve similar traits as a result of having to adapt to similar environments or ecological niches. ...

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) A protein primary structure is a chain of amino acids. ... In biochemistry, the tertiary structure of a protein is its overall shape. ...

Convergent evolution of antifreeze glycoproteins in Antarctic notothenioid fish and Arctic cod

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). Adsorption is a process that occurs when a gas or liquid or solute (called adsorbate) accumulates on the surface of a solid or more rarely a liquid (adsorbent), forming a molecular or atomic film (adsorbate). ... In chemistry, the term van der Waals force originally referred to all forms of intermolecular forces; however, in modern usage it tends to refer to intermolecular forces that deal with forces due to the polarization of molecules. ...

Binding to ice

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 at al, 2004). Molecular modelling is a collective term that refers to theoretical methods and computational techniques to model or mimic the behaviour of molecules. ... Molecular dynamics (MD) is a form of computer simulation where atoms and molecules are allowed to interact for a period of time under known laws of physics. ... Monte Carlo methods are a widely used class of computational algorithms for simulating the behavior of various physical and mathematical systems, and for other computations. ...

History

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. Classes Magnoliopsida - Dicots Liliopsida - Monocots The flowering plants (also angiosperms or Magnoliophyta) are one of the major groups of modern plants, comprising those that produce seeds in specialized reproductive organs called flowers, where the ovulary or carpel is enclosed. ...

Name Change

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. Ethylene glycol (monoethylene glycol (MEG), IUPAC name: ethane-1,2-diol) is an alcohol with two -OH groups (a diol), a chemical compound widely used as an automotive antifreeze. ...

Ice structuring proteins - a new name for antifreeze proteins

Commercial Use

Commercially, there appears to be infinite uses for antifreeze proteins. Numerous fields would be able to benefit for 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 Cryosurgery (cryotherapy) is the application of extreme cold to destroy abnormal or diseased tissue. ...

- enhancing preservation of tissues for transplant or transfusion in medicine

- a therapy for hypothermia

Currently two companies market AFPs: A/F Protein Inc and Ice Biotech Inc

Antifreeze proteins and their genes: From basic research to business opportunity

Recent News

One recent, successful business endeavor has been the introduction of AFPs into ice cream. 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. The Food and Drug Administration (FDA) is an agency of the United States Department of Health and Human Services and is responsible for regulating food (humans and animal), dietary supplements, drugs (human and animal), cosmetics, medical devices (human and animal) and radiation emitting devices (including non-medical devices), biologics, and...

There is concern from anti-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). As well, it is safe to conclude from the history of AFP consumption that their functional properties do not impart any toxicologically significant effect (Crevel et al, 2002). Furthermore, consumption data on fish AFPs suggest that there is no allergenic effect (Crevel et al, 2002). An allergen is any substance (antigen), most often eaten or inhaled, that is recognized by the immune system and causes an allergic reaction. ...

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. A genetically modified organism is an organism whose genetic material has been deliberately altered. ... Insulin (from Latin insula, island, as it is produced in the Islets of Langerhans in the pancreas) is a polypeptide hormone that regulates carbohydrate metabolism. ... This article is about the disease that features high blood sugar. ...

Currently AFPs are used by Unilever to make products like 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). Unilever (Euronext: UNA, LSE: ULVR, NYSE: UN) is an Anglo-Dutch company that owns many of the worlds consumer product brands in foods, beverages, cleaning agents and personal care products. ... Not to be confused with Dreyers. ...

Creamy, Healthier Ice Cream? What’s the Catch?

References

• (Crevel et al, 2002) - R.W.R Crevel, J.K. Fedyk and M.J. Spurgeon. (2002). Antifreeze proteins: characteristics, occurrence and human exposure (Review). Food and Chemical Toxicology 20, 899-903
• (Deng et al, 1997) - G. Deng, D.W. Andrews and R.A. Laursen. (1997). Amino acid sequence of a new type of antifreeze protein, from the longhorn sculpin Myoxocephalus octodecimspinosis. FEBS Lett. 402:1, 17-20.
• (De Vries and Wohlschlag, 1969) - A.L. De Vries and D.E. Wohlschlag. (1969). Freezing resistance in some Antarctic fishes. Science 163, 1074–1075.
• (De Vries et al, 1970) - A.L. De Vries, S.K. Komatsu and R.E. Feeney. (1970). Chemical and physical properties of freezing point-depressing glycoproteins from Antarctic fishes. J Biol Chem. 245:11, 2901-8.
• (Dortch, 2006) - Dortch, Eloise. (2006). Fishy GM yeast used to make ice-cream. Network of Concerned Farmers. Retrieved October 09, 2006
• (Duman and DeVries, 1976) - J. Duman and A.L. DeVries. (1976). Isolation, characterization and physical properties of protein antifreezes from the Winter Flounder Pseudopleunectus Americanus. Comp. Biochem. Physiol. B54, 375–380
• (Duman and Olsen, 1993) - J.G Duman and T.M. Olsen. (1993). Thermal hysteresis protein activity in bacteria, fungi and phylogenetically diverse plants. Cryobiology 30, 322–328.
• (Duman, 2001) - J.G. Duman. (2001). Antifreeze and Ice Nucleator Proteins in Terrestrial Arthropods. Annu. Rev. Physiol. 63, 327–57
• (Fletcher et al, 2001) - G.L. Fletcher, C.L. Hew, and P.L. Davies. (2001). Antifreeze Proteins of Teleost Fishes. Annu. Rev. Physiol. 63, 359–90
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• (Haymet et al, 1999) - A. Haymett, L. Ward and M. Harding (1999). Winter Flounder 'anti-freeze' proteins: Synthesis and ice growth inhibition of analogues that probe the relative importance of hydrophobic and hydrogen bonding interactions. J. Am. Chem. Soc. 121. 941-948
• (Jorov et al, 2004) - A. Jorov, B.S. Zhorov and D.S. Yang. (2004). Theoretical study of interaction of winter flounder antifreeze protein with ice. Protein Science. 13, 1524-1537
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• (Regand et al, 2006) - A. Regand, HD. Goff et al. (2006). Ice recrystallization inhibition in ice cream as affected by ice structuring proteins from winter wheat grass. J Dairy Sci. 89:1, 49-57.
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External links

• Cold, Hard Fact: Fish Antifreeze Produced in Pancreas