Antifreeze Proteins: From Nature to Industry
Monday, October 1, 2001 11:16 IST
ey were first discovered in the blood of Antarctic fish species, where they act in a true antifreeze role.A number of insect species, such as the spruce budworm, also resist freezing throughout the winter and accumulate AFPs to levels that lower their freezing points.In these species, the role of AFPs is analogous to those of fish.AFPs are also present in other insects and plants.AFPs are likely to play their role by preventing ice recrystallisation by binding to ice crystals and inhibiting crystal growth.
The recrystallisation of ice refers to the coalescence of small crystals into larger ones that can occur at high subzero temperatures. Laboratory studies have shown that AFPs are remarkably effective inhibitors of ice recrystallisation and at concentrations far lower than those required for detectable antifreeze activity.
Antifreeze protein studies were started out initially as a biological curiosity, and now have emerged as a valuable tool in food science, medicine, and biotechnology.AFPs can be added to living tissues, foods, and other materials to depress the freezing point non-colligatively or to allow freeze/thaw without ice recrystallisation.The studies of these proteins seek to answer fundamental questions concerning their molecular structures, mechanism of action, or roles in the survival of the species that produce them.
More recently, AFPs have been shown to have new uses in the laboratory that differ from their known roles in nature and those too have general interest in their applications.For this reason and others, AFPs show promise in several different areas and there is great interest in understanding precisely how they work.
Although AFPs show diverse structural features among certain closely related species, all have similar effects on ice-crystal growth.Individual AFP molecules appear to adsorb directly on to ice crystal surfaces. Crystal growth inhibition is assumed to result from the Kelvin effect, in which the curvature of an ice surface growing out in between two adsorbed AFP molecules, reaches a limit beyond which further ice growth is energetically unfavorable.Although antifreeze activities of different AFPs vary widely, each appears to reach its particular maximum with increasing protein concentrations.Moreover, all AFPs tested to date show both antifreeze activity and inhibition of ice recrystallisation, suggesting a common mechanism for these two effects mediated through ice binding.
Charles Knight of the National Center for Atmospheric Research, Colorado, US, with series of experiments, showed that binding of AFPs to ice is geometrically specific.Ice hemispheres were grown in the presence of low concentrations of different fish AFPs and planes of AFP binding to ice were identified through the patterns of surface roughening.The AFPs showed distinct binding planes.The precise interactions mediating AFP-ice binding are not yet well defined, though the crystal structures of several fish AFPs have been determined.The surfaces and specific amino acid residues mediating ice binding are now known. But the precise constellation of ice-protein interaction is not known in any AFP.
Applications in Food Science
Ice recrystallisation (also referred to as freeze ripening or freezer burn) compromises the value of foods that are frozen at high or variable temperatures.Food storage often requires costly measures, including quick freezing and maintenance of stable low temperatures, to prevent recrystallisation.The addition of AFPs to food products should inhibit recrystallisation at high and fluctuating subzero temperatures.The concentration of AFP required is normally very low ¾ of the order of 10-5M.Moreover, in some foods, AFPs are naturally present under appropriate conditions.Therefore, the cost of using AFPs for this purpose would, in some cases, be modest or negligible.
Stephen Payne and colleagues at Meat Industry Research Institute, New Zealand, investigated the effect of various fish AFPs on the quality of frozen meat, and found that drip loss ¾ a consequence of recrystallisation damage ¾ was reduced in meat from lambs injected with fish antifreeze to a final concentration of 0.1(g/Kg prior to slaughter.Soaking the meat in an antifreeze solution gave a similar result.
American scientists found that addition of AFP to various ice cream products also prevented the formation of large ice crystals and AFP genes are being introduced for the same purpose into microorganisms that are used to produce frozen yoghurt.
Applications In Medicine
AFPs can enhance the freeze stability of cells and tissues for medical or veterinary purpose in exactly the same way as they do for foods.For example, low concentrations of AFP increase the freeze survival of red blood cells, presumably by inhibiting ice recrystallisation.However, higher levels of AFP can cause tissue damage, which is believed to result from piercing of the cell plasma membrane by the ''sharp'' spicular or bipyramidal ice crystals that are formed at high antifreeze concentrations. This effect of cell destruction at high AFP concentrations (10-3 M range), which could be problematic in food or tissue preservation, may be valuable in cryosurgery. The objective in cryosurgery is to destroy all the targeted tissue without causing undue damage to surrounding tissues. The bipyramidal or spicular ice crystals that grow in solutions containing high concentrations of AFP or other effects of AFPs on cell freezing aid the killing of cells during cryosurgery. AFPs may prove to be an important tool in cryosurgery because, under the thermal conditions used in a group''s experiment, at temperatures as high as -20 C, cell death approximated 100%.
It is shown that AFPs from Antarctic fishes interact directly with liposomes and prevent damage over cooling and warming between 2 and 20o C, which spans the lipid transition temperature. This hypothermic protection of membrane integrity may be useful in cool temperature storage of cells and tissues.
Frozen Future of AFPs?
The successful application of AFPs in food preservation or cryosurgery have created an exceptional future for AFPs and further investigation in other applications, such as vitrification, or hypothermic membrane protection, is needed.For applications such as food preservation, natural AFPs, purified or partially purified, from fish blood may be a convenient source that is likely to meet consumer acceptance. Abundant AFPs could be generated as by-products of processing certain fish species for food, thereby enhancing the sustainability and profitability of fisheries and aquaculture industries.
For medical applications, such as commercial scale cell or tissue preservation, or cryosurgery, supply of large amounts of well-defined AFPs would be a requisite. This might be supplied by recombinant DNA technology, although high cost of expressed protein would be a limitation.
Although no mimetic has been made for AFPs to date, required for large-scale industrial application, synthetic and possibly improved AFPs could be prepared from relatively simple components. Therefore, AFPs will continue to find many valuable and sustainable uses, even before their mechanism of action becomes crystal clear.
About the author:
The author is with UDCT, Mumbai