Presence Of Antifreeze Proteins Research Articles

Freezing-enhanced oxidation of iodide by hydrogen peroxide in the presence of antifreeze proteins from the Arctic yeast Leucosporidium sp.AY30

Ice-binding proteins (IBPs), originating from Arctic or Antarctic microorganisms, have freeze-inhibiting characteristics, allowing these organisms to survive in polar regions. Despite their significance in polar environments, the mechanism through which IBPs affect the chemical reactions in ice by controlling ice crystal formation has not yet been reported. In this study, a new mechanism for iodide (I−) activation into triiodide (I3−), which is the abundant iodine species in seawater, by using hydrogen peroxide (H2O2) in a frozen solution with IBPs was developed. A significant enhancement of I− activation into I3− was observed in the presence of Arctic-yeast-originating extracellular ice-binding glycoprotein (LeIBP) isolated from Leucosporidium sp. AY30, and a further increase in the I3− concentration was observed with the introduction of H2O2 to the frozen solution (25 times higher than in the aqueous solution after 24 h of reaction). The reaction in the ice increased with an increase in LeIBP concentration. The in-situ pH measurement in ice using cresol red (CR) revealed protons accumulated in the ice grain boundaries by LeIBP. However, the presence of LeIBP did not influence the acidity of the ice. The enhanced freeze concentration effect of H2O2 by LeIBP indicated that larger ice granules were formed in the presence of LeIBP. The results suggest that LeIBP affects the formation and morphology of ice granules, which reduces the total volume of ice boundaries throughout the ice. This leads to an increased local concentration of I− and H2O2 within the ice grain boundaries. IBP-assisted production of gaseous iodine in a frozen environment provides a previously unrecognized formation mechanism of active iodine species in the polar regions.

Protective role of antifreeze proteins on sterlet (Acipenser ruthenus) sperm during cryopreservation.

The loss of sperm quality in sterlet (Acipenser ruthenus) due to freeze-thaw process in cryopreservation was investigated in the present study. Two antifreeze proteins (AFPI or AFPIII) were used at different concentrations of 0.1, 1, 10, and 100μg/mL. We compared motility, curvilinear velocity, and plasma membrane integrity of fresh, cryopreserved sperm, and sperm cryopreserved in the presence of antifreeze proteins. Fresh sperm (control) had 85 ± 4% motility and 160 ± 2μm/s curvilinear velocity, respectively. After cryopreservation, the motility of frozen-thawed sperm without addition of antifreeze proteins significantly decreased (44 ± 9%), compared to the control. The highest motility of frozen-thawed sperm was obtained in cryopreserved sperm with addition of 1μg/mL of AFPIII (58 ± 14%). No significant differences were observed in curvilinear velocity between fresh sperm and cryopreserved sperm with/without addition of AFPI or AFPIII. The flow cytometry analysis revealed that fresh sperm contained 94.5 ± 6% live cells, while the cryopreserved sperm only contained 26.6 ± 14% live cells. Supplementation of antifreeze proteins has significantly improved the percentage of live cells in frozen-thawed sperm, except 0.1μg/ml of AFPI group. No significant difference in percentage of live cells was detected in the sperm cryopreserved with 10μg/mL of AFPI or AFPIII, compared to fresh sperm. Thus, addition of antifreeze proteins to cryopreservation medium could be considered to improve the post-thawed sperm quality of sterlet.

Phase equilibria, kinetics and morphology of methane hydrate inhibited by antifreeze proteins: application of a novel 3-in-1 method

The action of three distinct recombinant antifreeze proteins (AFPs) as methane hydrate inhibitors was examined using a recently-developed reactor. Compared with traditional approaches, this reactor uses minimal reactant volumes and short experimentation times to assess phase equilibria, kinetics and morphology of a hydrate system in a single experiment (3-in-1). Two of the recombinant AFPs are considered highly active with respect to the inhibition of ice: ‘Maxi’, a fish AFP, and a beetle AFP (TmAFP). The third protein from a grass, is classified as a low activity AFP (LpAFP). ‘Maxi’, an AFP that has not been tested previously as a hydrate inhibitor, slowed hydrate growth rates up to an order of magnitude compared to pure water. TmAFP and LpAFP also exhibited kinetic inhibition, but were less effective than ‘Maxi’. In the presence of AFPs, hydrate films were thinner and showed a single growth mechanism compared to multiple crystal growth mechanisms observed in control experiments. The addition of TmAFP generated large irregular hydrate halos that propagated outside the original water boundary. Halo propagation was somewhat less prominent with LpAFP, and was not observed with ‘Maxi’. Although, none of the AFP’s showed thermodynamic inhibition properties, ‘Maxi’ appeared to form clusters of hydrate which remained metastable in the liquid–vapour region.

Antifreeze proteins in naturally cold acclimated leaves of Drimys angustifolia, Senecio icoglossus, and Eucalyptus ssp.

Summary Antifreeze proteins (AFPs) present in plants may inhibit ice recrystallization even at low concentrations, and show potential application to many frozen foods. This study evaluated the presence of antifreeze proteins in naturally cold acclimated and non-acclimated leaves of Drimys angustifolia, Senecio icoglossus and Eucalyptus ssp. No proteins were detected in apoplastic extracts of Eucalyptus ssp. Extracts of cold acclimated and non-acclimated S. icoglossus showed protein concentrations of 42.89 and 17.76 µg mL-1, both with bands between 25 and 37 kDa in the SDS-PAGE. However, they did not inhibit recrystallization. The extract of cold acclimated D. angustifolia contained a protein concentration of 95.17 µg mL-1, almost five times higher than the extract of non-acclimated D. angustifolia. In the extract of cold acclimated D. angustifolia, there was presence of ice recrystallization inhibitors. This extract showed a protein band just below 37 kDa and another more intense band between 20 and 25 kDa. It is the first time that the presence of antifreeze proteins in this species is being described.

Induced ice melting by the snow flea antifreeze protein from molecular dynamics simulations.

Antifreeze proteins (AFP) allow different life forms, insects as well as fish and plants, to survive in subzero environments. AFPs prevent freezing of the physiological fluids. We have studied, through molecular dynamics simulations, the behavior of the small isoform of the AFP found in the snow flea (sfAFP), both in water and at the ice/water interface, of four different ice planes. In water at room temperature, the structure of the sfAFP is found to be slightly unstable. The loop between two polyproline II helices has large fluctuations as well as the C-terminus. Torsional angle analyses show a decrease of the polyproline II helix area in the Ramachandran plots. The protein structure instability, in any case, should not affect its antifreeze activity. At the ice/water interface the sfAFP triggers local melting of the ice surface. Bipyramidal, secondary prism, and prism ice planes melt in the presence of AFP at temperatures below the melting point of ice. Only the basal plane is found to be stable at the same temperatures, indicating an adsorption of the sfAFP on this ice plane as confirmed by experimental evidence.

Dynamical mechanism of antifreeze proteins to prevent ice growth.

The fascinating ability of algae, insects, and fishes to survive at temperatures below normal freezing is realized by antifreeze proteins (AFPs). These are surface-active molecules and interact with the diffusive water-ice interface thus preventing complete solidification. We propose a dynamical mechanism on how these proteins inhibit the freezing of water. We apply a Ginzburg-Landau-type approach to describe the phase separation in the two-component system (ice, AFP). The free-energy density involves two fields: one for the ice phase with a low AFP concentration and one for liquid water with a high AFP concentration. The time evolution of the ice reveals microstructures resulting from phase separation in the presence of AFPs. We observed a faster clustering of pre-ice structure connected to a locking of grain size by the action of AFP, which is an essentially dynamical process. The adsorption of additional water molecules is inhibited and the further growth of ice grains stopped. The interfacial energy between ice and water is lowered allowing the AFPs to form smaller critical ice nuclei. Similar to a hysteresis in magnetic materials we observe a thermodynamic hysteresis leading to a nonlinear density dependence of the freezing point depression in agreement with the experiments.

Insights into the Behavior of Biological Clathrate Hydrate Inhibitors in Aqueous Saline Solutions

The performance of two antifreeze proteins (AFPs), type I and III (AFP I and III), as kinetic natural gas hydrate inhibitors in saline solutions was evaluated using a high pressure micro differential scanning calorimeter and a high pressure apparatus consisting of two crystallizers. Although AFP I and III were found to prolong crystallization time and reduce initial gas hydrate growth in saline solutions, the growth rate increased when hydrate started to form in the gas phase. Circular dichroism experiments suggested that the saline solution did not perturb AFP I and III structures. However, in the presence of saline, the inhibitory activity of AFP I to prolong induction time decreased while AFP III was more active. As a consequence, we propose that a decrease in hydrophobic forces and the neutralization of ion charges could explain AFP adsorption to the surface of hydrate crystals. Once the hydrate formed, melting was delayed, and consequently hydrate decomposition took longer in the presence of AFPs. We...

142 Ice growth in the presence of an antifreeze protein

Antifreeze proteins (AFPs) have evolved in cold-adapted organisms to control ice crystal growth when exposed to sub-zero temperature conditions. It has been suggested that the effect of the proteins results in small ice crystal size, thus avoiding the mechanical damage to frozen tissues and cells caused by large ice grains. The polar diatom Fragilariopsis cylindrus , a dominant species within sea-ice assemblages, produces AFPs. We expressed in E. coli a recombinant form of this protein and isolated it by affinity chromatography. We studied its effect on ice grain size after shock-freezing and subsequent annealing, and under slow freezing conditions. Shock-freezing (−40 °C) produced small sized crystals, and during annealing at −4 °C AFPs successfully inhibited recrystallization already at low concentrations (1.2 μM), as observed at light microscopy and using the Otago optical recrystallometer. However, slow ice growth at −5 °C, more likely to resemble natural freezing conditions, surprisingly resulted in the formation of larger crystals in the presence of AFPs than in the negative controls. Further characteristic microstructural features, such as gradual c -axis transition within individual grains and sublimation etching patterns, were observed under crossed polarizers and at light microscopy. These features are possibly due to the incorporation of proteins into the ice lattice during growth, causing local defects. Our observations remain to be clarified, but should be taken into account when considering the biological role of AFPs as well as potential industrial applications of the proteins. Source of funding: Public funding by German AiF/IGF. Conflict of interest: None declared. maddalena.bayer@awi.de

Antifreeze protein-induced selective crystallization of a new thermodynamically and kinetically less preferred molecular crystal.

The formation of a new, dihydrate crystalline form of 5-methyluridine (m(5)U) was selectively induced by a protein additive, antifreeze protein (AFP) in a highly efficient manner (in 10(-6) molar scale, whereas known kinetic additives need 0.1 molar scale). The hemihydrate form (form I, the only previously known crystalline form of m(5)U) and the dihydrate form of m(5)U (form II) obtained herein were characterized using X-ray crystallography and differential scanning calorimetry (DSC). Compared to form I, remarkably, form II is thermodynamically and kinetically less preferred. The presence of AFP can selectively inhibit the appearance of form I and hence allows the growth of form II, the pure form of which cannot grow directly from m(5) U supersaturated solutions under the same conditions. An explanation supported by both experimental and theoretical results is provided for the AFP-induced selection process. Implications on AFP-induced ice shape changes are also discussed. Control of crystallization from supersaturated solutions is of great interest in both fundamental research and practical applications in fields like chemistry, pharmacology and materials science. These findings suggest that crystallization processes with AFPs could be valuable for selective growth of hydrates and polymorphs of important pharmaceutical compounds.

Inhibition of Ice Growth and Recrystallization by Zirconium Acetate and Zirconium Acetate Hydroxide

The control over ice crystal growth, melting, and shaping is important in a variety of fields, including cell and food preservation and ice templating for the production of composite materials. Control over ice growth remains a challenge in industry, and the demand for new cryoprotectants is high. Naturally occurring cryoprotectants, such as antifreeze proteins (AFPs), present one solution for modulating ice crystal growth; however, the production of AFPs is expensive and inefficient. These obstacles can be overcome by identifying synthetic substitutes with similar AFP properties. Zirconium acetate (ZRA) was recently found to induce the formation of hexagonal cavities in materials prepared by ice templating. Here, we continue this line of study and examine the effects of ZRA and a related compound, zirconium acetate hydroxide (ZRAH), on ice growth, shaping, and recrystallization. We found that the growth rate of ice crystals was significantly reduced in the presence of ZRA and ZRAH, and that solutions containing these compounds display a small degree of thermal hysteresis, depending on the solution pH. The compounds were found to inhibit recrystallization in a manner similar to that observed in the presence of AFPs. The favorable properties of ZRA and ZRAH suggest tremendous potential utility in industrial applications.

February 2013: This Month in JoVE

Here's a look at what's coming up in the February 2013 issue of Journal of Visualized Experiments (JoVE). Since JoVE's first issue, biomedical research has comprised the predominate fraction of our content, and we've got sections for Neuroscience, Clinical and Translational Medicine, Bioengineering, and Immunology. In July 2012, we branched out to the physical sciences with the launch of JoVE Applied Physics. This month, we are pleased to release the first video-articles in JoVE Chemistry, which features subjects ranging from complex biochemistry, to assay development, to chemical engineering, and organic synthesis. In the chemical engineering department, Pandey et al. combine planar lithography with self-folding methods to create 3-dimensional structures inspired by the art of origami. Through exposure to heat or magnetic fields, these microstructures can be induced to fold over objects, and can be used to excise tissue - an application that could translate to taking nanoscale biopsies in vivo. Moving on to biochemistry, Braslavsky and Drori present an experiment designed to measure the effects of ice-binding proteins on ice crystal formation. Through video microscopy and subsequent video analysis, thermal hysteresis-the difference between freezing and melting temperatures-can be determined in the presence of antifreeze proteins, which protect many organisms like bacteria and plants from freezing temperatures. In JoVE Applied Physics, Baranec et al. aim for the stars in our very first astronomy article, which brings the visible universe into focus with the Robo-AO laser adaptive optics system. With the help of a high-power laser beam, this system corrects the incoming light waves that get distorted by atmospheric turbulence so objects in the sky can be seen with high resolution. The authors plan to use this technique to find planets beyond our Solar System, and make precise observations of astronomical phenomena. Looking back at Earth, Qi and Du examine how human movements in spatial and temporal dimensions can be used to study disease transmission. GPS-enabled devices can be used to automatically collect human trajectory data, which the authors process using a novel interactive interface. The trajectories can also be animated in Google Earth. These methods can help detect hot spots for the transmission of infectious diseases, such as influenza. In JoVE Clinical and Translational Medicine, Kim presents a non-invasive method for imaging fetal mouse hearts in utero using high-frequency ultrasonography. This procedure, also called echocardiography, can be used to study normal heart structure and function as well as the mechanisms of cardiovascular disease. It can even be used for the early diagnosis of heart defects, making this a powerful tool with many scientific and clinical applications. From visualizing the far reaches of space, to analyzing human movements in space and time, you've just caught a glimpse of JoVE's new content for February 2013. Like the universe, we continue to expand, and this month marks our arrival at a new frontier with the launch of Chemistry in JoVE: The Journal of Visualized Experiments.

Thermolabile antifreeze protein produced in Escherichia coli for structural analysis

The only hyperactive antifreeze protein (AFP) found to date in fishes is an extreme variant of the 3-kDa, alpha-helical, alanine-rich type I AFP, which is referred to here as type Ih. Purification of the 33-kDa homodimeric AFP Ih from a natural source was hampered by its low levels in fish plasma; by the need to remove the more abundant smaller isoforms; and by its extreme thermolability. Moreover, ice affinity as a purification tool was spoiled by the tendency of fish IgM antibodies to bind to ice in the presence of AFPs. In order to produce enough protein for crystallography we expressed AFP Ih as a recombinant protein in the Arctic Express® strain of Escherichia coli at 12°C, just below the thermal denaturation temperature of 16–18°C. His-tags were not useful because they compromised the activity and yield of AFP Ih. But in the absence of fish antibodies we were able to recover 10-mg quantities of the antifreeze protein using two cycles of ice affinity purification followed by anion-exchange chromatography to remove contaminating chaperones. The purified recombinant AFP Ih yielded diffraction-quality crystals with an extremely asymmetrical unit cell. By transferring the genes of the chaperones into a methionine auxotroph we were able to grow this host at low temperatures and produce sufficient selenomethionine-labeled AFP Ih for crystallography.

Construction of Time-Lapse Scanning Electrochemical Microscopy with Temperature Control and Its Application To Evaluate the Preservation Effects of Antifreeze Proteins on Living Cells

Antifreeze proteins (AFPs) can protect cells from hypothermic damage; however, their mechanism of action remains unclear. Scanning electrochemical microscopy (SECM) can evaluate the size and activities of cells, although long-term continuous monitoring has been unsuccessful. We constructed a novel, fully automated, time-lapse SECM system and investigated the cell preservation effect of AFPs by analyzing single cellular topography at low temperatures. From the SECM measurements, mammalian cells (HepG2), treated in Euro-Collins (EC) solution at 4 degrees C, began to swell at 8 h and then immediately ruptured. In AFP-containing EC solution, the cellular size did not change until 16 h and then gradually increased and finally ruptured. In addition, the cellular height at rupture point significantly increased in the presence of AFPs. These results suggest that AFPs stabilize the cellular membrane and protect cells from hypothermic damage. This SECM system allowed us to observe the single cellular response to hypothermia by long-term automatic scanning and will be applicable for analysis to other cellular activities and topographies.

Determination of the expression of fish antifreeze protein (AFP) in 7th generation transgenic mice tissues and serum

In this study, the presence of antifreeze protein (AFP) gene expression through successive generations in transgenic mice carrying the chimeric gene construct of the coding sequence for the AFP protein from ocean pout was investigated. AFP transgenic hemizygote mice were used for AFP gene expression. AFP genome expressions in transgenic mice were analyzed by Western blotting, and tissue location of AFP protein was shown by immunohistochemical and immunofluorescence techniques. Seventh transgenic mice from the established founders demonstrated the expression of AFP in organs such as the skin, oviduct, lung, kidney and liver tissues and serum except for the heart. Our results demonstrate successful expression of AFP gene products in several tissues and serum of transgenic mice, the association of in vivo expressed AFP protein, for the first time. These results indicate that the coding sequence for the AFP protein gene (ocean pout type III AFP gene) could be integrated and stably transcribed and expressed in the 7th generation of transgenic mice. In conclusion transgenic mouse lines would be a good model for the cryostudy of AFP and for the determination of AFP roles in several organs and tissues.

Sperm cryoconservation in Anarhichas sp., endangered cold-water aquaculture species with internal fertilization

This study contributes to the identification of an adequate cryopreservation medium for wolffishes sperm (Anarhichas minor and A. lupus). Our results also confirm the presence of antifreeze proteins in the seminal fluid of the Atlantic wolffish (Anarhichas lupus) that could facilitate the process of cryoconservation in association with increased post-thaw motility and fertilization rates. Cryo-Fish, a commercial diluent was the most efficient in our trials, with 80% motility post-thaw.

The mechanism by which fish antifreeze proteins cause thermal hysteresis

Antifreeze proteins are characterised by their ability to prevent ice from growing upon cooling below the bulk melting point. This displacement of the freezing temperature of ice is limited and at a sufficiently low temperature a rapid ice growth takes place. The separation of the melting and freezing temperature is usually referred to as thermal hysteresis, and the temperature of ice growth is referred to as the hysteresis freezing point. The hysteresis is supposed to be the result of an adsorption of antifreeze proteins to the crystal surface. This causes the ice to grow as convex surface regions between adjacent adsorbed antifreeze proteins, thus lowering the temperature at which the crystal can visibly expand. The model requires that the antifreeze proteins are irreversibly adsorbed onto the ice surface within the hysteresis gap. This presupposition is apparently in conflict with several characteristic features of the phenomenon; the absence of superheating of ice in the presence of antifreeze proteins, the dependence of the hysteresis activity on the concentration of antifreeze proteins and the different capacities of different types of antifreeze proteins to cause thermal hysteresis at equimolar concentrations. In addition, there are structural obstacles that apparently would preclude irreversible adsorption of the antifreeze proteins to the ice surface; the bond strength necessary for irreversible adsorption and the absence of a clearly defined surface to which the antifreeze proteins may adsorb. This article deals with these apparent conflicts between the prevailing theory and the empirical observations. We first review the mechanism of thermal hysteresis with some modifications: we explain the hysteresis as a result of vapour pressure equilibrium between the ice surface and the ambient fluid fraction within the hysteresis gap due to a pressure build-up within the convex growth zones, and the ice growth as the result of an ice surface nucleation event at the hysteresis freezing point. We then go on to summarise the empirical data to show that the dependence of the hysteresis on the concentration of antifreeze proteins arises from an equilibrium exchange of antifreeze proteins between ice and solution at the melting point. This reversible association between antifreeze proteins and the ice is followed by an irreversible adsorption of the antifreeze proteins onto a newly formed crystal plane when the temperature is lowered below the melting point. The formation of the crystal plane is due to a solidification of the interfacial region, and the necessary bond strength is provided by the protein “freezing” to the surface. In essence: the antifreeze proteins are “melted off” the ice at the bulk melting point and “freeze” to the ice as the temperature is reduced to subfreezing temperatures. We explain the different hysteresis activities caused by different types of antifreeze proteins at equimolar concentrations as a consequence of their solubility features during the phase of reversible association between the proteins and the ice, i.e., at the melting point; a low water solubility results in a large fraction of the proteins being associated with the ice at the melting point. This leads to a greater density of irreversibly adsorbed antifreeze proteins at the ice surface when the temperature drops, and thus to a greater hysteresis activity. Reference is also made to observations on insect antifreeze proteins to emphasise the general validity of this approach.

Activity and dormancy in relation to body water and cold tolerance in a winter-active springtail (Collembola)

Ceratophysella sigillata (Collembola, Hypogastruridae) has a life cycle which may extend for >2 years in a temperate climate. It exists in two main morphs, a winter-active morph and a summer-dormant morph in central European forests. The winter-active morph often occurs in large aggregations, wandering on leaf litter and snow surfaces and climbing on tree trunks. The summer-dormant morph is found in the upper soil layers of the forest floor. The cryobiology of the two morphs, sampled from a population near Bern in Switzerland, was examined using Differential Scanning Calorimetry to elucidate the roles of body water and the cold tolerance of individual springtails. Mean (SD) live weights were 62 +/- 16 and 17 +/- 6 mug for winter and summer individuals, respectively. Winter-active springtails, which were two feeding instars older than summer-dormant individuals, were significantly heavier (by up to 4 times), but contained less water (48% of fresh weight [or 0.9 g g(-1) dry weight]) compared with summer-dormant animals (70% of fresh weight [or 2.5 g g(-1) dry weight]). Summer-dormant animals had a slightly greater supercooling capacity (mean (SD) -16 +/- 6degreesC) compared with winter-active individuals (-12 +/- 3degreesC), and they also contained significantly larger amounts of both total body water and osmotically inactive (unfrozen) water. In the summer morph, the unfrozen fraction was 26%, compared to 11% in the winter morph. The ratio of osmotically inactive to osmotically active (freezable) water was 1 : 1.7 (summer) and 1 : 3.3 (winter); thus unfrozen water constituted 59% of the total body water during summer compared with only 30% in winter. Small, but significant, levels of thermal hysteresis were detected in the winter-active morph (0.15degreesC) and in summer-dormant forms (0.05degreesC), which would not confer protection from freezing. However, the presence of antifreeze proteins may prevent ice crystal growth when feeding on algae with associated ice crystals during winter. It is hypothesised that in summer animals a small decrease in freezable water results in a large increase in haemolymph osmolality, thereby reducing the vapour pressure gradient between the springtail and the surrounding air. A similar decrease in freezable water in winter animals will not have such a large effect. The transfer of free water into the osmotically inactive state is a possible mechanism for increasing drought survival in the summer-dormant morph. The ecophysiological differences between the summer and winter forms of C. sigillata are discussed in relation to its population ecology and survival.

Three-dimensional simulation of ice growth in the presence of antifreeze proteins

A Monte Carlo computational method for simulating the growth of entire ice crystals from the liquid phase has been developed specifically to study the inhibition of ice-crystal growth by antifreeze proteins (AFPs). AFPs are found in the fluids of certain organisms that inhabit freezing environments and constrain ice-crystal growth by adsorbtion to the ice surface, but their inhibition mechanism is still poorly understood. Thus, it was of interest to incorporate these molecules into the dynamic ice simulations to examine the inhibition phenomenon on a whole-crystal basis. We have undertaken simulations with AFPs from two different organisms that differ in activity; the insect AFP has up to 100 times the activity of the fish AFP on a molar basis. Simulations involving insect and fish AFPs have achieved ice-growth inhibition at simulation temperatures within reported activity ranges for both fish and insect AFPs, accompanied by resulting ice morphologies similar to those observed experimentally. These results, as well as other studies on ice-etching patterns and ice burst growth at temperatures below known AFP ice-growth inhibition abilities suggest that AFP activity is dominated by the AFP ice-binding position rather than AFP ice-binding strength. PACS No.: 07.05T

Hexagonal shaped ice spicules in frozen antifreeze protein solutions

In the presence of antifreeze proteins from both Antarctic and Arctic fishes, water freezes in the form of long c-axis spikes or spicular-like crystals. Transmission electron microscopy of the Pt/C replicas of the freeze fractured spicular ice in a small capillary revealed the presence of many hexagonally shaped structures whose cross-sectional dimensions were between 0.5 and 10 μm. Well-defined parallel faces were associated with most fractured and etched spicules. When fracture planes occurred near the tip of a spicule, well-defined pyramidal faces were apparent. Steps were sometimes associated with these pyramidal spicular crystal faces. On some of the replicas obvious roughening of certain crystal faces of the spicule was observed, suggesting that the antifreeze proteins may have adsorbed to those faces.

Freezing survival, body ice content and blood composition of the freeze-tolerant European common lizard, Lacerta vivipara.

To investigate the freeze tolerance of the European common lizard, Lacerta vivipara, we froze 17 individuals to body temperatures as low as -4 degrees C under controlled laboratory conditions. The data show that this species tolerates the freezing of 50% of total body water and can survive freezing exposures of at least 24-h duration. Currently, this represents the best known development of freeze tolerance among squamate reptiles. Freezing stimulated a significant increase in blood glucose levels (16.15+/- 1.73 micromol x ml(-1) for controls versus 25.06 +/- 2.92 micromol x ml(-1) after thawing) but this increase had no significant effect on serum osmolality which was unchanged between control and freeze-exposed lizards (506.0 +/- 23.8 mosmol x l(-1) versus 501.0 +/- 25.3 mosmol x l(-1), respectively). Tests that assessed the possible presence of antifreeze proteins in lizard blood were negative. Recovery at 5 degrees C after freezing was assessed by measurements of the mean time for the return of breathing (5.9 +/- 0.5 h) and of the righting reflex (44.8 +/- 4.5 h). Because this species hibernates in wet substrates inoculative freezing may frequently occur in nature and the substantial freeze tolerance of this lizard should play a key role in its winter survival.