Compartmentalization of NaCl in frozen solutions of antifreeze glycoproteins.

The freezing behavior of NaCl solutions containing antifreeze glycoproteins from an Antarctic fish has been investigated to determine whether the glycoproteins prevent concentration of NaCl during freezing. Frozen NaCl solutions containing glycoproteins exhibit greater resistance to releasing their brine during centrifugation than NaCl solutions containing other cryoprotectants. With the aid of calorimetry this is shown to be caused not by an incorporation of the NaCl into the ice but by compartmentalization of the brine pockets. The compartmentalization is attributed to an unusual spicular structure that is imposed on the ice by glycoproteins.     

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 microm. 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.     

Structurally diverse disaccharide analogs of antifreeze glycoproteins and their ability to inhibit ice recrystallization

The β-d-galactosyl-(1,3)-α-N-acetyl-d-galactosamine disaccharide is present in antifreeze glycoproteins (AFGPs). Analogs of this disaccharide including the β-linked (1,3)-, (1,4)-, and (1,6)-galactosyl-N-acetyl galactosamine and the β-(1,3)-galactosyl-galactoside were synthesized and evaluated for ice recrystallization inhibition (IRI) activity. The results from this study demonstrate that the β-linked-(1,4) disaccharide exhibits more potent IRI activity than the native β-linked-(1,3) disaccharide. The C2 N-acetyl group of the disaccharide does not affect IRI activity but in monosaccharides, the presence of the C2 N-acetyl group decreases IRI activity. The current study will facilitate the design of potent small-molecule ice recrystallization inhibitors.     

Analysis of antifreeze glycoproteins in fish serum

A procedure utilizing high-pressure size-exclusion chromatography that permits rapid screening for both the types of components present in and the quantity of antifreeze glycoprotein in fish serum or solution is described. The applicability of the method is demonstrated by a comparative study of five different fish species, four of which contain the antifreeze glycoprotein and one which does not contain this protein. The antifreeze glycoprotein compositions of two fish of the same species, collected at different locations or under different environmental conditions, are also compared. A linear molecular-weight versus elution-volume function is established for both standard native proteins and the antifreeze glycoproteins, but these two lines do not coincide. The differences in tertiary structure between the antifreeze glycoproteins and normal proteins are presented as an explanation for the nonequivalence of calibration lines.     

Impact of antifreeze proteins and antifreeze glycoproteins on bovine sperm during freeze-thaw

There are no reports on the use of antifreeze proteins (AFP) and antifreeze glycoproteins (AFGP) for the use of bull sperm cryopreservation despite studies in the ram, mouse and chimpanzee. The effect of freezing and thawing on bull sperm viability, osmotic resistance and acrosome integrity were observed following the addition of AFP1, AFPIII and AFGP at four concentrations (0.1, 1, 10 and 100 microg/ml). In a second part of the experiment, fluorescein was conjugated to the AFPs and AFGP and observations were made using fluorescence microscopy to determine whether binding occurred between the sperm cell membranes and the proteins. In the final part of the study the cryopreservation media were cooled in the presence of the AFPs and AFGPs at the four concentrations on a cryomicroscope to mimic similar cooling curves as those used in the presence of sperm. Following freeze-thaw, AFPI resulted in increased osmotic resistant cells at 0.1-10 microg/ml compared to the control (P<0.01). AFPI and AFPIII did bind to the sperm cells. There was no visual difference in ice structure between the control, AFPIII and AFGP but AFPI resulted in parallel crystals at 0.1, 1 and 10 microg/ml. We suggest that the increased osmotic resistance in the spermatozoa cryopreserved in AFPI is due to the cells orientating between the ice crystals, reducing mechanical stress to the cell membrane. Previous research has shown that osmotic resistance correlates with bull fertility, suggesting that bull spermatozoa cryopreserved in the presence of AFPI may have increased fertility in vivo.     

Raman spectra of a solid antifreeze glycoprotein and its liquid and frozen aqueous solutions.

Raman spectroscopy was used to study the anomalous decrease in the freezing temperature of water produced by an antifreeze glycoprotein obtained from the sera of an Antarctic fish. An active fraction of this glycoprotein has a molecular weight of approximately 18,000 by equilibrium sedimentation compared to an apparent weight of 20 by freezing temperature depression. The Raman spectra of water present in a 1% antifreeze glycoprotein solution and of ice frozen from this solution were indistinguishable from the spectra of pure water and ice, respectively. These results indicate that the bulk properties of water and ice are unaffected by the presence of the antifreeze glycoprotein. Raman measurements on ice grown slowly, using as seed an oriented single crystal of ice in contact with 1% glycoprotein solutions, showed that the active glycoprotein was not excluded from the ice phase. On the other hand, we found that a smaller, inactive glycoprotein was excluded. Comparison of the Raman spectra of active and inactive glycoprotein components as solids, in 5% solutions, and rapidly frozen 5% solutions, showed that the two components differ in conformation and possibly in the environment of their carbohydrate hydroxyls. These observations suggest that hydrogen bonding of the carbohydrate hydroxyls of the active glycoprotein at the ice-solution interface may physically prevent growth of the ice lattice.     

Inhibition of recrystallization in ice by chimeric proteins containing antifreeze domains

Using synthetic DNA, we assembled a gene encoding a protein identical in sequence to one of the antifreeze proteins produced by the fish Pseudopleuronectes americanus (winter flounder). To address the relationship between structure and function, we also assembled genes encoding proteins varying in sequence and length. The synthetic genes were cloned into a bacterial expression vector to generate translational fusions to the 3' end of a truncated staphylococcal protein A gene; the chimeric proteins encoded by these fusions, varying only in their antifreeze domains, were isolated from Escherichia coli. The antifreeze domains conferred the ability to inhibit ice recrystallization, which is characteristic of naturally occurring antifreeze proteins, on the chimeric proteins. The chimeric proteins varied in their effectiveness of inhibiting ice recrystallization according to the number of 11-amino acid repeats present in the antifreeze moiety. A protein with only two repeats lacked activity, while the inhibitory activity increased progressively for proteins containing three, four, and five repeats. Some activity was lost upon removal of either the salt bridge or the carboxyl-terminal arginine, but surprisingly, not when both features were absent together.     

Structure of rapidly frozen gap junctions

The structure of gap junctions in the rabbit ciliary epithelium, corneal endothelium, and mouse stomach and liver was studied with the freeze-fracturing technique after rapid freezing to near 4 degrees K from the living state. In the ciliary epithelium, the connexons were randomly distributed, separated by smooth membrane matrix. In the corneal endothelium, both random and crystalline arrangements of the connexons were observed. In the stomach and liver, the connexons were packed but not crystalline. Experimental anoxia or lowered pH caused crystallization of the connexons within 20-30 min. In the ciliary epithelium, the effects of prolonged anoxia or low pH could not be reversed . In addition, invaginated or annular gap junctions increased in number, but their connexons were usually distributed at random. Rapid freezing thus demonstrates that gap junctions of different tissues are highly pleiomorphic in the living state, and this may explain their variations in structure after chemical fixation. The slow time-course and irreversibility of the morphological changes induced by prolonged anoxia or low pH suggest that connexon crystallization may be a long-term consequence rather than the morphological correlate of the switch to high resistance.     

Measurement of the pH of frozen buffer solutions by using pH indicators.

A method was established to estimate the pH change of several buffers solutions on freezing by using a combination of pH indicators. Among more than 30 buffers solutions examined, almost half exhibited a pH change in the temperature range between freezing point and 220 degrees K; the results were tabulated. Glycerol was found to suppress the pH changes because of its "salt buffer" effect.     

Calorimetric analysis of antifreeze glycoproteins of the polar fish, Dissostichus mawsoni

Solutions of antifreeze glycoproteins 1 through 5 and 8 were analyzed for activity by differential scanning calorimetry. With a scan rate of 1 degree C min-1, antifreeze glycoproteins 1-5 (20 mg/ml) revealed antifreeze activity with a delay in the freeze exotherm during cooling in the presence of ice. Antifreeze glycoprotein 8 (60 mg/ml), however, did not reveal antifreeze activity. When a 0.1 degree C min-1 scan rate was used, glycoproteins 1-5 again yielded a delay in the freeze onset, but the exotherm consisted of multiple events. At the slower scan glycoprotein 8 revealed an initial freeze followed by multiple exothermic events resembling those of glycoproteins 1-5. Thermograms exhibiting antifreeze activity had an initial shoulder in the exotherm direction upon cooling followed by a delay before the exotherm. The shoulders were correlated with c-axis ice growth observed in visual methods. The glycoprotein antifreezes had a linear increase in activity with decreased ice content.     

Survival of rapidly frozen hatched mouse blastocysts

The objective of the present study was to examine the effect of rapid freezing on the in vitro and in vivo survival of zona-pellucida-free hatched mouse blastocysts. Hatched blastocysts were rapidly frozen in a freezing medium containing either ethylene glycol (EG) or glycerol (G) in 1.5 M or 3 M concentration. Prior to freezing, embryos were equilibrated in the freezing medium for 2 min, 10 min, 20 min or 30 min at room temperature. To freeze them, embryos were held in liquid nitrogen vapour [approximately 1 cm above the surface of the liquid nitrogen (LN2)] for 2 minutes and then immersed into LN2. After thawing, embryos were transferred either to rehydration medium (DPBS + 10% foetal calf serum +0.5 M sucrose) for 10 minutes or rehydrated directly in DPBS supplemented with foetal calf serum. In vitro survival of embryos frozen with EG was higher than those frozen with G. The highest survival was obtained with 3 M EG and 2 min or 10 min equilibration prior to freezing, combined with direct rehydration after thawing. Frozen blastocysts developed into normal foetuses as well as unfrozen control ones did, with averages of 30% (control), 26% (EG) and 15% (G). The results show that hatching and hatched mouse blastocysts can be cryopreserved by a simple rapid freezing protocol in EG without significant loss of viability. Our data indicate that the mechanical protection of the zona pellucida is not needed during freezing in these stages.     

Calorimetric determination of inhibition of ice crystal growth by antifreeze protein in hydroxyethyl starch solutions.

Differential scanning calorimetry and cryomicroscopy were used to investigate the effects of type I antifreeze protein (AFP) from winter flounder on 58% solutions of hydroxyethyl starch. The glass, devitrification, and melt transitions noted during rewarming were unaffected by 100 micrograms/ml AFP. Isothermal annealing experiments were undertaken to detect the effects of AFP-induced inhibition of ice crystal growth using calorimetry. A premelt endothermic peak was detected during warming after the annealing procedure. Increasing the duration or the temperature of the annealing for the temperature range from -28 and -18 degrees C resulted in a gradual increase in the enthalpy of the premelt endotherm. This transition was unaffected by 100 micrograms/ml AFP. Annealing between -18 and -10 degrees C resulted in a gradual decrease in the premelt peak enthalpy. This process was inhibited by 100 micrograms/ml AFP. Cryomicroscopic examination of the samples revealed that AFP inhibited ice recrystallization during isothermal annealing at -10 degrees C. Annealing at lower temperatures resulted in minimal ice recrystallization and no visible effect of AFP. Thus, the 100 micrograms/ml AFP to have a detectable influence on thermal events in the calorimeter, conditions must be used that result in significant ice growth without AFP and visible inhibition of this process by AFP.     

Comparison of the solution conformation and dynamics of antifreeze glycoproteins from Antarctic fish

The (1)H- and (13)C-NMR spectra of antifreeze glycoprotein fractions 1-5 from Antarctic cod have been assigned, and the dynamics have been measured using (13)C relaxation at two temperatures. The chemical shifts and absence of non-sequential (1)H-(1)H NOEs are inconsistent with a folded, compact structure. (13)C relaxation measurements show that the protein has no significant long-range order, and that the local correlation times are adequately described by a random coil model. Hydroxyl protons of the sugar residues were observed at low temperature, and the presence of exchange-mediated ROEs to the sugar indicate extensive hydration. The conformational properties of AFGP1-5 are compared with those of the previously examined 14-mer analog AFGP8, which contains proline residues in place of some alanine residues (Lane, A. N., L. M. Hays, R. E. Feeney, L. M. Crowe, and J. H. Crowe. 1998. Protein Sci. 7:1555-1563). The infrared (IR) spectra of AFGP8 and AFGP1-5 in the amide I region are quite different. The presence of a wide distribution of backbone torsion angles in AFGP1-5 leads to a rich spectrum of frequencies in the IR spectrum, as interconversion among conformational states is slow on the IR frequency time scale. However, these transitions are fast on the NMR chemical shift time scales. The restricted motions for AFGP8 may imply a narrower distribution of possible o, psi angles, as is observed in the IR spectrum. This has significance for attempts to quantify secondary structures of proteins by IR in the presence of extensive loops.     

Ice recrystallization in a model system and in frozen muscle tissue

Recrystallization produces modifications on ice crystal sizes during storage and transport of frozen foods, reducing the advantages obtained by quick freezing and inducing physicochemical changes which alter their quality and shorten their shelf life. This process involves the growth of the larger crystals at the expense of the smaller ones, being the interfacial energy, the driving force of the phenomenon. In the present work recrystallization was analyzed using direct microscopic observation of ice crystals in a model solution (0.28 N NaCl) and indirect observation of frozen muscle tissue. The model solution allowed visualization of the interface behavior; from the analysis of the ice crystal frequency distributions, relationships between shape and size of the grains were established. A kinetic model based on the average system curvature was proposed obtaining a satisfactory fitness of the experimental data. Values of the kinetic constants determined at different temperatures allowed the estimation of the process activation energy. In muscle tissues isothermal freeze-substitution was used to observe the holes left by the ice in frozen semitendinous beef muscle stored at -5, -10, -15, and -20 degrees C during long periods of time. A different evolution of the mean ice crystal diameter was observed with respect to the model system. In meat samples, at long storage times, a limit diameter value was reached; this situation has been proved to be independent of temperature and initial size (freezing rate); a theoretical expression based on tissue characteristic parameters was proposed for its evaluation. Activation energy for recrystallization in muscle tissue was also determined, being comparable to values for protein denaturation and quality losses.