Inhibition of Ice Crystal Growth by Fish Antifreezes - ACS Symposium

Chapter 19

Inhibition of Ice Crystal Growth by Fish Antifreezes Downloaded by STANFORD UNIV GREEN LIBR on August 2, 2012 | http://pubs.acs.org Publication Date: November 26, 1991 | doi: 10.1021/bk-1991-0444.ch019

1

2

James A. Raymond and Arthur L. DeVries Alaska Department of Fish and Game, Fairbanks, AK 99701 Department of Physiology and Biophysics, University of Illinois, Urbana, IL 61801 1

2

Polar and subpolar marine fishes have peptide and glycopeptide antifreezes in their body fluids that protect them from freezing. The antifreezes have a high affinity for ice and prevent it from growing by a non-colligative process. Despite wide variations in composition and structure, the antifreezes cause ice single crystals to develop unusual and strikingly similar habits. This indicates that the antifreezes have affinities for similar crystal faces of ice. In many parts of the world's oceans, water temperatures fall to as much as 1 ° below the equilibrium freezing point offishes'body fluids. If a temperate water fish is placed in these waters, it will die immediately from freezing. One can actually see the ice propagating through its tissues. However, many fishes inhabit the polar and subpolar regions where freezing occurs. Avoidance of freezing in these fishes has been linked to the presence of a class of serum peptides and glycopeptides (1). These antifreezes are not found in temperate water fishes and they disappear in summer in fishes that experience warmer summer temperatures (1-3). Some polarfishesdo not have an antifreeze, and avoid freezing by existing in a supercooled state in ice-free deeper waters (4). Fishes that have an antifreeze are usually found in shallow waters where ice particles are abundant. Composition and Structure The first antifreezes to be identified were a series of glycopeptides in the antarctic fishes that consisted of a repeating tripeptide-disaccharide unit (1): -Ala-Ala-Thrgalattose N-acetylgalactosamine The glycopeptides are found in eight sizes ranging in molecular mass from 2300 to 34,000. In the smaller glycopeptides, glycopeptides 6-8, thefirstalanine is occasionally replaced with proline. 0097-6156/91/0444-0249$06.00/0 © 1991 American Chemical Society In Surface Reactive Peptides and Polymers; Sikes, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

Downloaded by STANFORD UNIV GREEN LIBR on August 2, 2012 | http://pubs.acs.org Publication Date: November 26, 1991 | doi: 10.1021/bk-1991-0444.ch019

250

SURFACE REACTIVE PEPTIDES AND POLYMERS

The freezing point depressing activity of the glycopeptide antifreezes is shown as a function of concentration (Figure 1). There are a number of features in this figure that show the antifreezes are unusual freezing point depressants. First, the curve is non-linear. Most solutes, such as NaCl, produce linear freezing point depressing curves. Second, the freezing point depression is, for a given molar concentration, about two orders of magnitude greater than one would expect. Another unusual feature is a difference in the freezing and melting curves; if a 10mg/ml solution freezes at -0.6 °C, the temperature would have to be raised to close to 0°C before it would melt. This is called a melting point-freezing point hysteresis. These features indicate that the antifreezes are quite different from normal solutes in their effect on ice. Do the antifreezes merely slow down the growth of ice, or do they completely halt it? We have placed ice crystals in glycopeptide antifreeze solutions at subzero temperatures for up to 13 days and have not detected any growth at 40X magnification (5). The glycopeptide antifreeze thus appears to completely stop growth of ice. One approach to understanding the antifreeze mechanism was to compare antifreezes from different species of fish. If different antifreezes were found, an analysis of their common features would help to focus on the active part of the molecules. Two of the first non-antarctic fish to be examined were the saffron cod, Eleginus gracilis and the sculpin Myoxocephalus verrucosus. The antifreeze of the saffron cod was found to be almost identical to glycopeptides 6-8 (6,7). One difference was the occasional substitution of arginine for threonine. This was quite surprising because the cod family and the antarctic nototheniid fishes, from which glycopeptides 1-8 were obtained, are widely separated, both taxonomically and geographically. Circular dichroism spectra of the glycopeptide antifreezes indicated either an extended coil configuration or a 3i helix configuration similar to that found in collagen. In order to distinguish between the two spectra, a solution of glycopeptides 1-5 was gradually heated from 0 to 95°. An abrupt loss of C D signal at around 40 °C would have indicated the melting of a helix. However, only a gradual loss in signal was observed, indicating that the extended coil was a more likely configuration (8). The antifreezes of two other northern fishes, the sculpin Myoxocephalus scorpius and the winter flounder, Pseudopleuronectes americanus, were found to be very similar to one another (1,9). Like the glycopeptide antifreezes, these antifreezes consisted of more than 60% alanine, repeating sequences of amino acids, and were of relatively small molecular mass. On the other hand, the repeats consisted of 11 amino acids and the molecules did not contain a sugar moiety. Although not sequenced, the antifreeze of M. verrucosus was similar to these peptides in amino acid composition. Circular dichroism spectra of the antifreeze of M. verrucosus and P. americanus were very different from the glycopeptide CD spectra. The CD spectra

In Surface Reactive Peptides and Polymers; Sikes, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

19.

RAYMOND AND DEVRIES

Fish Antifreezes

251


1.4

Concentration (mg/ml) Figure 1. Freezing and melting point depressions of aqueous solutions of glycopeptide antifreezes.



252


indicated that approximately 85% of these molecules were in the alpha-helical configuration (8,10). More recently, other antifreezes from the sculpin and zoarcid families have been characterized and have been found to differ from the above glycopeptide and peptide antifreezes. Antifreezes from three different zoarcids from the arctic and the antarctic have nonrepeating sequences, with between 61 and 64 amino acid residues, and have homologies ranging from 56 to 69% (11-13). C D analysis of one of these antifreezes indicated a distinct tertiary structure that lacks both alpha-helical and beta components (12). The antifreeze of the sea raven, a northern sculpin, has a molecular mass of 17,000 Da and a nonrepeating sequence that differs from the zoarcid sequences (14). Its secondary structure includes both beta and alpha-helical components. Physical Chemical Properties How do the antifreezes affect the growth of ice? Normally, when ice grows from solution, it grows in a direction parallel to the a-axes and forms large plates. In the presence of most of the antifreezes, however, ice grows in long spicules that are parallel to the ice c-axis (15). In other words, growth in the direction of the a-axes is inhibited. Another unusual property of the antifreezes is that they are not completely excluded from ice as it grows, as are most solutes. The distribution coefficient quantifies the exclusion, with a value of zero corresponding to complete exclusion and a value of 1.0 corresponding to no exclusion, i.e., equal concentrations in ice and liquid. The antifreezes have distribution coefficients ranging from 0.17 to 0.89 (15). In general, the larger the molecular mass of the antifreeze, the greater is its incorporation in ice. For comparison, the distribution coefficient for bovine serum albumin was 0.10. Because of the affinity of the antifreezes for ice, it is reasonable to ask whether the structure of the ice is in any way changed by the antifreeze. However, an x-ray diffraction pattern of spicular ice grown in the presence of the glycopeptide antifreeze showed normal hexagonal ice (5). Ideas underlying the antifreeze mechanism were developed over a hundred years ago when Lord Kelvin found that the equilibrium of a system depended not only on the bulk phases but also on the surface that separated them. Working from Kelvin's equation, Kuhn (16) derived an expression for the lowering of the equilibrium freezing temperature of water due to surface effects. We applied Kuhn's equation to the case of adsorbed antifreeze molecules on an ice surface (15). Because the growth of crystals occurs mainly through the advancement of steps, adsorbed molecules will force the steps to grow between them, thus increasing the curvature and surface area of the steps. The result is a freezing point depression that is proportional to the square root of the product of the antifreeze concentration and the antifreeze's distribution coefficient. The predicted



19.

RAYMOND AND DEVRIES

Fish Antifreezes

253

freezing point depressions for various concentrations agree well with the observed freezing point depressions. Various mechanisms have been proposed to account for the binding of the antifreezes to ice. Sequences of the alpha-helical antifreezes show that they are amphiphilic, i.e., polar and nonpolar residues are on opposite sides of the molecule (1,17). If the polar groups are spaced a distance that corresponds to a fundamental repeat distance in the ice lattice, as has been shown for the antifreeze of the winter flounder (1), the polar side of the antifreeze would have a higher affinity for water molecules in the lattice than water molecules in solution. This type of affinity is called lattice matching. In addition, the nonpolar groups on the antifreeze would project out into the water and tend to keep other water molecules from joining the lattice. The existence of amphiphilicity and lattice matching in other, non-alpha helical antifreezes has not yet been confirmed or ruled out. Another binding mechanism recently proposed for the alpha helical antifreezes is a dipole- dipole interaction (18). In this model, an electric dipole associated with the alpha helix induces an opposite dipole in the surface of the ice and is then attracted to it. In an attempt to see more clearly how the antifreezes bind to ice, recently we have used large single crystals of ice with well-defined basal (0001) and prism (10Ï0) faces (19). We found a number of unusual growth features in the ice. The antifreezes completely inhibit growth on the prism faces, but allow limited growth on the basal plane. As new layers are deposited on the basal plane, pyramidal planes develop on the outside of the crystal, and large, hexagonal pits form within the basal plane (Figure 2). This growth eventually leads to the disappearance of the basal plane, at which point all growth ceases. An unusual feature of the pits was that their orientation is rotated 30° with respect to the normal orientation of hexagonal ice crystals. These features were found to occur with 6 antifreezes having widely varying compositions and structures. The unusual faces that develop in ice single crystals indicate that they are sites of binding by the antifreezes. As each new layer is deposited on an adjacent faster growing face, the retarded face increases slightly in area. The result is a crystal that is dominated by its slowest growing faces. That the crystal habits were similar for different antifreezes suggests that they share a common binding mechanism. Some features of the pits suggested that they were associated with dislocations: spiral steps occasionally occurred in the pits and rows of pits often were aligned with low-index crystal planes. The number density of the pits was also inversely proportional to the temperature at which the basal plane growth occurred. These observations suggest the possibility that dislocations may play a role in the binding of the antifreezes to ice. However, further study is needed to confirm this.



254


Figure 2. A single crystal of ice grown in the presence of the glycopeptide antifreeze of the antarctic fish Dissostichus mawsoni. In this view, the basal plane, shown as small flat areas, is deeply pitted with hexagonal pits. The pit faces are likely sites of antifreeze adsorption. The antifreeze inhibits growth on the pit faces but not on the basal plane. Growth ceases when the pit faces completely cover the basal plane. Width of pits is approximately 500 μ.


19.

RAYMOND AND DEVRIES

Fish Antifreezes

255

Literature Cited


1. DeVries, A. L. Ann. Rev. Physiol. 1982, 45, 245-60. 2. Duman, J. G.; DeVries, A. L. J. Exp. Zool. 1974, 190, 89-97. 3. Fletcher, G. L.; Haya, K.; King, M . J.; Reisman, H. M . Mar. Ecol. Prog. Ser. 1985, 21, 205-12. 4. Scholander, P. F.; van Dam, L.; Kanwisher, J. W.; Hammel, H. T.; Gordon, M . S. J. Cell Comp. Physiol. 1957, 49, 5-24. 5. Raymond, J. A. Ph.D. Thesis, University of California, San Diego 1976. 6. Raymond, J. Α.; Lin, Y.; DeVries, A. L. J. Exp. Zool. 1975, 193, 12530. 7. O'Grady, S. M.; Schrag, J. D.; Raymond, J. Α.; DeVries, A. L. J. Exp. Zool. 1982, 224, 177-85. 8. Raymond, J. Α.; Radding, W.; DeVries, A. L. Biopolymers 1977, 16, 25758. 9. Hew, C. L.; Fletcher, G. L.; Ananthanarayanan, V. S. Can. J. Biochem. 1980, 58, 377-83. 10. Ananthanarayanan, V. S.; Hew, C. L. Biochem. Biophys. Res. Commun. 1977, 74, 685-9. 11. Schrag, J. D.; Cheng, C.-H. C.; Panico, M.; Morris, H. R.; DeVries, A. L. Biochim. Biophys. Acta 1987, 915, 357-70. 12. Li, X . M.; Trinh, Κ. Y.; Hew, C. L.; Buettner, B.; Baezinger, J.; Davies, P. L. J. Biol. Chem. 1985, 260, 12904-9. 13. Cheng, C.-H. C.; DeVries, A. L. Biochim. Biophys. Acta 1989, 997, 5564. 14. Hew, C. L.; Joshi, S.; Wang, N . C.; Kao, M . H.; Ananthanarayanan, V. S. Eur. J. Biochem. 1985, 151, 167-172. 15. Raymond, J. Α.; DeVries, A. L. Proc. Natl. Acad. Sci. USA 1977, 74, 258993. 16. Kuhn, W. Helv. Chim. Acta 1956, 39, 1071-86. 17. Hew, C. L.; Fletcher, G. L. In Proceedings in Life Science; Gilles, R., Ed.; Springer-Verlag, Heidelberg, 1985; pp 553-563. 18. Yang, D. S.; Sax, M . ; Chakrabartty, Α.; Hew, C. L. Nature 1988, 333, 232-7. 19. Raymond, J. Α.; Wilson, P.; DeVries, A. L. Proc. Natl. Acad. Sci. USA 1989, 86, 881-5. RECEIVED August 27, 1990


Inhibition of Ice Crystal Growth by Fish Antifreezes - ACS Symposium

Recommend Documents