solution, between the melting and freezing points where novisible ice growth occurs, a 5- to 6-fold increase in light scatter-ing was observed over that from an ice-water interface. Theincreased scattering is indicative of increased surface rough-ness, which is consistent with the adsorption-inhibition mecha-nism. Whether the increased surface roughness is due to sub-microscopic growth of the small curved fronts remains to beelucidated. These results are being presented at the 19th Sym-posium on the Chemistry and Physics of Ice, Grenoble, France,September 1986.

This research was supported by National Science Foundationgrant DPP 84-15266.

References

Ahlgren, J.A., and A.L. DeVries. 1984. Comparison of antifreezeglycopeptides from several antarctic fish. Polar Biology, 3, 93-97.

DeVries, A. L. 1984. Role of glycopeptides and peptides in inhibition ofcrystallization of water in polar fishes. Philisophical Transaction of theRoyal Society of London, 13304, 575-588.

Raymond, J.A., and A.L. DeVries. 1977. Adsorption inhibition as amechanism of freezing resistance in polar fishes. Proceedings of theNational Academy of Sciences, U.S.A., 74, 2589-2593.

Turner, J.D., J.D. Schrag, and A.L. DeVries. 1985. Ocular freezingavoidance in antarctic fish. Journal of Experimental Biology, 118,121-131.

Structure-function studies onantifreeze glycoproteins

from blood serums of antarctic fish

R.E. FEENEY, D.T. OSUGA, D.S. REID,and T.S. BURCHAM

Department of Food Science and TechnologyUniversity of CaliforniaDavis, California 95616

W.L. KERR and Y. YEH

Department of Applied ScienceUniversity of CaliforniaDavis, California 95616

Our laboratory started studying the blood serum proteins ofantarctic penguins and fish in 1964. During the past decade ourlaboratory has been procuring blood serums of fish from Ant-arctica (Pagothenia borchgrevinki and Dissostichus mawsoni), theBarents Sea (Boreogadus saida), off Kotzebue, Alaska (Elecinusgracilis), and off Mombetsu, Japan (Eleginus gracilis). Antifreezeglycoproteins (AFGP) were prepared from all of these serumsand their properties studied. During the last 2 years the use ofseveral different experimental techniques has shown that theantifreeze glycoproteins must function at the ice-solution inter-face (Feeney, Burcham, and Yeh 1986).

Tools for characterizing structure have included chemicalprocedures such as sequencing of the polypeptide chains, deg-radations, and chemical modifications of the amino and carbox-yl terminal groups and the carbohydrate chains; and physicalprocedures like ultracentrifugal analyses, circular dichroism,Raman spectroscopy, laser-light scattering, surface second-har-monic generation, direct visual observations of crystal growth,and surface activity by microscopic observation of growing crys-tal fronts (Feeney, Burcham, and Yeh 1986).

All of the antifreeze glycoproteins appear to be functional,although the extent and nature of the activity are not identical.

An abbreviated summary of the characteristics is as follows:(1) Lowers solution freezing temperature noncolligatively:

freezing temperature is 500 times lower than predictedfrom solution colligative properties.

(2) Lowers solution freezing temperature additively with salt.(3) No evidence for functioning in liquid or solid phases.(4) Functions in the presence of ice.(5) Not excluded from bulk ice phase.(6) Ice has a normal hexagonal structure as determined by X-

ray diffraction.(7) Many observations indicate that AFGP functions at the ice-

solution interface:(a) Growing edge of crystal disrupted.(b) Surface energy at ice-solution interface is lowered

(Kerr et al. 1985) (table 1).(c) Crystals growing into AFGP solution have unusual

form:(i) With a linear temperature gradient, growth is

transverse to the gradient (Kerr et al. 1985)(ii) On free growth, with minimal supercooling (be-

low the freezing point), growth is along the basaldirection (c-axis), while growth along the pris-matic face (a axis) is inhibited (cooperative with J.Hallett).

(d) When growth of performed ice crystals is studied,small AFGP peptides do not function if ice is highly

Table 1. Interfacial energies between ice and AFGP solutions

ConcentrationSL(in milligrams(in ergs perStandard

Sample per milliliter)square centimer)deviation

Water - 30 ±5AFGP 1-5

(high activity)2 26 ± 84 21 ±9

11 9 ±4AFGP 7-8

(weak activity)5 30 ± 326 26 ±772 20 ±10

Ovomucoid 10 28 ± 3

1986 REVIEW 199

Table 2. Effect of ice crystalline structure on AFGP activitya

Freezing temperature of solutionin the presence of ice made at:

Sample -10C- 6°C

AFGP 1-4 (4 milligramsper milliliter)-0.39°C ± 0.02-0.36°C ± 0.03

AFGP 7-8 (10 milligramsper milliliter)-0.30°C ± 0.01-0.05°C ± 0.02

AFGP 1-4 (4 milligramsper milliliter) +AFGP 7-8 (10 milligramsper milliliter)-0.51°C ± 0.02-0.51°C ± 0.02

a From Burcham et al. (1982).

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LiLj IL0LLIL< .E< <

Normalized intensity derived from surface second harmonic gener-ation versus glycoprotein solution. Intensity increase is measuredin photons detected per 2,000 laser pulses and is normalized withrespect to the square of the baseline intensity. Numbers in paren-thesis are concentrations in milligrams per milliliter. From Brown etal. (1985).

dendritic, but will function additively in the presence oflarge AFGP (cooperatively) (Burcham et al. 1984) (table2).

(e) Direct evidence for AFGP adsorbing to an ice surface:AFGP-ice systems exhibit large inversion asymmetryas detected by surface second-harmonic generation(Brown et al. 1985) (figure).

(f) Analysis of inhibition data indicates Langmurian ad-sorption on ice surface (Burcham et al. 1986).

Only references to our most recent studies are included. Forother studies from our laboratory see Feeney, Burcham, andYeh (1986) and from other laboratories see DeVries (1984),Knight, DeVries, and Oolman (1984), Hew and Fletcher (1985),and Ananthanarayanan, Slaughter, and Hew (1986).

This research has been supported by National Institutes ofHealth grant GM 23817 and by National Science Foundationgrant CHE-84-05390.

References

Ananthanarayanan, VS., D. Slaughter, and C.L. Hew. 1986. Antifreezeproteins from the ocean pout, Macrozoarces a pncricanus: Circular di-chroism spectral studies on the native and denatured states. Bio-chimica et Biophysica Acta, 870, 154-159.

Brown, R.A., Y. Yeh, T.S. Burcham, and R.E. Feeney. 1985. Directevidence for antifreeze glycoprotein adsorption onto an ice surface.Biopolyiners, 24(7), 1265-1270.

Burcham, T.S., M.J. Knauf, D.T. Osuga, R.E. Feeney, and Y. Yeh. 1984.Antifreeze glycoproteins: Influence of polymer length and ice crystalhabit on activity. Biopolymers, 23(7), 1379-1395.

Burcham, T.S., D.T. Osuga, Y. Yeh, and R.E. Feeney. 1982. Antifreezeglycoprotein activity as a function of ice crystalline habit. Cryo-Letters,3, 173-176.

Burcham, T.S., D.T., Osuga, Y. Yeh, and R.E. Feeney. 1982. A kineticdescription of antifreeze glycoprotein activity. The Journal of BiologicalChemistry, 261(14), 6390-6397.

DeVries, A. L. 1984. Role of glycopeptides and peptides in inhibition ofcrystallization of water in polar fishes. Philosophical Transactions of theRoyal Society of London, Series B, 304, 575-588.

Feeney, R.E., T.S. Burcham, and Y. Yeh. 1986. Antifreeze glycoproteinsfrom polar fish blood. Annual Review of Biophysics and BiophysicalChemistry, 15, 59-78.

Hew, CL., and G.L. Fletcher. 1985. Biochemical adaptation to thefreezing environment-structure, biosynthesis and regulation of fishantifreeze polypeptides. In R. Gilles (Ed.), Circulation, respiration, andmetabolism. Berlin: Springer-Verlag.

Kerr, W.L., R.E. Feeney, D.T. Osuga, and D.S. Reid. 1985. Interfacialenergies between ice and solutions of antifreeze glycoproteins. Cryo-Letters, 6(6), 371-378.

Knight, CA., A.L. DeVries, and L.D. Oolman. 1984. Fish antifreezeprotein and the freezing and recrystallization of ice. Nature, 308,295-296.

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200 ANTARCTIC JOURNAL