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Ann. Rev. Physiol 1983. 45:245-60

Copyright 1983 by Annual Reviews Inc. All rights reserved
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ANTIFREEZE PEPTIDES.AN'D
GLYCOPEPTIDES IN
COLD-WATER FISHES

A. L. De Vries
The University of lllinois, Urbana, Illinois 61801
Annu. Rev. Physiol. 1983.45:245-260.

INTRODUCTION
During the winter season the polar oceans and the near-shore waters of the
north temperate oceans are at the freezing point of seawater (-1.9C), a
temperature well below the freezing point of a' typical marine teleost
(-O.8C) (5). This one degree difference between environment and body
fluid freezing point is large enough to result in freezing because supercooling
in the presence of ice cannot occur (74). Freezing of the body fluids or
tissues of fishes has been shown in all cases to lead to death, although several
hours may intervene between thawing and death. Two different strategies
by which fishes avoid freezing when their habitats are cooled below their
freezing points are recognized. The first concerns behavioral responses.
Some shallow-water summer inhabitants migrate offshore to warm water
(15) while others move into deep (100 m) cold water and overwinter at a
temperature of -1.9C in a supercooled state. One degree of supercooling
is small and appears to be metastable at these depths where the waters are
ice free.
In the shallow ice-laden waters of the' polar oceans many fishes spend
their entire lives beneath thick ice cover (11, 12, 14) yet do not appear to
freeze. In fact some even use the ice crystal formations associated with the
year-round thick ice cover as a habitat in which to forage for food yet appear
to be immune to freezing (3, 16). The body fluids of such fishes will not
freeze until the temperature is lowered below -2C (16). 'Lowering of the
environmental temperature below -2C in the presence of ice results in
freezing and death (16, 74).
In most temperate marine fishes, sodium chloride is the principle elec
trolyte present in the blocid and is responsible for 85% of the freezing-point

245
, 0066-4278/83/0315-0245$02.00
 246 DEVRIES

depression (40). The remainder of the freezing-point depression is due to

small amounts of potassium, calcium, urea, glucose, and the free amino
acids (6 7). In fishes inhabiting freezing environments, concentrations of
sodium chloride in the body fluids are elevated relative to temperate forms
(30, 32, 59). They are, however, not high enough to account for the low
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free1 zing
fre1ezing-point
small organic solutes are nearly the same as those found in temperate-water
fishes (15). In these cold-water fishes over half of the freezing-point depres
sion has been shown to be associated with the colloidal fraction of the blood,
and it is retained by a dialysis membrane with a cutoff of 3000 daltons (15,
16). The large freezing-point depression associated with the colloidal frac
tion indicates that relatively large molecules are involved and also implies
that they exert their effect by a noncolligative mechanism. In most antarctic
fishes and many north-temperate fishes these molecules are glycopeptides,
Annu. Rev. Physiol. 1983.45:245-260.

while in some arctic and north-temperate fishes they are peptides ( 43).
These glycopeptides and peptides range between 2,400 and 34,000 da1tons
(14, 27, 29). On a weight basis they appear to be as effective as sodium
chloride in depressing the freezing point of water (19). On a molal basis they
depress the freezing point 200-300 times more than expected on the basis
of colligative relationships (13). These colloidal solutes can be characterized
as having "antifreeze" properties where it is stressed that the freezing point
is lowered in a noncolligative manner with little effect on the melting point
of the solid phase.

BIOLOGICAL ANTIFREEZES
Glycopeptides
The glycopeptide antifreezes were first isolated from the blood of notothen
tid fishes inhabiting McMurdo Sound, Antarctica (19). They make up ap
proximately 3.5% (WIV) of the blood, and electrophoretic analyses
indicate 8 separate glycopeptides in the blood of most of the antarctic
nototheniids (13, 1 8, 20). They range from 2400 to 34,000 daltons. Glyco
peptides 1-5 are composed of repeating units of glycotripeptides in which
the disaccharide ,8-D-galactopyranosyl-(1-+-3)-2-acetamido-2-deoxy-a.-D
gallactospyranose is linked to the threonine residue of the tripeptide, alanyl
threonyl-alanine (21 , 49 , 78, 79). Glycopeptides 6 , 7, and 8 differ from 1-5
in that the amino acid proline replaces some of the alanines beginning at
position seven and appears at every third position until the C-terminal is
reached (52, 56). Glycopeptide 8 appears to be a mixture of three identical
sized molecules in which the prolines occupy different positions in the
polypeptide (52, 56). The same 8 glycopeptides have been isolatedfrom the
 BIOLOGICAL ANTIFREEZE AGENTS 247

northern gadids including the Greenland cod, Gadus ogac from Labrador
(83),the arctic polar cod, Boreogadus saida (62),and the Atlantic cod from
Newfoundland, Gadus morhua (47). The positions occupied by proline in
glycopeptide 8 in some of these forms differ from those reported for the
antarctic nototheniids (36,47,61). The saffron cod, Eleginus gracilis, from
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the Bering Sea and the Atlantic tomcod, Microgadus tomcod, also have
glycopeptide antifreezes that differ slightly (15, 36, 71). The antifreezes in
the saffron cod and rock cod are both glycopeptides but they differ substan
tially in composition and size, indicating considerable variability in the
same family. No variability among families has been noted in the glycopep
tides of three antarctic nototheniid families studied so far. It appears re
markable that fishes belonging to unrelated families inhabiting opposite
hemispheres have evolved identical antifreeze glycopeptides (83) while sym
patric species of the same family have evolved glycopeptides that show
considerable variation in composition and size.
Annu. Rev. Physiol. 1983.45:245-260.

Peptides
Peptide antifreezes have been identified in and isolated from several north
temperate and arctic fishes (Table 1). They vary in size and composition,
and only a few have been completely characterized. Three separate peptides
have been isolated from the winter flounder, Pseudopleuronectes america
nus. They are composed of 8 amino acids in which alanine accounts for
60% of the residues (23, 25). Most of the remainder are polar residues such
as aspartate, glutamate,lysine,serine, and threonine. A partial sequence of
each of the three peptides shows a repeat pattern of the two polar residues
aspartic and threonine separated by two alanines with each polar sequence
separated by 7 nonpolar residues, usually a leucine and 6 alanines (17). The
Alaskan plaice, Pleuronectes quadritaberulatus, has evolved peptide anti
freezes similar to those of the winter flounder and possesses a similar polar
sequence separated by long stretches of alanine (15). In this peptide, the
positions of threonine and aspartate are conserved but leucine is absent (15).
Antifreeze peptides have also been isolated from the Bering Sea sculpin,
Myoxocephalus verrucosus (71); the several electrophoretic variants may,
however, be of similar size. The composition of these peptides resembles
that of those isolated from the short horn sculpin, M. scorpius, from the
waters of Newfoundland and Ellsmere Island (34, 46). This is not entirely
unexpected since these species are taxonomically almost indistinguishable.
Like the flounder peptides, these contain about 60% alanine and are rich
in the polar residues aspartate,threonine,glutamate,and lysine. They differ
in that they contain the nonpolar amino acids isoleucine, glycine, methio
nine, and proline (34, 46). The peptide isolated from the polar eel pout,
Lycodes polaris, is similarly rich in polar residues, but some of the alanines
 248 DEVRIES

ha'e been replaced':by nonpolar residues such as leucine.and.valine. (15),

Re<:ently an antifreeze peptide has been isolated from the sea raven; Hemi
tripterus americanus, which differs substantially" from the other peptide
antifreezes. It has less alanine and relatively large amounts 'of glycine"and
some of the aromatic amino acids (81). None of the other peptide antifreezes
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contains the aromatic amino acids and the large amounts of glycine. Their
functional role is unexplained. The only antarctic fish that possesses a
peptide antifreeze is the eel pout, Rhigophila dearborni. This peptide con
tains twelve amino acids (primarily alanine); in addition to the nonpolar
residues. mentioned above, it contains valine (15).

CONFORMATION Only limited information about the secondary struc

turl of the antifreezes exists. Dialysis, viscosity, and circular 'dichroism
measurements indicate that both the glycopeptide and peptide antifreezes
are expanded molecules (2eY, 39, 72). X-ray diffraction, detaiU:d circular
Annu. Rev. Physiol. 1983.45:245-260.

diclhroism studies, and natural'aoundance C-13 nuclear magnetic resonance

(NMR) studies have not given" definitive' information on the secondary
structure of the glycopeptides (1, 4, 8). Circular dichroism studies and
vis(,,osity measurements indicate that the peptide antifreezes that have been
studied thus far are helical rods (2, 72). The significance of:this conforma
tion is that the polar residues aspartic and threonine, which are generally
separated by two alanine residues, are separated by a distance of 45 A (17).
The existence of this repeat spacing of these polar residues in the peptide.
is of paramount importance for recognition of the ice lattice and the binding'
to the oxygen atoms in it (17).

Co/ligative Freezing Points

Thle freezing point of a solution is defined as the temperature at which the
vapor pressure over the liquid phase is equal to that over the solid phase
(64). If the system is in thermal equilibrium, then by defuiiiionthe freezing
point of the solution will be the same as the melting point ofihe,solid phase.
In practice this "equilibriUm freezing .point" can be estimated by determin
ing the melting temperature of a small seed ice crystal in a small volume
of the solution. In such a system, raising the temperature by 0.01(; or
lowering it by 0.0 1 C results in melting of the-solid phase and freezing of
a small amount of the liquid, respectively. Thus the melting point and
frelzing point can be assumed to be the same. In order to obtain the freezing
point in salt solutions and biological 'solutions underconditions approach
ing thermal equilibrium the size ofthe ice crystal must be kept small relative
to the volume ofthe solution. In addition,' the rate of warming or cooling
necessary to observe'melting or freezing must be slow so that the system
approaches therm:alequilibrium. These rates are on the order ofO.0 1C per
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'
Table 1 Comprison of sugar and amino acid content of antifreezes in northern and an tarctic fishes a

Pagothenia
borchgrevinki (20) Eleginus
Boreogadus saida (62) gracilis (71) Pseudo Pieuronectes Myoxo Myoxo Hemi
" i
Amino acid Gadusmorhua (47) Mierogadus pleuroneetes quadrita cepha/us eepha/us Lycodes tripterus
or sfgar Gadus ogac (83) lomeod (15, 36) americanus bercu/atus (15) verrucosus (71) seorpius (46) po/ars (15) amerieanus (81)

Aspartic" aid 3 3 3 3 3 4
Threori;n 8 6 6 3 4 3 3
Annu. Rev. Physiol. 1983.45:245-260.

Serine 1 1 1
Glutamic acid 3 2 5 2

Glycine 2 2 6
Proline
Alanine 18 16 32 34 31 38 28 12
Half-cystine 6
Methionine 4

Isoleucine 1 2
Leucine 2 3 2 5 4

Tyrosi e
Phenylalanine
Lysine 2
Histidine
Tryptophan 2
Arginine
N-acetylgalac-
tosamine 8
Galactose 8

a Values are number of resi,ru es per 5,000 grams antifreeze.

 250 DEVRIES

2 min for one-microliter samples. Since the depression of the freezing point
oflbody fluids lacking antifreeze is dependent on the number of particles in
solution, an indirect estimate of this freezing point can be obtained by
determining the vapor pressure lowering, or by determining the freezing
point with a freezing osmometer (15). The latter device has a strong depend
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an(:e on the rate of freezing and therefore must be standardized against

known freezing points. With such a device it is assumed that the unknowns
freeze the same way the standards do.

Noncolligative Freezing Points

Because relatively large molecules in the blood of polar fishes produce a
substantial depression of the freezing point, the depression must occur via
a noncolligative mechanism. Determinations of the equilibrium freezing
points of the blood and solutions of the purified antifreezes have revealed
a most unusual freezing-melting behavior. The melting point of the solid
phase (seed ice crystal) occurs at a temperature predicted by colligative
Annu. Rev. Physiol. 1983.45:245-260.

relationships; however, the freezing point (temperature of ice crystal propa

gation) is much lower than the melting point (13, 28, 70, 73, 82). In
antarctic nototheniid blood serum,the seed crystal melts at approximately
- l .OC while ice begins to propagate rapidly from the face of the seed at
-2.2C (13,42). Ice in a 2% glycopeptide or peptide antifreeze solution will
melt at -O.02C and will not propagate from the face of the seed until the
temperature is lowered below -1.2C. Growth, then, as in the blood, is in
the form of long thin spicules (Figure 1) (13,70). Most of the glycopeptide
and peptide antifreezes exhibit the same depression of the freezing point of
water on a weight basis except for some of the smaller glycopeptides isolated
from the antarctic nototheniids (52) and northern cods (36, 61). Solutions
of the low-molecular-weight glycopeptides produce only about half the
noncolligative lowering of the freezing point than the larger glycopeptides
and peptides do when compared on a weight basis (15). Even though some
of the lower-molecular-weight antifreezes of the same size have occasional
arginines substituted for threonines, the activity is the same (77). When
compared on a molar basis this antifreeze effect of the various antifreezes
increases with the size (77). The antifreezes also affect the crystal habit.
When the seed crystal grows, spicules form parallel to the c-axes (70). The
size of the seed crystal does not change at temperatures intermediate be
tween melting and freezing (68).
Freezing-point estimates obtained under conditions where substantial
supercooling (4-6C) occurs results in very different freezing points (14,
69). With the northern hemisphere cads that possess glycopeptide anti
freezes, estimates of the blood freezing points, obtained with a modified
freezing-point osmometer (80), are much higher than those obtained by
 BIOLOGICAL ANTIFREEZE AGENTS 251

-1.2
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-1.0
U
0

w -0.8
0::
C R YSTAL GROWTH
::>
I-
ANTI FREEZE
-0.6
0:: GLYCOPEPTIDES

W 7 and 8 SEED

CRYSTAL

-0.4
w
I- ICE
SPICULES
Annu. Rev. Physiol. 1983.45:245-260.

----
-0.2
"\
MELTING POINTS
OF ALL ANTI FREEZES

0
0 10 20 30 40
(mg/ml)
Figure 1 Freezing and melting points of aqueous solutions of the peptide and glycopeptide
antifreezes as a function of concentration. The high-molecular-weight g1ycopeptides and pep
tides have essentially the same antifreeze activity while the low-molecular-weight glycopep
tides have much less. The melting points are the same for all sizes. The inset shows the spicular
ice growth that propagates from the seed crystal when freezing occurs.

determining the temperature of crystal growth in the same fishes (14, 15).
Use of the freezing-point osmometer where glycopeptide solutions are
supercooled by 4C before freezing is initiated reduces the freezing point
depression of glycopeptides 1-5 by 25%; in glycopeptides 7 and 8 the
depression is reduced to values expected on the basis of colligative relation
ships (14, 75). Addition of small amounts of the large glycopeptides to the
small ones leads to a significant increase in the freezing-point depression of
the small ones and has been referred to as a potentiation (57,63). However,
when freezing points are determined in the presence of a small seed crystal
and the freezing point is approached slowly (O.OIC/min), no potentia
tion of the antifreeze activity of the small by the large glycopeptides is
observed (75). Thus prevention of supercooling prior to freezing is of the
utmost importance because freezing points obtained in this manner give
misleading results. Evidence for this interpretation comes from examination
of the biological role of the antifreezes. Fishes that inhabit freezing oceans
 252 DEVRIES

experience only small changes in temperature when the water is freezing.

Slow freezing conditions employed for freezing-point detennination more
closely approximate those observed in nature. Thus the distinction between
active and inactive antifreeze components (29, 63) is an artifact of the
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method and does not accurately describe the freezing behavior of glycopep
tidc 7 and 8. The facts that they have recently been shown to be the only
antifreeze components present in intestinal1luid of antarctic fishes and that
they lower the 1luid's freezing point to _2C (60) support this thesis.

MECHANISM OF ANTIFREEZE ACTIVITY

Water Structuring
Thc expanded structures of the antifreeze and the abundance of side chains
rich in hydroxyls and polar groups suggest that these peptides may struc
turc the water that surrounds them. However, recent NMR studies (41)
Annu. Rev. Physiol. 1983.45:245-260.

indicate that the amount of water bound is small. Isopiestic determinations

of water binding under equilibrium conditions reveal that these peptides
bind only slightly more water than other proteins of a similar size when in
solution (26). It would appear that the amount of "bound" water is much
too small to explain the antifreeze effect.

ADSORPTION Considerable information indicates that adsorbed impuri

ties can inhibit the crystallization or the growth of small crystals (7). Such
inhlbitors are usually characterized by a specificity for a particular kind of
crystal, and large polymers composed of repeating units are more effective
than small nonrepetitive ones. It is currently thought that adsorption of an
impurity inhibits crystal growth by interfering with the propagation of steps
across the face of the crystal. In many cases, adsorption of impurities also
causes a change in the type of crystal growth, or habit, observed when the
supersaturation point is exceeded (9). The inhibition of the freezing of water
in the presence of the antifreezes, at temperatures below the expected
freezing point when the system is in thermal equilibrium, appears to be
another example of the adsorption-inhibition phenomenon.
Studies of the freezing behavior of solutions of the glycopeptide and
peptide antifreezes indicate that they adsorb to ice (22, 48, 70, 82). At very
low concentrations the antifreezes (a) alter the direction in which water
freezes fastest on the faces of an ice crystal and (b) prevent recrystallization
(48). Thus the mechanism of antifreeze activity involves the ice-water inter
face. The affinity of these peptides for ice varies with molecular weight; the
small molecules bind less than the large (70). The affinity for ice disappears
if these molecules are chemically modified and their antifreeze activity is
then lost as well (22, 78). In the case of glycopeptides, alteration of the
 BIOLOGICAL ANTIFREEZE AGENTS 253

hydroxyls of the carbohydrate moiety leads to loss of activity (54) as well

as limited cleavage of the polypeptide backbone (49). Reduction in the size
of the glycopeptides by sequential degradation results in decreased binding
and antifreeze activity (77). This is consistent with the observation that
large polymers made up of repeating subunits are better inhibitors of crys
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tallization than is the subunit alone. Modifications of the carboxyl groups

of the aspartic and glutamic acid residues of the peptides result in loss of
activity (25) and, presumably, of binding. Specific modification of the scul
pin peptide antifreeze by attachment of tluorocene to its 4 lysine residue
results in complete loss of activity (76). Loss of antifreeze activity resulting
from modification of polar side chains appears to be correlated with a loss
in the affinity for ice (70). Since all of the polar side chains are potential
hydrogen bonders, it would appear that the antifreezes probably attach to
ice through hydrogen bonding between the polar side chains and water
molecules in the ice lattice. In order for maximal hydrogen bonding to occur
it can be predicted that the potential hydrogen bonding residues should
Annu. Rev. Physiol. 1983.45:245-260.

occupy positions in the molecule that would align them opposite the oxy
gens in the ice lattice.
Space-filling models of the glycopeptides reveal that many of the hydrox
yls of the disaccharide side chain are spaced approximately 4.5 A apart, a
distance that also separates the oxygens in the ice lattice parallel to the
a-axis. The secondary structure of the glycopeptides remains to be eluci
dated (4,21). However it is instructive to speculate how the spacings of the
hydroxyl groups and other hydrogen bonding groups in the various glyco
peptide conformations might lead to binding with ice. The carbohydrate
moieties might keep the polypeptide backbone of the antifreeze in a com
pletely extended conformation. In such a conformation,alternate carbonyl
groups project from the same side of the polypeptide; in the completely
extended conformation they are separated by -7.3 A (15,65). This distance
also sarates alternate oxygens along the c-axis in the ice lattice (38). This
7.36 A spacing of the oxygens in the lattice is also a repeat spacing. Some
evidence for the existence of a compltely extended conformation exists (39)
but is not overwhelming. With the glycopeptides, the presence of a repeat
spacing may not be necessary for binding. The important requirement may
be the. presence of several groups on the glycopeptides that can attach to
several oxygens in the ice lattice, which may not necessarily be regularly
arranged. Within the .disaccharide side chain, for example, many of the
hydroxyl groups:are separated by distances that closely approximate the 4.5
A spacing between the oxygens of the water molecules in the ice lattice,as
well as other spacings such as 7.36 A.
Sequence studies of the flounder antifreeze peptides indicate the presence
of clusters of polar amino acids separated by long sequences of nonpolar
 254 DEVRIES

aloimine residues (Figure 2). The polar clusters usually contain threonine and
aspartate separated by two alanines (17, 45). In the Alaskan plaice peptide,
a similar arrangement of polar and nonpolar residues is present (15). Physi
cal-chemical studies indicate that these peptides are all a.-helixes (2, 70). In
such a conformation, the polar side chains are located on one side of the
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helix while the nonpolar side chains face the other side. Aspartate and
threonine residues in such a conformation are separated by 4.5A., a repeat
distance that also separates adjacent oxygens in the prism faces of hexagonal
ice that are parallel to the a-axes. This lattice match between the polar
residues and the oxygens in the ice lattice suggests that the peptides orient
themselves on the lattice and bind to it through hydrogen bonding. Figure
3 illustrates how the flounder peptide might be aligned parallel to one of
thc a-axes of the lattice and how they might hydrogen-bond to it. In such
a model, every third row of oxygens does not participate in hydrogen
bonding. This may be important because an uninterrupted 4.5A repeat
Annu. Rev. Physiol. 1983.45:245-260.

spacing of the polar residues might result in nucleation.

Thus far complete sequences have been determined for a winter flounder
and an Alaskan plaice antifreeze peptide. The repeat spacing of the polar
threonines and aspartates found in the flounder peptide are conserved in
that of the plaice. Preliminary sequences of fragments of the sculpin pep
tides also indicate the presence of threonines or serines separated from
either aspartate or glutamate by 4.51.
Recently it has been suggested that the polar clusters participate in
,8-turns between the helical segments of alanine (55). In such turns, a

ALA-ALA-THR-ALA-ALA-THR-ALA-ALA-THR-ALA-ALA-THR-ALP.
I . I I I
-ALA-THR-ALA-ALA
I
NAGA NAGA NAGA NAGA NAGA
GAL GAL GL GAL GL

ASP-THR-ALA-SER-ASP-ALA-ALA-ALA-ALA-ALA-ALA-LEU-THR-ALA-ALA-ASP
ALA-ALA-ALA-ALA-ALA-ALA-LEU-THR-ALA-ALA-ASP
ALA-ALA-ALA-ALA-ALA-ALA-ALA-THR-ALA-ALA X -

r-45 Al
Figure 2 The primary structure of gJycopeptides 1-5 isolated from the antarctic nototheniid,
Dissostichus mawsoni. The basic glycotripeptide is repeated in the different sizes except glyco
peptides 6, 7, and 8 where prolines replace some of the alanines. The lower panel shows the
stmcture of one of the antifreeze peptides isolated from the blood of the winter flounder,
PseiUdopleuronectes americanus. In the conformation of an a-helix, the aspartic and threonine
residues are separated by 4.5 A. a distance that also separates the oxygens along the a-axes
. .
of the ice lattice.
 BIOLOGICAL ANTIFREEZE AGENTS 255

e; e;

:I:
I- <C
I I
u.o u-o
I
'"
0
" :I: "
1 0
I 0
1 1 i .... 11,5A --1
HYDROGEN BOND- : = I = = c:p c::D = =1 =1=
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I c:::> I <::)
c:::>
cp cP c:::>
<:::1 c:::> I

;\ .-'
I
Ole)O cb cb 0
ICE LATTICE 5 0 dD 0
..
I 1 u>
1
0 cD 0 0 @ 0 0 cD 0
0 0 0 0 0 0 0 0 0

0000 00000
A-AXIS
I
4,51
Annu. Rev. Physiol. 1983.45:245-260.

Figure 3 Model of flounder antifreeze hydrogen bonded to prism face of hexagonal ice,
parallel to the a-axes, Darker circles represent oxygens in the ice lattice that participate in
hydrogen bond formation with the hydroxyl of the threonine residue and carboxyl of the
aspartic acid residue. These two residues are separated by 4,5 A, a distance that also separates
the oxygens in the ice lattice parallel to the a-axes,

4.5A spacing between aspartate and threonine could still be conserved;

however, recent fluorescence polarization studies indicate that the peptides
are rigid rods and therefore probably lack -tums (76). Further studies are
needed in order to verify the underlying structural requirements necessary
for binding to ice.

INHIBITION Anomalously low freezing points of water in gels and tissues

have been explained on the basis of increases in surface free energy resulting
from a high ratio of surface area to volume. In gels, physical constraints
result in the formation of microcrystals with high surface free energies
(6, 50). For freezing to occur in such systems, energy must be removed
from the system. This is done by lowering the temperature. The appear
ance of microcrystals at the lower temperature would show that in
effect the freezing point of the water in which they formed had been
lowered.
Adsorption of the antifreezes to ice crystals could lead to an increase in
surface area with only a small increase in volume and could result in a
lowered freezing point. The evidence for this hypothesis and the mathemati
cal analysis are reviewed in depth elsewhere (68); only a qualitative over
view is set forth here, where it is assumed that the adsorption of the
antifreeze to ice leads to an increase in surface free energy of the ice crystal
resulting in depression of the freezing point. Ice crystal growth occurs as
 256 DEVRIFS

water molecules join the crystal at steps on the basal planes. It is thought
trutt adsorbed antifreeze molecules force growth to occur in the regions
between them. The small distances between the adsorbed antifreezes result
in lthe growth of many highly curved individual fronts with a large surface
area compared to their volume and as a consequence a high surface free
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em:rgy. Growth between the molecules stops when the ratio of surface area
to volume exceeds a critical point. This point is related to the radius of
curvature of the front. When the radius is equal' to one half of the spac
ing between two adsorbed adjacent antifreeze molecules, growth stops.
If the spacing between the antifreeze molecules is reduced then theunder
cooling required to allow the' step to propagate through the spacing
must be increased. Stated another way, the freezing point of water is
.

lowered.
The spacing between the antifreeze molecules appears to be a function of
their concentration and size (68). If certain assumptions are made about the
Annu. Rev. Physiol. 1983.45:245-260.

detllsity and randomness of the antifreeze molecules on the crystal face, the
undercooling (or freezing-point depression) is proportional to the square
root of the concentration. Good agreement exists between freezing point
depression curves obtained experimentally and those derived from the
above relationship (70).

SEASONAL OCCURRENCE OF ANTIFREEZES

Compared to the high latitude waters of the antarctic and arctic, those of
the north-temperate regions show extreme temperature variation. As a
consequence, blood levels of antifreeze in the winter flounder, Atlantic cod,
tomcod, and short homed sculpin all show seasonal changes (23, 24, 30, 32,
33, 37, 66). Antifreeze is synthesized in the liver during the autumn; its
biosynthesis is apparently controlled at the level of transcription and possi
bly translation ( 10, 45, 5 1, 53). Seasonal changes in the level of antifreeze
are correlated with temperature (24, 32, 33, 66), and photoperiod may be
involved (24, 32). There is some evidence for an endogenous cycle (32, 33)
with some pituitary influence (31, 35, 44).
.Antarctic and high arctic waters are near their freezing point throughout
the year and their fishes always have high levels of antifreeze in their blood
( 14-- 16). The antifreeze is synthesized by the liver (58). Warm acclimation
at +4C for 60 days does not alter the levels of glycopeptide antifreeze in
these fishes ( 16), indicating that temperature has little effect on the control
of its synthesis. The control of the annual cycle of antifreeze production
dest.arves further attention.
 BIOLOGICAL ANTIFREEZE AGENTS
ACKNOWLEDGMENTS
Much ofthe research described here was supported by NSF PCM 77-25166
and NSF DPP 78-23462 to ALD.
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