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First published online December 3, 2004
Journal of Experimental Biology 207, 4633-4649 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01312

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Cold-stable eye lens crystallins of the Antarctic nototheniid toothfish Dissostichus mawsoni Norman

Andor J. Kiss¹, Amir Y. Mirarefi², Subramanian Ramakrishnan³, Charles F. Zukoski^2,3, Arthur L. DeVries¹ and Chi-Hing C. Cheng^1,*

¹ Department of Animal Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, 61801, USA
² Centre for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, 61801, USA
³ Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, 61801, USA

^* Author for correspondence (e-mail: c-cheng{at}uiuc.edu)

Accepted 22 September 2004

	Summary

TOP Summary Introduction Materials and methods Results Discussion References

 
The eye lenses of the Antarctic nototheniid fishes that inhabit the perennially freezing Antarctic seawater are transparent at –2°C, whereas the cold-sensitive mammalian and tropical fish lenses display cold-induced cataract at 20°C and 7°C, respectively. No cold-cataract occurs in the giant Antarctic toothfish Dissostichus mawsoni lens when cooled to temperatures as low as –12°C, indicating highly cold-stable lens proteins. To investigate this cold stability, we characterised the lens crystallin proteins of the Antarctic toothfish, in parallel with those of the sub-tropical bigeye tuna Thunnus obesus and the endothermic cow Bos taurus, representing three disparate thermal climes (–2°C, 18°C and 37°C, respectively). Sizing chromatography resolved their lens crystallins into three groups, /ß_H, ß and , with crystallins being the most abundant (>40%) lens proteins in fish, in contrast to the cow lens where they comprise only 19%. The upper thermal stability of these crystallin components correlated with the body temperature of the species. In vitro chaperone assays showed that fish crystallin can protect same-species crystallins from heat denaturation, as well as lysozyme from DTT-induced unfolding, and therefore are small Heat Shock Proteins (sHSP) like their mammalian counterparts. Dynamic light scattering measured an increase in size of crystallin mixtures upon heating, which supports formation of the complex as an integral part of the chaperone process. Surprisingly, in cross-species chaperone assays, tuna crystallins only partly protected toothfish crystallins, while cow crystallins completely failed to protect, indicating partial and no interaction, respectively. Toothfish was likely to be the component that failed to interact, as the supernatant from a cow plus toothfish incubation could chaperone cow crystallins in a subsequent heat incubation, indicating the presence of uncomplexed cow . This suggests that the inability of toothfish crystallins to fully complex with tuna , and not at all with the cow crystallins, may have its basis in adaptive changes in the protein that relate to the extreme cold-stability of the toothfish lens.

Key words: lens crystallins, chaperone, Antarctic toothfish, Dissostichus mawsoni, bigeye tuna, cold adaptation, cold cataract, dynamic light scattering, alpha crystallin, gamma crystallin

	Introduction

TOP Summary Introduction Materials and methods Results Discussion References

 
The fish fauna that inhabits the freezing (–2°C) coastal waters of the Antarctic continent in the Southern Ocean is dominated by members of the endemic teleost suborder Notothenioidei. Their ability to survive in the frigid Antarctic marine environments is due to the evolution of blood-borne antifreeze glycoproteins, which allowed them to avoid freezing (Chen et al., 1997b; Cheng and Chen, 1999; DeVries, 1971) and to undergo subsequent adaptive radiation to become the predominant fish Antarctic fish taxon (Eastman, 1993; Eastman and McCune, 2000). Since cold temperatures depress the rates of biochemical reactions as well as affect the stability of protein structure (Hochachka and Somero, 2002), these fishes also exhibit a suite of biochemical and physiological adaptations to low temperature, including cold-efficient catalytic enzymes (Fields and Somero, 1998), cold-effective protein translocation (Romisch et al., 2003), membrane phospholipid unsaturation (Cossins et al., 2002; Romisch et al., 2003), and tubulins that polymerise at subzero temperatures (Detrich et al., 2000; Williams et al., 1985). An important physiological system in notothenioid fish that has not been characterised in any detail with regards to protein property and function at freezing seawater temperature is the lens. Although studies indicate that the structure of the eye and retina are the same to those of sub-tropical fishes, it is not known whether the lens and their constituent proteins differ from temperate and tropical species (Eastman and Lannoo, 2001, 2003).

The model system for studies of the vertebrate lens has been that of the cow. Bovine lens consists of a small insoluble albuminoid fraction, and a large soluble crystallin fraction (Mörner, 1864), which comprises three groups of proteins: , ß and crystallins (Bloemendal, 1986). In mammals, the most abundant protein is crystallin, a large oligomeric structure composed of two polypeptides, A and B, which belong to the small Heat Shock Protein (sHSP) family with chaperone-like ability to protect unrelated proteins from heat or chemical-induced protein denaturation (Horwitz, 2003; Narberhaus, 2002). The ß and crystallins are members of the ß gene superfamily of crystallins, which all possess a Greek-key fold protein motif, but whose non-refractive functions remain undetermined (Liaw et al., 1992; Slingsby and Clout, 1999; Wistow, 1993). Depending on the species, between four and six of the known ß and crystallin genes are expressed (Chiou et al., 1986; Norledge et al., 1997).

When the cow lens is cooled from the body temperature of 37°C to about 19°C, it begins to develop an opacity, a phenomenon known as cold-cataract, which has been attributed to the cold instability of some of the constituent crystallins, resulting in a liquid–liquid phase transition within the lens (Delaye et al., 1982; Gulik-Krzywicki et al., 1984; Norledge et al., 1997; Siezen et al., 1985). Cold-cataract has also been reported in a few other endothermic mammals such as the rat (Zigman and Lerman, 1964, 1965). There are very few studies on the temperature response of the lens crystallins of ectothermic vertebrates, but one study indicates that for the sub-tropical striped mullet Mugil cephalus, a cold-insoluble precipitate appears to form when the lens homogenate is cooled from 24°C to 7°C (Ferguson et al., 1971). In marked contrast to these warmer bodied species, polar fishes, particularly the Antarctic notothenioids that live in perennially freezing temperatures of –2°C, maintain complete lens transparency, suggesting a high degree of cold stability of their constituent lens proteins.

In this study, we report the first detailed characterisations of the whole lens and the lens crystallin proteins of the giant Antarctic nototheniid fish Dissostichus mawsoni, in terms of thermal response, protein composition, and various physical and biochemical properties. For comparison, we also examined the thermal, physical and biochemical properties of the crystallin proteins of the equally large lens of a mesophilic fish, the bigeye tuna Thunnus obesus and the endothermic cow Bos taurus, which collectively span the vertebrate body temperature range. This comparative analysis has allowed us to gain insights into the adaptive changes in protein properties that may have ensured transparency of the Antarctic notothenioid eye lens in constantly freezing seawater temperatures.

	Materials and methods

TOP Summary Introduction Materials and methods Results Discussion References

 
Lens collection
Lenses of the giant Antarctic toothfish Dissostichus mawsoni Norman were collected from live specimens caught in McMurdo Sound, Antarctica; cow Bos taurus lenses were obtained from Allen's Farm Quality Meats, Homer, Illinois; lenses of the small tropical marine fish blackbar soldierfish Myripristis jacobus were from specimens purchased at a local pet store; and the lenses of bigeye tuna Thunnus obesus were a generous gift of Timothy Ke (Luen Thai Fishing Venture, Ltd., Hong Kong and LTFV-USA) from specimens caught off the coast of the Marshall Islands. Some lenses were analysed when fresh, and others were stored frozen at –80°C until analysis.

Cold-cataract formation
The effect of temperature on the transparency of the lens was determined using fresh lenses. Bovine lenses, either within or removed from the eye capsule, were wrapped in plastic and embedded in a bucket of wet ice (0°C). Lenses of the tropical blackbar soldierfish M. jacobus were placed in test tubes with a small amount of vitreous humour and incubated at 15°C or in wet ice (0°C). Lenses of the Antarctic toothfish D. mawsoni were kept at –2°C (body temperature of toothfish and ambient temperature of Antarctic seawater) or at –12°C for a period of 48 h in a vial of High Temperature Silicon Oil (Aldrich 17,563-3). The appearance of each lens after these various temperature incubations was assessed and photographed with a digital camera (Fig. 1).

View larger version (59K): [in this window] [in a new window]   Fig. 1. Lens shapes and cold-cataract cooling experiment on the lenses of three species (Bos taurus, Myripristis jacobus and Dissostichus mawsoni) from three different physiological temperatures (37°C, 25°C and –2°C, respectively). (a–d) Schematics of the shapes (a,c) and a picture (b,d) of a D. mawsoni (a,b) and B. taurus (c,d) lens. (e,f) Cold-cataract experiment results showing lenses from the cow, B. taurus. (e) A fresh lens at room temperature (25°C); (f) a bovine lens that had been packed in ice for approximately 1.5 h, from which it was removed and allowed to warm. This image was taken during the warming process after the cortex had rapidly clarified and the nucleus was still opaque, showing the cold-cataract (arrow). (g–i) The eye lens from the tropical marine blackbar soldierfish M. jacobus held at (g) 15°C for 6 h; (h) 0°C for 6 h; (i) 0°C for 48 h, showing a definite inner nuclear region that is more opaque that the cortex region (arrow). (j–k) Images of the Antarctic toothfish D. mawsoni eye lens. (j) Endogenous clear –2°C lens contrasted to a lens held at –12°C for 6 h (k). The still clear toothfish lens after 48 h at –12°C (l) has a thin sheen of opacity. It is important to note that the opacity is restricted to the surface and not to the inner portions of the lens, as in the cow (f) and the soldierfish (i). Scale bars, 1.2 cm (e), 0.4 cm (g), 1.0 cm (j).

Size-exclusion chromatographic separation of the soluble crystallin proteins
Size-exclusion chromatography (SEC) using Sephacryl 200 High Resolution (S200HR) resin (Sigma, St Louis, MO, USA) was performed to fractionate the soluble lens protein components of D. mawsoni, T. obesus and B. taurus. Fresh or frozen lenses were first solubilised by stirring overnight at 8°C in three volumes of solubilisation buffer (10 mmol l^–1 potassium phosphate, 100 mmol l^–1 KCl, 0.05% NaN₃, pH 7.6). The potassium phosphate buffer pH was adjusted as per the method of Gomori (1955) and the complete buffer suction filtered through a Buchner funnel with No. 54 Whatman (hardened) filter paper. Solubilised lens homogenate was centrifuged at 20 198 g (13 000 revs min^–1) in an SS-34 rotor (Sorval RC-5B, Asheville, NC, USA) for 20 min set at 4°C to sediment the insoluble components. Between 0.5 and 1.0 g of soluble lens proteins (supernatant) were loaded (maximum volume 5 ml) on a S200HR column (2.5 cm i.d. x116 cm length) equilibrated with the solubilisation buffer, and protein elution was carried out with the same buffer at room temperature with a flow rate of 36 ml h^–1. Tube fractions (5 ml) were collected, and protein absorbance at 280 nm of each fraction was measured. Integration of the area under protein absorption peaks in the elution chromatogram was performed using the programme ORIGIN 6.0.4 (OriginLab, Northampton, MA, USA).

SDS-polyacrylamide gel electrophoresis and immunoblot detection of , ß and crystallin polypeptides
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using BioRad Mini-PROTEAN 3 system (BioRad Laboratories, Hercules, CA, USA). Crystallin protein (10–20 µg per sample) was loaded on a discontinuous SDS-polyacrylamide gel (4% stacking gel, 15% resolving gel) and electrophoresed for 1 h and 15 min at a constant voltage of 200 V. Gels were stained with 0.1% Coomassie R-250 in 10% acetic acid, 40% methanol, and destained with 10% acetic acid, 40% methanol.

For immunoblotting, SDS-PAGE separated proteins were transferred electrophoretically (BioRad Mini-Trans Blot Electrophoretic Cell) from un-stained gels to nitrocellulose membrane (Hybond ECL, Amersham Biosciences, Corp., Piscataway, NJ, USA) at 90 V for 3.5 h at 8°C. Membranes were incubated for 1 h at room temperature with the blocking solution 1 x TBS (20 mmol l^–1 Tris-HCl, pH 7.5, 150 mmol l^–1 NaCl) containing 5% non-fat milk powder (Carnation, Nestlé USA Inc., Solon, OH, USA) and 0.05% Tween-20, and then probed with polyclonal rabbit anti-bovine crystallin primary antibodies (gift of Dr J. Sam Zigler, National Eye Institute, NIH, USA) in antibody (Ab) buffer (1 x TBS, 5% non-fat milk powder, 0.25% Triton-X100). The anti-bovine crystallin (, ß, ) antisera were able to react with fish crystallins in this study. Bovine crystallins were detected colorimetrically, while the fish crystallins required more sensitive chemiluminescence detection. For colorimetric detection, the primary Ab stock (1 mg ml^–1) was diluted 1:500 for , ß and , and for chemiluminescence detection, 1:2000 for , 1:2500 for ß, and 1:5000 for crystallin, respectively. Unbound primary Ab was removed by washing in 1 x TBS with 0.05% Tween-20. Secondary antibody, horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H+L) (1.5 mg ml^–1, ZYMAX 81-6120, Zymed, San Francisco, CA, USA) was diluted 1:5000 in Ab buffer for colorimetric detection, and 1:50 000 forchemiluminescence detection. Bovine crystallin blots were incubated in colormetric reagent (BioRad HRP Conjugate Substrate Kit) for approximately 10 min, washed with distilled water and air-dried. Fish crystallin blots were developed with chemiluminescence reagent (Pierce SuperSignal WestPico Chemiluminescent Substrate, Pierce, IL, USA) per manufacturer's instructions and autoradiographed on Kodak BioMax film for 10 s to 5 min, as appropriate.

Determination of upper limits of thermal stability of crystallins
The upper limit of thermal stability or T_S is defined as the maximum temperature that a crystallin fraction can be subjected to without precipitation of the protein. T_S values for the SEC purified and crystallin fractions from toothfish, bigeye tuna and cow were empirically determined to establish the appropriate range of test temperatures for the chaperone-like activity assays (next section). The protein concentrations of the crystallin fractions were calculated from their A₂₈₀ values (cow) using published (Siezen et al., 1986) and our gravimetrically determined extinction coefficients (fish). To determine these gravimetrically, , ß and fish crystallin solutions were collected from the SEC separations (separately), diluted down below 1.0 A₂₈₀ units, and the absorbance at 280 nm recorded. Exactly 10 ml was lyophilised for 24 h in a pre-weighed, pre-lyophilised clean glass vial. Vials containing the lyophilised crystallins were weighed immediately on an analytical scale with 0.001 mg resolution. All measurements for fish crystallin were made twice. All extinction coefficients are expressed as A₂₈₀ in 1 cm path length of a 1% solution (Table 1). Samples of each crystallin (800 µl at 1 mg ml^–1) were incubated at various temperatures in a UV-transparent polystyrene microcuvette placed in a spectrophotometer equipped with a water-jacketed 7-cuvette holder (Helios Gamma, Spectronic Instruments Inc., Rochester, NY, USA). The incubation temperature was controlled using a circulating water bath (HaakeFS) and the actual sample temperature was monitored using a digital thermocouple thermometer equipped with a fine wire probe (Cole-Parmer, Vernon Hills, IL, USA) in an isothermal adjacent microcuvette containing sample buffer. Heat-induced protein aggregation results in static light scattering that can be followed by monitoring turbidity (increased absorbance) at 360 nm(Jaenicke and Seckler, 1997). A₃₆₀ of the sample was automatically measured at 5 min intervals by a computer interfaced with the spectrophotometer using the program HYPERTERMINAL (Hilgraeve, Monroe, MI, USA). The highest incubation temperature at which A₃₆₀ remained at or very near zero for at least 1 h was taken as the T_S.

Assays for chaperone-like activity of crystallin
Lens crystallins are known sHSPs and have the chaperone-like ability to protect other proteins from stress-induced denaturation (Horwitz et al., 1998). The chaperone-like function of the fish and bovine crystallins in protecting against heat-induced aggregation of crystallins, and from chemically induced (by DL-dithiothreitol) aggregation of lysozyme was assayed. Since SEC fractionation of crystallins does not achieve perfect separation of the individual classes of crystallins (see Results), the crystallin fractions for these chaperone assays were chosen as follows. The SEC crystallin peak from SEC separation of crystallins of all three species (see Results and Fig. 2A–C, peak III) was predominantly crystallin, based on immunoblots (see Results and Fig. 3), and thus the five highest A₂₈₀ fractions of this peak were used in the assays. The _S fraction (Fig. 2C, peak IIIa) was not used in chaperone assays or dynamic light scattering (DLS) studies. For bovine crystallin, the first 2–3 fractions of the ascending slope of the SEC peak I (Fig. 2C, peak I) are near homogeneous for crystallin (see Results), so these were used for the assays. For fish, none of the fractions from the SEC crystallin peak (Fig. 2A,B, peak I) are homogeneous for crystallin, but contain various amounts of ß crystallins (see Results). We found that the first 2–3 fractions of the ascending slope of the elution peak have the highest and most consistent chaperone activity, indicating the greatest concentration of crystallins, and these were selected for all subsequent chaperone assays. Thus, the terms crystallin and crystallin in the described chaperone experiments refer to these particular SEC elution fractions. Because of this protein heterogeneity, the stated concentrations of crystallin used in the assays represent the upper bound values. In other words, the observed chaperone activity is achieved by crystallin concentrations at or lower than the stated values. Beta crystallins are not known to be molecular chaperones (or sHSPs) and therefore their presence in the crystallin fractions is not expected to contribute to the observed chaperone activity in the assays.

View larger version (16K): [in this window] [in a new window]   Fig. 2. Size-exclusion chromatography using Sephacryl 200 High Resolution resin of crystallins from three species, D. mawsoni (A), T. obesus (B) and B. taurus (C). Peaks are labelled as /ßH (peak I), ßH (peak IIa), ßL (peaks II or IIb), or S and (peaks IIIa and III). Numbers labelled on each individual peak correspond to the lanes of the SDS-PAGE and immunoblots presented in Fig. 3. Fraction volume sizes are 5 ml each.

View larger version (72K): [in this window] [in a new window]   Fig. 3. SDS-PAGE (A,E,I) and immunoblot analysis (B–D, F–H, J–L) of SEC elution profiles of crystallins from D. mawsoni (A–D), T. obesus (E–H) and B. taurus (I–L). The primary antibodies used for detection are denoted on the left. Molecular mass markers (Lane M; kDa) are denoted on the left of each panel. Lane numbers correspond to the locations of the numbered positions on Fig. 2. for each species. All immunoblots are independent replicas of SDS-PAGE presented in Fig. 3A,E,I.

Assays of chaperone protection against heat aggregation were performed similar to previously published methods (Horwitz, 1992; Liao et al., 2002). Gamma crystallin (final concentration of 1 mg ml^–1) of each species was mixed with different amounts of crystallin (final concentration of 62.5 µg ml^–1 to 1.0 mg ml^–1) of the same species in a total assay volume of 800 µl. The incubation temperature was 47°C, 55°C and 60°C for toothfish, tuna and cow, respectively, and 5 min A₃₆₀ measurements were carried as described above. T_S of crystallin is lower than T_S of crystallin (see Results), thus no increase in A₃₆₀ at these incubation temperatures was interpreted to be the result of chaperone protection by crystallin against thermal aggregation of crystallin. All assays were repeated three times.

The ability of crystallin to protect DTT-induced aggregation of lysozyme at 37°C, a common approach used for testing the chaperone-like activity of crystallins (Abgar et al., 2000; Vanhoudt et al., 2000), was used for the three species in this study. Various amounts (0.2–2.2 mg) of crystallin fraction were added to 200 µg of chicken egg white lysozyme (EC 3.2.1.17, Sigma) in a final volume of 1 ml. Freshly made 1 mol l^–1 DTT (Sigma) was added to a final concentration of 20 mmol l^–1 to initiate lysozyme unfolding, and A₃₆₀ was measured at 5 min intervals. Three replicates of each condition were performed for each species.

Cross-species chaperone assays
To assess whether crystallins from cold-adapted and warm-adapted organisms could interact, and crystallins of toothfish, tuna and cow, representing three disparate thermal environments (polar, –2°C; subtropical, 18°C; endothermal, 37°C), were used in pairwise cross-species chaperone protection assays similar to the same-species assays described above. Pairwise combinations of and crystallin from D. mawsoni, T. obesus and B. taurus at a 1:1 mass ratio (1 mg:1 mg in 0.8 ml), were incubated at the T_S of the crystallin component, and A₃₆₀ recorded at 5 min intervals. For the combination of the bovine crystallin and toothfish crystallin, an additional two-part assay was performed as follows. Bovine crystallin (final concentration mg ml^–1) and toothfish crystallin (final concentration 1 mg ml^–1) were incubated for 1 h at 60°C (final volume of 0.8 ml). Aggregated protein that was observed was then sedimented by centrifugation at 21 000 g for 10 min at 4°C. The supernatant was combined with bovine crystallin (final concentration 1 mg ml^–1) in a total volume of 1 ml or less, and incubated for a second 1 h period at 60°C. A₃₆₀ was recorded at 5 min intervals for all assays.

Estimation of effective crystallin size by dynamic light scattering
The effective molecular size in solution of individual crystallins of cow and fish lens, and of interacting -crystallin complexes, were measured by dynamic light scattering (DLS) with the Brookhaven Instruments goniometer system (BI-200SM, Brookhaven Instruments Corp., Holtsville, NY, USA) equipped with a Lexel argon-ion laser (model 95, Cambridge Lasers Lab., Fremont, CA, USA) operating at 514 nm. Individual and crystallin fractions, freshly purified by SEC, were first centrifuged at 18 400 g at 4°C for 20 min and the supernatant was filtered through 0.050 µm SPI-Pore polycarbonate filters (SPI Supplies, West Chester, PA, USA) to remove dust and other particulates before sizing. We found that the filtration step was necessary to obtain reproducible DLS sizes, especially for crystallins, which form increasingly larger high molecular mass (>1000 kDa) oligomers with storage time. For unheated samples, the filtered crystallin fraction was sized in a clean DLS cell at room temperature. Heated samples (1 h incubation at the desired temperature) were first cooled to room temperature, centrifuged to pellet any heat-aggregated protein, and the supernatant was transferred to a clean DLS cell and sized. Light intensity at 90° angle was measured by a photomultiplier tube and the output signal was processed by a Brookhaven 9000AT correlator. Crystallin size was calculated by Brookhaven Instruments DLS Software Version 2.17 and expressed as the hydrodynamic diameter in nm.

	Results

TOP Summary Introduction Materials and methods Results Discussion References

 
Lens shape and structure
A schematic of the shape and corresponding photographs of Antarctic toothfish D. mawsoni and cow B. taurus lens are shown in Fig. 1a–d. Fish lenses are spherical (Fig. 1a) and denser than bovine lens. The lens of a 27 kg Antarctic toothfish is approximately 1.5 cm in diameter and weighs about 3.5 g (Fig. 1b). Lenses of similar-sized bigeye tuna (T. obesus) are larger, about 3 cm in diameter and weigh about 10–12 g (not shown). The adult bovine lens is approximately 2 cm indiameter and has a mass of about 4.5 g (Fig. 1c,d). It is not spherical, but biconvex in shape, with the less convex side facing the cornea and the more convex side facing the retina. All lenses are enveloped in a collagen lamellar structure called the capsule (Davson, 1990). In contrast to the dense fish lens, the cow lens is much softer and once decapsulated, its outer cortical layers readily slough off.

Cold-cataract formation
Fig. 1e–l illustrates the effect of temperatures below normal body temperature on the transparency of the lens of cow and fish. Fig. 1e is a freshly dissected bovine lens at room temperature (25°C) showing complete transparency. Cold temperature induced opacity or cold-cataract in a cow lens after cooling in ice (0°C) for 1.5 h (Fig. 1f). When the cooled lens was retrieved for photographing at room temperature, the slightly opaque outer cortex rapidly clarified, while the nucleus remained opaque, as indicated by the arrow (Fig. 1e). The nucleus completely clarified later, after 2 h at room temperature. Lenses from the small tropical (24°C) marine fish, the blackbar soldierfish Myripristis jacobus (Fig. 1g–i), are approximately 6 mm in diameter and have the same structural morphology as the D. mawsoni and T. obesus lenses. Transparency of the lens was maintained on cooling to 15°C (Fig. 1g). A second lens incubated at 0°C (in ice) for 6 hbecame less transparent (Fig. 1h) than the lens at 15°C. Further incubation at 0°C (48 h total) resulted in further opacity throughout the lens. Retrieval from ice for photography at room temperature partially clarified the cortex, but the nucleus remained opaque, as indicated by the arrow (Fig. 1i). Further equilibration at room temperature for several hours did not clarify the nucleus.

Antarctic toothfish lenses are completely transparent at –2°C, the ambient environmental temperature and normal body temperature of this ectotherm (Fig. 1j). A second lens after 6 h and 48 h incubation at –12°C is shown in Fig. 1k and l, respectively. The lens was immersed in a silicon oil to permit incubation temperature as low as –40°C without the oil freezing or losing optical clarity. In contrast to cold-cataract development in cooled bovine and soldierfish lens, the toothfish lens remained internally clear even after 48 h at–12°C (Fig. 1l), with only a surface veneer of slight opacity, probably due to the reaction of the silicon oil with the outer layers of the collagen capsule. The silicon oil did become slightly translucent itself, but the lens remained clear. Cooling the lens below –12°C was not possible as the lens invariably froze. It can be concluded, however, that cold-cataract formation does not occur in the Antarctic toothfish lens at temperatures as low as –12°C.

SEC fractionation of lens crystallins
Fig. 2 shows that the chromatograms of the crystallins from toothfish, bigeye tuna and cow fractionated on S200HR size-exclusion resin are all similar in pattern. The bovine elution chromatogram (Fig. 2C) is similar to that reported in the literature (Björk, 1961; Bloemendal, 1986; Siezen et al., 1986; van Dam, 1966) using G75 Sephadex and 200HR Sephacryl resins. Prior characterisations of bovine lens showed that the three predominant peaks I, II and III (or IIIa and III) containing primarily , ß and crystallin, respectively (Bloemendal, 1986). Using the S200HR resin, the high molecular mass crystallin and ß_H (ß_High) crystallin both elute in peak I, which is designated as the `/ß_H' peak in Fig. 2. (The constituents of these elution peaks are given here, with the verification of their specific identities detailed in the next Results section.) Fractionation on Sepharose 6B resin provided better separation of cow and ß_H crystallins (data not shown), but much poorer separation of ß_L (ß_Low) from the crystallins for all three species. Therefore, the S200HR resin was used in this study to allow optimal comparisons of SEC chromatograms and subsequent analyses.

Although similar in pattern, the SEC elution profiles of fish crystallins differ from the cow in the relative size of the ß and peaks. The bovine ß crystallins partitioned into ß_H (part of peak I, apex and descending slope; Fig. 2C) and a prominent distinct ß_L peak (Fig. 2C, peak II). While ß_H is present in peak I of tuna and toothfish, there are also distinct small ß_H and ß_L peaks in the bigeye tuna chromatogram (Fig. 2B, peaks IIa and IIb). The toothfish profile (Fig. 2A) has no ß_H peak and only a very minor ß_L peak (Fig. 2A, peak II). In contrast, the crystallin peak of both fish species (Fig. 2 A, B peak III) is much larger than the bovine crystallin peaks (Fig. 2C, peaks IIIa and III combined). Fig. 2C, peak IIIa contains cow _S, which elutes ahead of the rest of the cow crystallins in peak III.

Estimations of relative protein abundance based on peak areas (Fig. 2) for each species are tabulated in Table 2. Toothfish (52% /ß_H, 5% ß_L and 43% ) is similar to bigeye tuna (47% /ß_H, 6% ß_H, 6% ß_L and 41% ) in relative percentage abundance of the three classes of crystallins. Both fish differ from the cow (54% /ß_H, 27% ß_L and 19% ) in having a substantially higher percentage of crystallins, over 40% as opposed to 19%, making it the predominant crystallin fraction in the fish lens. Separate estimates of percentage abundance of and ß crystallins are not possible because of their overlapping elution within peak I (Fig. 2A,B), as revealed by SDS-PAGE analysis and immunoblotting with , ß, antisera (next section). The SEC peaks, peak III of fish and peaks IIIa and III for cow, however, are near homogeneous for crystallins, based on immunoblots (next section), and therefore their percentage abundance estimations and comparisons are reliable.

SDS-PAGE and immunoblot analyses of crystallin fractions
The identities of the class of crystallins and the extent of protein heterogeneity in the SEC elution peaks (Fig. 2) were assessed by SDS-PAGE and immunoblots (Fig. 3). The lane numbers in the SDS gels and the immunoblots (western blots) (Fig. 3) correspond to the number of selected fractions from the SEC elution (Fig. 2, labelled next to the peaks). The results of the SEC fractionation, SDS gels and immunoblots collectively show the following. For all three species, the crystallin polypeptides range in size from about 18 kDa to 35 kDa. Rabbit antisera against the three bovine crystallins could crossreact with the respective fish crystallins. SEC separation achieved near-homogeneous crystallin fractions in all species (Fig. 3A,E,I, fractions), but did not adequately resolve and ß_H crystallins from each other in the two fish species, such that SEC peak I of both fish species encompasses considerable protein and size heterogeneity (Fig. 3A,E,I, /ß_H fractions).

More specifically, the selected fractions 1–5 from the bovine peak I (Fig. 2C) contain A and B crystallins at 21 and 23 kDa, and an additional 18 kDa band for fractions 1–4 (Fig. 3I, lanes 1–5), which is probably an A crystallin truncation product previously reported in the literature (Augusteyn et al., 1992; Horwitz et al., 1998). These protein bands are all immunopositive to anti-bovine crystallin polyclonal antibodies in the immunoblot (Fig. 3J, lanes 1–5), and of these five fractions, the first two (indicated with arrow at peak I, Fig. 2C) are predominantly crystallin, as indicated by strong anti- immunopositive bands (Fig. 3J, lanes 1,2), and little or no immunopositivity to anti-ß (Fig. 3K, lanes 1,2). The subsequent fractions 3–5 of bovine SEC peak I (Fig. 3I, lanes 3–5), all contain ß_H crystallins, as indicated by the two sets of anti-ß crystallin immunopositive bands close to the 31.1 kDa and 28.4 kDa and markers in the immunoblot (Fig. 3K, lanes 3–5).

The SEC peak I fractions of the two fish (Fig. 2A,B) contain substantially more protein heterogeneity on SDS-PAGE (Fig. 3A,E, lanes 1–5) than their bovine counterpart and none of the selected fractions examined is homogeneous for crystallin. Chemiluminescence immunodetection using anti-bovine crystallin polyclonal antibodies show crystallins of 21 kDa in peak I fractions 1–4 for toothfish (Fig. 3B, lanes 1–4), and peak I fractions 1-4 for bigeye tuna (Fig. 3F, lanes 1–4, signal weak for lane 4). All five selected fractions of SEC peak I of both fish species contain two sets of anti-ß immunoreactive bands, close to the 28.4 kDa and 31.1 kDa markers (Fig. 3B, F, lanes 1–5). These sizes are consistent with the reported values (26–35 kDa) for mammalian ß crystallins (Slingsby and Clout, 1999) and known fish ß crystallins (Wistow, 1995; Zigler and Sidbury, 1976). The presence of the 31.1 kDa immunopositive bands indicates that ß_H coeluted with crystallin in peak I of fish. For both fish and cow, some fractions of the /ß_H peak contain several weakly staining, large-sized protein bands, close to the 49.9 kDa marker (and above in the toothfish) in the SDS gel (Fig. 3A,E,I). These 49.9 kDa bands are likely oligomeric ß or /ß aggregates since they are weakly immunoreactive to anti-ß (Fig. 2C,G,K), and to anti- (Fig. 2J). The bands greater than 49.9 kDa of the toothfish (Fig. 3A) are not immunoreactive to any antisera, and thus their identity remains to be investigated.

For both fish and cow, the SEC peak II (peaks IIa and IIb for bigeye tuna) contains only ß crystallin. SEC peak II of toothfish and cow, and peak IIb of the bigeye tuna (Fig. 2A–C) appears homogeneous for ß_L in immunoblots (Fig. 3C,G, lane 7, and K, lane 6).

For all three species, SEC peak III (Fig. 2) is homogeneous for crystallins on immunoprobing (Fig. 3D,H, lanes 8,9, L, lane 8). In the case of the cow, _S (Fig. 3L, lane 7), formerly ß_S in older literature, eluted as a discrete peak ahead of the other crystallins (Fig. 2C, peaks IIIa and III). Because the SEC crystallin fractions do not have or ß crystallins within them, their relative percentage abundance in the lens, estimated on the basis of protein elution peak areas (Table 2), is reliable. Gamma crystallins constitute the predominant crystallin in fish lens (43% of toothfish crystallins, 41% of bigeye tuna crystallins), which is over twofold more abundant than its bovine counterpart (19% of total crystallins).

Upper limit of temperature stability (T_S) of and crystallin from D. mawsoni, T. obesus and B. taurus
Fig. 4 shows A₃₆₀ versus time for a series of incubations at increasing temperatures for toothfish crystallin and crystallin. The highest temperature at which the A₃₆₀ remained at or near zero for at least 1 h was defined as the T_S value, and was found to be 47°C and 33°C for toothfish and crystallins, respectively. The T_S value for the and crystallins of bigeye tuna and cow were similarly determined. T_S values of the crystallins of bigeye tuna and cow were 55°C and 68°C, respectively, and T_S of their crystallins were 39°C and 50°C respectively (Table 3). Thus the crystallins are much more heat-labile relative to crystallins, by 14°C, 16°C and 18°C for toothfish, tuna and cow, respectively. Additionally, there was a direct correlation between the T_S of the crystallins and the body temperature of each animal, highest for the endothermic cow (mean body temperature 38.3°C; Piccione et al., 2003), followed by the sub-tropical ectothermic bigeye tuna (mean body temperature 18°C; Holland et al., 1992), and lowest for the ectothermic Antarctic toothfish, which inhabits the perennially freezing seawater of the Southern Ocean (mean body temperature –1.9°C; Hunt et al., 2003).

View larger version (19K): [in this window] [in a new window]   Fig. 4. Determination of the upper limit of thermal stability TS for the (A) and (B) crystallins from D. mawsoni, measured as the change in turbidity (A360) over time at various incubation temperatures. Concentration of crystallins was 1 mg ml–1 in all assays. (A) Upper limit of TS for crystallin. At temperatures above 47°C, turbidity increases with time. at 47°C (x); 55°C (x); 60°C (x). (B) Upper limit of TS for crystallin. at 30°C (x); 33°C (x); 35°C(x); 42.5°C (x); 44°C (x); 45°C (x); 47°C (x); 57.5°C (x). The TS was determined as 47°C for the and 33°C for the crystallin.

View this table: [in this window] [in a new window]   Table 3. Empirically determined upper limit of thermal stability for and crystallins from S200HR size fractionations

Chaperone-like activity of crystallin from D. mawsoni, T. obesus and B. taurus
In the absence of crystallin, heat aggregation of Antarctic toothfish crystallin occurred rapidly, after 10 min at 47°C. Turbidity as measured by A₃₆₀ increased sharply to 0.8 in the first 30 min, and reached a plateau of 1.0 in the next 30 min (Fig. 5A). At mass ratios of 1:1 and 1:2 of : crystallin, aggregation of the crystallin was prevented during the 1 h assay time (Fig. 5A). Protection decreased as crystallin concentration was decreased, but even with 1:16 ratio of to crystallin, there was still significant protection compared to unchaperoned crystallin (Fig. 5A).

View larger version (35K): [in this window] [in a new window]   Fig. 5. Chaperone protection by crystallin of heat-aggregated crystallin (A–C) along with protection of DTT-denatured lysozyme (D–F). (A,D) D. mawsoni; (B,E) T. obesus; (C,F) B. taurus. Temperatures for the assay are indicated in each panel. Values are means ± S.E.M. (N=3). In A–C the crystallin and crystallin are both from the same species, with fractional amounts of crystallin added based on a 1:1 mass ratio of to crystallin at a final assay concentration of 1 mg ml–1 for both crystallins (i.e. x in A–C). In D–F the amount of crystallin added was based on a 1:1 mass ratio of crystallin to lysozyme, where in all assays (D–F) there was 0.2 mg ml–1 of lysozyme containing 20 mmol l–1 DTT. For A–C: x ; x 1:16 :; x 1:8 :; x 1:4 :; x 1:2 :; x :. For D–F: x 0:1 :lyso; x 1:1 :lyso; x 2:1 :lyso; x 3:1 :lyso; x 2.5:1 :lyso; x 5:1 :lyso; x 7.5:1 :lyso; x 10:1 :lyso; x 11:1 :lyso.

For comparison, chaperone assays were performed using crystallin of the bigeye tuna T. obesus and the cow B. taurus and same-species crystallin, at 55°C for bigeye tuna and at 60°C for the cow (Fig. 5B,C). By itself, crystallin from either species increased in turbidity rapidly on heating, while mass to ratios of 1:1, 1:2 and 1:4 displayed effective protection of crystallin (Fig. 5B,C). At the lower to mass ratios of 1:8 and 1:16, very effective chaperone protection of crystallin was observed in the case of the tuna, while much less chaperone protection was observed in the case of cow (Fig. 5A–C).

Chaperone protection of lysozyme by crystallin from D. mawsoni, T. obesus and B. taurus
In the absence of crystallin, lysozyme rapidly unfolded in 20 mmol l^–1 DTT at 37°C, with A₃₆₀ kinetics similar to heat denaturation, plateauing at A₃₆₀1.1 over 1 h (Fig. 5D–F). Protection against DTT-induced denaturation of lysozyme by crystallin (calculated by dividing the reduction in A₃₆₀ between the unchaperoned and chaperoned assays by the A₃₆₀ of the unchaperoned assay at the 1 h end point) increased with increasing amounts of crystallin added. A 10:1 mass ratio of D. mawsoni crystallin to lysozyme resulted in 79% protection, 66% at a 5:1 mass ratio, and 25% at a 2.5:1 mass ratio (Fig. 5D). For T. obesus crystallin, an 11:1 mass ratio provided 88% protection, 75% at a 7.5:1 mass ratio, but only 9% at a 5:1 mass ratio of T. obesus crystallin to lysozyme (Fig. 5E). The B. taurus crystallin exhibited much greater chaperone ability than either fish, achieving 95% protection of lysozyme at a mass ratio of only 3:1 crystallin to lysozyme (Fig. 5F).

Dynamic light scattering (DLS) sizing of and crystallins
To determine whether the chaperone effect is related to a stable interaction between the and crystallins, the sizes of individual crystallins, and of and together before and after incubation at the T_S of the crystallin of each species, were measured by DLS (Table 4). The hydrodynamic diameters of crystallin at room temperature were 27.6 nm, 29.1 nm and 31.7 nm for toothfish, bigeye tuna and cow, respectively, while that of crystallin was about 4–5 nm for all three species. The major bovine crystallin fraction from Sepharose 6B fractionation (data not shown) was found to be 19.4 nm in diameter, which is smaller than the 31.7 nm of the S200HR bovine crystallin fraction. This discrepancy is probably due to the presence of residual high molecular mass aggregate in the S200HR crystallin fraction, which partitioned as a small peak in Sepharose 6B elution ahead of the major peak (data not shown). Except for the cow, the crystallin increased in size after 1 h incubation at its T_S, consistent with `activation' of the crystallin chaperone complex (Putilina et al., 2003), and perhaps also due to association of with the ß crystallin present in the heterogeneous crystallin fraction used. A mixture of and crystallin (1:1 mass ratio) at room temperature has a similar size to crystallin alone, probably because there is no molecular interaction between and crystallin before heating, and thus the DLS size of the mixture essentially reflects that of the much bigger crystallins. After a 1 h incubation at the respective crystallin T_S, the hydrodynamic diameter of the of and crystallin mixture increased by 94% (27.4–53.4 nm) for D. mawsoni, 142% (24.9–60.3 nm) for T. obesus, and 55% increase (from 30.3 nm to 47.2 nm) for B. taurus (Table 4), indicative of molecular association of the two crystallins forming a larger size complex.

View this table: [in this window] [in a new window]   Table 4. Hydrodynamic diameters of and crystallins determined by dynamic light scattering

Cross-species chaperone protection of crystallin by crystallin
The ability of crystallin of each species to protect the crystallin of a different species from heat aggregation was evaluated (Fig. 6). The chaperone assays were carried out at the T_S of the crystallin, for pairwise combinations in which the crystallin has a lower T_S than that of the crystallin. The combination of toothfish plus cow at 47°C, though not meaningful as a chaperone assay since cow with a T_S of 50°C is more heat stable, was included as a `non-interacting' control. Protection was observed for bigeye tuna crystallin plus bovine at 55°C, toothfish crystallin plus tuna at 47°C, and bovine plus tuna at 68°C, with A₃₆₀ reaching only 0.05, 0.10 and 0.25 (respectively) at the 1 h end point. Tuna crystallin did not completely protect toothfish at 55°C, and an A₃₆₀ of 0.75 was reached at 1 h; however, the slope of the turbidity increase was much less steep (tenfold) than that of toothfish alone at 55°C, indicative of partial interaction of the two crystallins leading to partial protection. The most notable and unexpected result is that cow crystallin offered no protection at all to toothfish at 68°C, resulting in a rate of turbidity increase identical to that of toothfish alone at 55°C, indicating that all or most of the toothfish crystallins had precipitated (Fig. 6).

View larger version (19K): [in this window] [in a new window]   Fig. 6. Cross-species chaperone protection assay of crystallin by crystallin from the three species D. mawsoni, T. obesus and B. taurus. Chaperone assay temperature was at TS for the crystallin in the assay. Final concentration of both and crystallin in the assay was 1 mg ml–1. Combinations of and crystallin are indicated. Values are means ± S.E.M.; bars are obscured by symbols (N=3).

DLS measurements of heated heterologous combinations showed that chaperone protection was associated with a size increase of the crystallins, indicating the formation of a complex (Table 5). Assuming the pre-incubation DLS size of the pairwise + combinations is similar to that of the component, 20–29 nm (Table 4), the post-incubation DLS size of the cross-species + complex increased significantly, to 43–73 nm for the combinations that exhibited chaperone protection (Table 5). In contrast, the hydrodynamic diameter of the heated cow and toothfish combination (the supernatant after removal of protein aggregates) was the smallest, at 38.5 nm, indicating the least extent of interaction.

View this table: [in this window] [in a new window]   Table 5. Cross-species mixtures of and crystallins

The extent of non-interaction of cow with toothfish was assessed in a two-part chaperone assay (Fig. 7). Fig. 7A shows the A₃₆₀ of a 1 h incubation of bovine plus toothfish at 60°C, and the control incubation of bovine plus bovine , both at mass ratio mg to 1 mg in 1 ml final volume ( mg of cow crystallin is the estimated minimal amount that would completely protect 1 mg of bovine crystallin; Fig. 5). The 60°C incubation of bovine crystallin plus toothfish crystallin resulted in rapid turbidity increase, with the same kinetics and peak A₃₆₀ as the 68°C incubation (Fig. 6), while bovine was fully protected by bovine , as expected (Fig. 7A). After centrifugation, additional bovine crystallin was added to the supernatant of both incubations to the same final concentration (1 mg ml^–1) as the first 1 h incubation, for a second 1 h incubation at 60°C (Fig. 7B). The supernatant from the cow crystallin plus toothfish crystallin incubation showed substantial chaperone protection of added cow crystallin in the second hour, reaching an A₃₆₀ of only 0.486. This result indicates that most of the cow crystallin did not complex with toothfish crystallin during the first 1 hincubation, and therefore was free to interact with the added cow crystallin in the second 1 h incubation. In contrast, the supernatant from cow crystallin plus cow crystallin provided no protection of added cow crystallin in the second hour, reaching an A₃₆₀ of 1.5 at 50 min. This indicates that little or no free cow crystallin was available to chaperone the added cow crystallin, probably because most or all of the cow crystallin was saturated or complexed to cow crystallin during the first 1 h incubation.

View larger version (13K): [in this window] [in a new window]   Fig. 7. Functional assessment of B. taurus crystallin after 1 h incubation at 60°C in the presence of D. mawsoni crystallin. (A) Experimental mg B. taurus crystallin + 1 mg D. mawsoni crystallin (x) and control reaction of mg B. taurus crystallin + 1 mg B. taurus crystallin (x). After 1 hsamples were removed from the cuvette and any precipitate was removed by centrifugation at 21 000 g at 4°C in a benchtop microcentrifuge. (B) Supernatants (750 µl) were then transferred to a new cuvette and a further 1 mg of B. taurus crystallin was added. B. taurus crystallin was concentrated such that the total volume in the second assay (after addition of the B. taurus crystallin) was not greater than 1 ml. Final composition of assay was (B. taurus + D. mawsoni ) supernatant + B. taurus crystallin (x) for the experimental and (B. taurus + B. taurus ) supernatant + B. taurus crystallin (x). Second assay incubation was for 1 h at 60°C.

	Discussion

TOP Summary Introduction Materials and methods Results Discussion References

 
The giant nototheniid fish Dissostichus mawsoni Antarctic toothfish is endemic to the Antarctic region of the Southern Ocean, where water temperatures are perennially at or near the freezing point of seawater (–2°C), the coldest known extreme for marine ectotherms. Its eye lens is transparent at this freezing temperature, indicating that the lens and its protein constituents are highly cold-stable. The whole lens cooling experiments presented in this study revealed that the toothfish lens remained clear even when cooled to temperatures as low as –12°C (Fig. 1j–l). This low temperature stability is in contrast to the endothermic mammalian lens, which undergoes rapid cold-induced lens opacity (cold-cataract) below 20°C, as was shown for the bovine lens in this study (Fig. 1e,f) and by others (Banh and Sivak, 2004; Clark and Benedek, 1980a; Delaye et al., 1982). Investigations of the phenomenon with intact teleost fish lenses have been sparse, limited to temperate and subtropical species (Hikida and Iwata, 1985, 1986; Loewenstein and Bettelheim, 1979). We have demonstrated that the cold stability of the Antarctic toothfish lens is far greater when compared to the lens of other ectothermic teleost fish of warmer clime such as the tropical blackbar soldierfish, M. jacobus, which had a body temperature 24°C prior to experimentation (Fig. 1g–i). Though soldierfish lens develops irreversible cold-cataract, it is less cold-sensitive than bovine lens as prolonged exposure (x24 h) at 0°C was required, as opposed to the rapid onset of bovine cold-cataract at much higher temperature (Delaye et al., 1982). Thus, there is a direct correlation between body temperature and lens cold-stability (low temperature limit of lens clarity), with the cryophilic toothfish having the most cold-stable lens.

Cold-cataract formation in bovine lens is attributed to a liquid–liquid phase separation of one or more of the cold-labile crystallin isoforms (Broide et al., 1991; Siezen et al., 1985). Fish lenses are much denser than mammalian (bovine) lenses (Jagger and Sands, 1996, 1999; Pierscionek and Augusteyn, 1995), thus a plausible hypothesis might be that the greater cold stability of fish lens could be due to protein density-constrained mobility of the crystallin molecules, reducing the propensity for the kind of cold-induced structural rearrangement of the small crystallins that occurs in the much softer cow lens, leading to cold-cataract (Gulik-Krzywicki et al., 1984). However, while the increased density could account for the slower onset of the cold-cataract in the blackbar soldierfish, the toothfish lens is as compact and protein-dense as other teleost fish lens (slightly less dense than tuna), but is far more cold stable (Ferguson et al., 1971; Smith, 1972). Therefore, the basis of cold stability in ectothermic fish lens is likely to lie in the adaptive protein properties of the constituent crystallins for function in lower habitat temperatures (synonymous with body temperatures for ectotherms) than the warm-bodied mammals. The extraordinary cold stability of Antarctic toothfish lens exemplifies the preservation of normal protein function at the coldest known extreme of marine ectothermic vertebrate life, the freezing point of seawater. Moreover, initial results showing that the toothfish crystallin fraction at concentration of 58 mg ml^–1 remains clear to temperatures as low as –10°C, which is 14°C lower than similarly concentrated cow crystallin fraction (Siezen et al., 1985), further implicates the cold-adapted crystallins as the basis of toothfish whole lens cold stability in vivo.

In order to examine the thermal, biochemical and physical properties of the individual classes of lens crystallins of Antarctic toothfish, we first fractionated whole lens homogenate by Sephacryl 200HR size-exclusion chromatography (SEC) (Fig. 2). This was also performed for the subtropical bigeye tuna T. obesus and the endothermic cow in parallel, to allow comparison of the relationship between crystallin protein properties and organismal body temperature. The three species have lenses of comparable size, and collectively they span the range of vertebrate body temperatures – the very cold-bodied toothfish (–2°C), the cool-bodied bigeye tuna (18°C), and the warm-bodied cow (37°C).

Incomplete SEC separation of individual classes of fish crystallin, especially between and ß crystallin, is a commonly reported difficulty (Chiou et al., 1987; Posner et al., 1999; Wistow, 1995), which also occurred for Antarctic toothfish and bigeye tuna in this study (Figs 2, 3). Sepharose 6B, which provided good separations of bovine crystallins (Horwitz et al., 1998), is even less adequate than Sephacryl 200HR for fish crystallins separation (data not shown). While S200HR could not achieve complete separation of