Exp. Eye Res. (1990) 50, 715-718
Hydration FREDERICK
Properties
A. BETTELHEIM
Chemistry
Department,
Adelphi
AND
of Lens Crystallins NESABRAVKA
University,
POPDIMITROVA”
Garden City, NY 11530, U.S.A.
The water binding properties of bovine lens crystallins, a, p,, 8, and low molecular weight (LMW), were investigated with different techniques. The water sorptive capacity was obtained in high vacuum sorption experiments volumetrically, and also gravimetrically in controlled atmosphere experiments. The two methods gave reproducible isotherms. The strongly sorbed water (bound water) was evaluated from the B.E.T. monolayer and from the values of isosteric heats. The bound water content of the crystallins were also measured by combined differential scanning calorimetry and thermogravimetric analysis. The nonfreezable water contents calculated as % of the total water content and also as g g-l crystallin, were used as an indicator for bound water. In all three different techniques, the following order was obtained: p, > a > /I, = LMW. Key words: bound water: crystallins: freezable water: non-freezable water: water vapor sorption.
1. Introduction
198 7) and cataractogenesis (Racz, Tompa and Pocsik, 19 79 ; Siew et al., 198 1; Bettelheim, Siew and Chylack 1981; Racz et al., 1983; Bettelheim et al., 1986; Wang and Bettelheim, 1988). Thus it is important to know the hydration properties of the individual
Transparency of the normal lens depends on the short range order that exists in the supramolecular organization which provides interference effect (Benedek, 1971; Bettelheim and Siew, 1983: Delaye and
crystallins
Tardieu, 1983). Cataract formation or turbidity, on the other hand, is enhanced by two cytoplasmic factors: (1) an increase in the size of the scattering
change with different supramolecular organization homo or heteroaggregation. The following reports on the hydration behavior of the individual crystallins.
units, i.e. crystallin
aggregates
aging (Siew, Opalecky and Bettelheim.
00144835/90/060715
1981; Lahm,
1985; Castor0 and Bettelheim,
* Permanent address: Department Medical Academy. Sofia. Bulgaria f04
the hydration
behavior
may
(Benedek, 1971), and
(2) an increase in the refractive index difference between the scattering units and their environment (Bettelheim and Paunovic, 1979; Bettelheim, 1985). Between these two factors, the size change is more influential since it enters the turbidity expression as an exponential term in the correlation function. For that reason, early on, an aggregation process was proposed as the main contributor to cataractogenesis (Harding andDilley, 1976: Lorand, Conrad and Velasco, 1985). The definitive work of Kinoshita and his coworkers (Dvornik et al., 19 73 ; Kinoshita, 1974 ; Varma and Kinoshita, 19 74 ; Obazawa, Merola and Kinoshita, 1974) on sugar cataracts, however, inspired us to think more closely on the hydration properties of lenses, not just in terms of lake formation due to the influx of water driven by osmotic gradient, but also in terms of more subtle continuous changes. Thus, the syneretic hypothesis was born (Bettelheim, 1979) in which changes in secondary, tertiary or quaternary structure of crystallin cause the release of water from the bound state (hydration layers) into the bulk. Such a change sets up an increase in the refractive index difference between the protein moieties and their environment. Although this change affects the turbidity only as a square term, nevertheless in many experiments it has been found to be operative, both in Lee and Bettelheim,
and how
Physics
$03.00/O
and
Biophysics,
2. Materials
and Methods
Bovine eyes were obtained from a local slaughterhouse. Lenses were removed and were processed immediately or were stored frozen until needed. Lenses were homogenized in about 7 volumes of 0.05 M Tris, pH 7-5, containing 0.1 M KCl, 1 mM EDTA, 10 mM 2mercaptoethanol. and 0.02% NaN,. After centrifugation (30000 g 30 min, 4’C) the supernatant was applied to a Sephadex G-200 column (2% x 85 cm) equilibrated with the same buffer. Five-milliliter fractions were collected and analyzed by SDS electrophoresis as previously described (Zigler, Horowitz and Kinoshita. 1980). Fractions containing the different crystallins were pooled, dialyzed vs. H,O and lyophilized. In this way four fractions, 51, fi,. /I,, and low molecular weight (LMW) crystallins, were obtained. The last refers to the group of monomeric proteins with apparent molecular weights in the range of 24000 and below. This includes the polypeptides classically referred to as y-crystallin and P,-crystallin. The partially purified preparations were subsequently redissolved in small volumes of buffer and rechromatographed on the same column of Sephadex G-200. Fractions containing only the desired crystallins were pooled, dialyzed exhaustively
vs. H,O. lyophilized and
the protein stored at - 20°C until used. Concentrated (7 20%) crystallin solutions were made by dissolving the purified crystallin in distilled water and adjusting the pH to 7.0 by dilute NaOH. Sequential dilutions 0 1990 Academic Press Limited
716
with distilled water at pH 7.0 gave concentration varying from 1 to 18 y0 crystallins. Each sample was hermetically sealed into a preweighed, coated aluminum sample pan, and stored at - 30°C until the actual measurements. For the measurements of the freezable water content, differential scanning calorimetry (DSC) was used. Total water of the solutions were measured by thermogravimetric analysis (TGA). Both techniques have been described previously (Wang and Bettelheim, 1988). The non-freezable water content as a function of concentration was obtained by the difference between the total and freezable water contents. It was expressed as a percentage of the total water content. Water binding capacity of crystallins was also investigated by vapor sorption techniques albeit in a different concentration range. The water uptake of solid dry crystallins were measured in a high vacuum vapor sportion apparatus, as described previously (Block and Bettelheim, 1970; Bettelheim, Zigler and Reddy, 1987). The water sorption was measured volumetrically as a function of increasing water vapor pressure. The desorption was obtained when the vapor pressure was diminished. The water vapor sorbed or desorbed was calculated from the pressure-volume data assuming ideal gas law. The water vapor uptake was also measured gravimetrically. The vacuum dried (10m3 torr, 29°C 3 days) crystallins were placed in weighing bottles, and their dry weight obtained by weighing. The samples were exposed to a set water vapor pressure by placing them in dessicators with set concentrations 1O-7 1% (w/w) of aqueous sulfuric acid. The sorption was allowed to proceed at constant temperature for lo-14 days and the samples were reweighed to obtain the water uptake. 3. Results The non-freezable water content of the different crystallins are presented in two forms. In Fig. 1 we obtained the non-freezable water content as a percentage of the total water as a function of crystallin concentration. The non-freezable water increases with crystallin concentration and the curves fit an S-shaped function approximated with a 3rd order polynomial, y = bx+cx2+dx3. On the other hand, when the non-freezable is presented as g, g-’ crystallin, the concentration dependence indicates a decreasing function. This can be fitted to a simple regression expression, y = a - bx + cx” (Fig. 2). Both modes of presentation of non-freezable water show that the order of hydration among the crystallins is ljH > 01> pL = LMW. The water vapor sorption isotherms obtained in the high vacuum apparatus volumetrically corresponded fairly well with those obtained gravimetrically over different sulfuric acid concentrations in vacuum dessicators (Fig. 3).
F.A.
BETTELHEIM
AND
N. POPDIMITROVA
% FIG. 1. Non-freezable water content as percent of the total water as a function of crystallin concentration. a (Jr-*). B, (O-a), pL (O-O) and y (a--m) (low molecular weight) crystallin.
+ 15a
I, s
IO-
z
5-
0
I IO
I 5
I 15
% FIG. 2. Non-freezable water content as g g-’ crystallin as a function of crystallin concentration. a(*-*), pII ( l --•), p, (0-O) and y (m--m) (low molecular weight) crystallin.
The sorption isotherms have been obtained at two temperatures and the isosteric heat of sorption was calculated from the ClausiusClapeyron equation at low vapor pressures where the water vapor can be assumed to behave close to ideal (Bettelheim and Volman, 19 5 7). Finally the ‘B.E.T. ’ monolayer values (Brunauer, Emmett and Teller, 1938) have been also calculated. These data are given in Table I. The sorption experiment covers a different concentration range, 100-2 5 % crystallin. The strongly bound water as represented by the B.E.T. monolayer or by the sorption at low vapor pressure before ‘monolayer ’ completion occurred, or by the calculated isosteric heat, is conceptually different from the bound water represented by the non-freezable water content.
HYDRATION
PROPERTIES
OF
LENS
600
P/P*
FIG. 3. Water vapor sorption by crystallins as % (g water vapor per 100 g crystallin) as a function of relative vapor pressure of water at 37°C. OL (*-*), /I, (a--•), /?, (0-O) and y (m--m) (low molecular weight) crystallin. The small symbols represent data obtained volumetrically. The large symbols, those obtained gravimetrically. TABLE I
Water sorptiorl parameters
Sample
Percentage sorption at 5 torr 31 63 35 26
B.E.T. monolayer %
717
CRYSTALLINS
Isosteric heat at maximum kcal molm’
31 87 30 26
In spite of that, the order of water binding
4.9 9.8 6.3 4.1
capacity
among the crystallins remains the same. 4. Discussion
The definition of bound water depends on the method of measurements (Ali and Bettelheim, 1985). Therefore, it is reassuring that two different, albeit closely related measurements, volumetric and gravimetric water vapor sorption, essentially gave the same isotherms (Fig. 3). The bound water defined as strongly sorbed water from the vapor sorption isotherms is conceptually quite different from the bound water defined as the non-freezable water obtained from DSC and TGA measurements. Not only are these definitions
different but, by the nature of measurements. they were obtained at different concentration ranges when the extent of bound water was investigated as a function of crystallin concentration. In spite of these differences, we find that the different crystallin fractions have the same order in terms of water immobilization, fi, being the most efficient followed by CL.The p,, fraction binds water more strongly than the low molecular weight fraction (y) (Table I. Fig. 3). but in the thermal studies it immobilizes water to a somewhat lesser extent than y (Figs 1 and 2). However, both fi,, and y have less water binding capacities than ,4,., or Z. This water immobilization and binding may have to do with the openness or compactness of the quaternary structures. We know from X-ray diffraction studies (Lindley et al., 1985) that y-crystallins are compact and it was also proposed that the /I-crystallins have the same compact core with extended chains protruding (Slingsby. 1985: Slingsby et al.. 1988). Thus, is seems that in the more dilute concentration range of O-18% crystallin. the non-freezable water content of /J’, is highest, because it has an open quaternary structure. This can immobilize water to a greater extent than the layered or other proposed compact quaternary structures of a-crystallin (Siezen, Bindels and Hoenders, 1980: Tardieu et al.. 1986; Augusteyn and Koretz, 1987; Chiou and Azari. 1989). The compactness of y- and p,,-crystallins is derived from having lower aggregation in the case of ,4’,, and monomeric form in y. Thus, they bind and immobilize less water. The fact that, in the more concentrated range (SO-lOOo/, crystallin) of the vapor sorption studies. the y-crystallin has the least water binding capacity may indicate the strong self-aggregating tendencies of these monomeric crystallins. The ammonia gas accessibility (Bettelheim and Zigler, 1988) indicated preferential self-aggregation via hydrophobic domains and the crystallographic studies indicate that at least in the crystals, strong electrostatic interactions may also operate (Lindley et al.. 1985). Thus the present study of the low hydration tendency demonstrated by the low molecular weight (y) fraction is in agreement with previous structural and accessibility studies. Furthermore, y-crystallin is implicated in cold cataract Zigman and Lerman, 1965: Siezen et al., 1985) and in local separation in nuclear cytoplasm (Tanaka, Ishimoto and Chylack. 19 77 : Clark, Mengel and Benedek. 1980). This tendency is in agreement with the low water binding capacity of ?I-crystallin. Acknowledgments I would like to pay tribute to Dr Kinoshita, who with his pioneering studies on cataracts opened up new research fields and with his probing questions and interest, enticed us to look into diverse fields for answers. Thus, his influence on eye research was and is both extensive and intensive. This research was supported by a National Eye Institute grant. EY-0157 1.
718
References Ali. S. and Bettelheim, F. A. (1985). Non-freezing water in protein solutions. Co/bid Polymer Sci. 203. 396-8. Augusteyn. K. C. and Koretz. J. F. (1987). A possible structure for a-crystallin. FEBS. Lett. 222. I-5. Benedek, G. G. (1971). Theory of transparency of the eye. Appl. Optics 10, 459-73. Bettelheim, F. A. (1979). Syneresis and its possible role in cataractogenesis. Exp. Eye Res. 28, 189-97. Bettelheim, F. A. (1985). Physical basis of lens transparency. In The Oculnr Lens, Structure, Function and Pathology (Ed. Maisel. H.). Pp. 265-300. Marcel Dekker, Inc.: New York. Bettelheim, F. A., Castoro. J. A., White, 0. and Chylack, Jr.. L. T. ( 1986). Topographic correspondence between total and non-freezable water content and the appearance of cataract in human lenses. Cum. Eye Res. 5, 925-32. Bettelheim, F. A. and Paunovic, M. (19 79). Light scattering of normal human lens I. Application of random density and orientation fluctuation theory. Biophys. I. 26. 85-100. Bettelheim, F. A. and Siew, E. L. (1983). The effect of change in concentration upon lens turbidity as predicted by the random fluctuation theory. Biophys. 1. 41, 29-33. Bettelheim, F. A., Siew, E. L. and Chylack, Jr,, L. T. (1981). Studies on human cataracts III. Structural elements in nuclear cataracts and their contribution to the turbidity. Invest. Ophthalmol. Vis. Sci. 20. 348-54. Bettelheim, F. A. and Volman, D. (1957). Pectic-substanceswater II. Thermodynamics of water vapor sorption. I. Polymer Sci. 24, 445-54. Bettelheim. F. A. and Zigler, Jr., J. S. (1988). Preferential interaction among lens proteins as evidenced from accessibility of crystallins to ammonia gases. Exp. Eye Res. 47, 227-36. Bettelheim, F. A., Zigler, Jr., J. S. and Reddy, V. N. ( 198 7). Accessibility of low-molecular weight crystallins to ammonia and hydrogen chloride gases. Erp. Eye Res. 45. 271-9. Block, A. and Bettelheim, F. A. ( 19 70). Water vapor sorption by hyaluronic acid. Biochem. Biophys. Acta 210.29-75. Brunauer, S.. Emmett, P. H. and Teller, E. ( 1938 ). Adsorption of gases in multimolecular layers. 1. Am Chem. Sot. 60. 309-19. Castoro. J. A. and Bettelheim, F. A. (1987). Water gradients across bovine lenses. Exp. Eye Res. 45, 191-5. Chiou, S. H. and Azari, P. ( 1989). Physio-chemical characterization of x-crystallins from bovine lenses: Hydrodynamic and conformational studies. J. Protein Chem. 8. l-l 7. Clark, J., Mengel, I,. and Benedek, G. B. (1980). Scanning electron microscopy of opaque and transparent states in reversible calf lens cataracts. Ophthalmic Res. 12. 16-33. Delaye. M. and Tardieu, A. (1983). Short range order of crystallin proteins accounts for eye lens transparency. Nature 302, 415-7. Dvornik. D., Simard-Duquesne. N., Krami, M., Sestanj. K., Gabbay. K. H.. Kinoshita. J. H., Varma, S. D. and Merola. L. A. ( 1973). Polyol accumulation in galactosemic and diabetic rats: control by an aldose reductase inhibitor. Science 182, 1146-8. Harding, J. J. and Dilley. V. J. ( 19 76). Structural proteins of mammalian lens: A review with emphasis on changes
F.A.
BETTELHEIM
AND
N. POPDIMITROVA
in development, aging and cataract. h[~. I+~ ks. 22. l--73. Kinoshita. J. H. I 19 71). Mechanisms initiating c’ataract formation. Invrst. Ophthnlmol. 1 3. 71 3-24. Lahm. D.. Lee. I,. K. and Bettelheim. F. A. I 1985). Ape dependence of freezable and non-freezable water corltent of normal human lenses. /r?vrst. ~~phthr/n~o/. Vis. Ski. 26. 11 h2-5. Lindley. P. F.. Narebor, M. E.. Summers, I,. J. and Wistow. G. J. ( 1985). Structure of Lens Proteins. In The Ocular Lens Structure, Function and Pathology (Ed. Maisel. H.). Chapter 4. Pp. 123-67. M. Dekker: N.Y. Lorand. L.. Conrad. S. M. and Velasco. P. ‘I’. ( 198 5). Formation of a 5 5.000 weight cross-linked /i’-crystallin dimer in the Ca?+ treated ions. A model for cataract. Biochemistry 24, 152 5-3 1. Obazawa, H., Merola, I,. 0. and Kinoshita. J. H. ( 1974). The effect of xylose on the isolated lens. Invest Ophthalmol. 13, 204-9. Racz. P.. Tompa, K. and Pocsik. 1. ( 19 79). The state of water in normal and senile cataractous lenses studied by nuclear magnetic resonance. Exp. Eye Res. 28. 12 9- 3 5. Racz, P., Tompa. K., Pocsik. I. and Banki. P. ( 198 3). NMR spectroscopy of the ocular lens. Lens Res. 1, 199-206. Siew. E. L., Bettelheim, F. A.. Chylack. Jr.. L. T. and Tung. W. H. ( 1981). Studies on human cataracts 11. Correlation between the clinical description and the light scattering parameters of human cataracts. Invest. Ophthalmol. Vis. Sci. 20, 334-47. Siew. E. L., Opalecky, D. and Bettelheim. F. A. ( 1Y 8 1). Light scattering of normal human lens II. Age dependence of light scattering parameters. Exp. Eye Res. 33, 603-14. Siezen. R. J.. Bindels, J. and Hoenders. H. J. (1980). The quaternary structure of bovine a-crystallin: Effects of variations in alkaline pH. ionic strength, temperature and calcium ion concentration. Eur. 1. Biochem. 111, 43 5-44.
Siezen, K. J., Fish, M. K.. Slingsby. C. and Benedek, G. B. ( 198 5). Opacification of y-crystallin solutions from calf lens in relation to cold cataract formation. Proc. Nntl. Acad. Sci U.S.A. 82. 1701-5. Slingsby. C. ( 198 5). Structural variations in lens crystallins. Trends Biochem. Sri. 10. 28 1-4. Slingsby, C., Driessen, H. P. C.. Mahadevan, D.. Bax. B. and Blundell, T. L. (1988). Evolutionary and functional relationships between the basic and acidic /Gcrystallins. Exp. Eye Res. 46, 375-403. Tanaka, T., Ishimoto, C. and Chylack, Jr., T. I,. (1977). Phase separation of a protein water mixture in cold cataract in the young rat lens. Science 197, 1010-2. Tardieu. A., Laporte. D.. Licinio, P., Krop, B. and Delaye. M. (1986). Calf lens a-crystallin quaternary structure. A three-layer tetrahedral model. 1. Mol. Biol. 192. 71 1-4. Varma. S. D. and Kinoshita, J. H. ( 1974). Sorbitol pathway in diabetic and galactosemic rat lens. Biochim. Biophys. Acta 338, 632-40. Wang, X. and Bettelheim. F. A. ( 1988). Distribution of total and non-freezable water contents of galactosemic rat lenses. Cum. Eye Res. 7. 771-h. Zigler. Jr., J. S., Horwitz. J. and Kinoshita. J. ( 1980). Human /%crystallin I. Comparative studies on the /I,, /j, and ,I],crystallins. Exp. Eye Res. 31. 41-55. Zigman, S. and Lerman, S. (1965). Properties of a coldprecipitable protein fraction in the lens. Exp. Eye Res. 4. 24-30.
Hydration FREDERICK
Properties
A. BETTELHEIM
Chemistry
Department,
Adelphi
AND
of Lens Crystallins NESABRAVKA
University,
POPDIMITROVA”
Garden City, NY 11530, U.S.A.
The water binding properties of bovine lens crystallins, a, p,, 8, and low molecular weight (LMW), were investigated with different techniques. The water sorptive capacity was obtained in high vacuum sorption experiments volumetrically, and also gravimetrically in controlled atmosphere experiments. The two methods gave reproducible isotherms. The strongly sorbed water (bound water) was evaluated from the B.E.T. monolayer and from the values of isosteric heats. The bound water content of the crystallins were also measured by combined differential scanning calorimetry and thermogravimetric analysis. The nonfreezable water contents calculated as % of the total water content and also as g g-l crystallin, were used as an indicator for bound water. In all three different techniques, the following order was obtained: p, > a > /I, = LMW. Key words: bound water: crystallins: freezable water: non-freezable water: water vapor sorption.
1. Introduction
198 7) and cataractogenesis (Racz, Tompa and Pocsik, 19 79 ; Siew et al., 198 1; Bettelheim, Siew and Chylack 1981; Racz et al., 1983; Bettelheim et al., 1986; Wang and Bettelheim, 1988). Thus it is important to know the hydration properties of the individual
Transparency of the normal lens depends on the short range order that exists in the supramolecular organization which provides interference effect (Benedek, 1971; Bettelheim and Siew, 1983: Delaye and
crystallins
Tardieu, 1983). Cataract formation or turbidity, on the other hand, is enhanced by two cytoplasmic factors: (1) an increase in the size of the scattering
change with different supramolecular organization homo or heteroaggregation. The following reports on the hydration behavior of the individual crystallins.
units, i.e. crystallin
aggregates
aging (Siew, Opalecky and Bettelheim.
00144835/90/060715
1981; Lahm,
1985; Castor0 and Bettelheim,
* Permanent address: Department Medical Academy. Sofia. Bulgaria f04
the hydration
behavior
may
(Benedek, 1971), and
(2) an increase in the refractive index difference between the scattering units and their environment (Bettelheim and Paunovic, 1979; Bettelheim, 1985). Between these two factors, the size change is more influential since it enters the turbidity expression as an exponential term in the correlation function. For that reason, early on, an aggregation process was proposed as the main contributor to cataractogenesis (Harding andDilley, 1976: Lorand, Conrad and Velasco, 1985). The definitive work of Kinoshita and his coworkers (Dvornik et al., 19 73 ; Kinoshita, 1974 ; Varma and Kinoshita, 19 74 ; Obazawa, Merola and Kinoshita, 1974) on sugar cataracts, however, inspired us to think more closely on the hydration properties of lenses, not just in terms of lake formation due to the influx of water driven by osmotic gradient, but also in terms of more subtle continuous changes. Thus, the syneretic hypothesis was born (Bettelheim, 1979) in which changes in secondary, tertiary or quaternary structure of crystallin cause the release of water from the bound state (hydration layers) into the bulk. Such a change sets up an increase in the refractive index difference between the protein moieties and their environment. Although this change affects the turbidity only as a square term, nevertheless in many experiments it has been found to be operative, both in Lee and Bettelheim,
and how
Physics
$03.00/O
and
Biophysics,
2. Materials
and Methods
Bovine eyes were obtained from a local slaughterhouse. Lenses were removed and were processed immediately or were stored frozen until needed. Lenses were homogenized in about 7 volumes of 0.05 M Tris, pH 7-5, containing 0.1 M KCl, 1 mM EDTA, 10 mM 2mercaptoethanol. and 0.02% NaN,. After centrifugation (30000 g 30 min, 4’C) the supernatant was applied to a Sephadex G-200 column (2% x 85 cm) equilibrated with the same buffer. Five-milliliter fractions were collected and analyzed by SDS electrophoresis as previously described (Zigler, Horowitz and Kinoshita. 1980). Fractions containing the different crystallins were pooled, dialyzed vs. H,O and lyophilized. In this way four fractions, 51, fi,. /I,, and low molecular weight (LMW) crystallins, were obtained. The last refers to the group of monomeric proteins with apparent molecular weights in the range of 24000 and below. This includes the polypeptides classically referred to as y-crystallin and P,-crystallin. The partially purified preparations were subsequently redissolved in small volumes of buffer and rechromatographed on the same column of Sephadex G-200. Fractions containing only the desired crystallins were pooled, dialyzed exhaustively
vs. H,O. lyophilized and
the protein stored at - 20°C until used. Concentrated (7 20%) crystallin solutions were made by dissolving the purified crystallin in distilled water and adjusting the pH to 7.0 by dilute NaOH. Sequential dilutions 0 1990 Academic Press Limited
716
with distilled water at pH 7.0 gave concentration varying from 1 to 18 y0 crystallins. Each sample was hermetically sealed into a preweighed, coated aluminum sample pan, and stored at - 30°C until the actual measurements. For the measurements of the freezable water content, differential scanning calorimetry (DSC) was used. Total water of the solutions were measured by thermogravimetric analysis (TGA). Both techniques have been described previously (Wang and Bettelheim, 1988). The non-freezable water content as a function of concentration was obtained by the difference between the total and freezable water contents. It was expressed as a percentage of the total water content. Water binding capacity of crystallins was also investigated by vapor sorption techniques albeit in a different concentration range. The water uptake of solid dry crystallins were measured in a high vacuum vapor sportion apparatus, as described previously (Block and Bettelheim, 1970; Bettelheim, Zigler and Reddy, 1987). The water sorption was measured volumetrically as a function of increasing water vapor pressure. The desorption was obtained when the vapor pressure was diminished. The water vapor sorbed or desorbed was calculated from the pressure-volume data assuming ideal gas law. The water vapor uptake was also measured gravimetrically. The vacuum dried (10m3 torr, 29°C 3 days) crystallins were placed in weighing bottles, and their dry weight obtained by weighing. The samples were exposed to a set water vapor pressure by placing them in dessicators with set concentrations 1O-7 1% (w/w) of aqueous sulfuric acid. The sorption was allowed to proceed at constant temperature for lo-14 days and the samples were reweighed to obtain the water uptake. 3. Results The non-freezable water content of the different crystallins are presented in two forms. In Fig. 1 we obtained the non-freezable water content as a percentage of the total water as a function of crystallin concentration. The non-freezable water increases with crystallin concentration and the curves fit an S-shaped function approximated with a 3rd order polynomial, y = bx+cx2+dx3. On the other hand, when the non-freezable is presented as g, g-’ crystallin, the concentration dependence indicates a decreasing function. This can be fitted to a simple regression expression, y = a - bx + cx” (Fig. 2). Both modes of presentation of non-freezable water show that the order of hydration among the crystallins is ljH > 01> pL = LMW. The water vapor sorption isotherms obtained in the high vacuum apparatus volumetrically corresponded fairly well with those obtained gravimetrically over different sulfuric acid concentrations in vacuum dessicators (Fig. 3).
F.A.
BETTELHEIM
AND
N. POPDIMITROVA
% FIG. 1. Non-freezable water content as percent of the total water as a function of crystallin concentration. a (Jr-*). B, (O-a), pL (O-O) and y (a--m) (low molecular weight) crystallin.
+ 15a
I, s
IO-
z
5-
0
I IO
I 5
I 15
% FIG. 2. Non-freezable water content as g g-’ crystallin as a function of crystallin concentration. a(*-*), pII ( l --•), p, (0-O) and y (m--m) (low molecular weight) crystallin.
The sorption isotherms have been obtained at two temperatures and the isosteric heat of sorption was calculated from the ClausiusClapeyron equation at low vapor pressures where the water vapor can be assumed to behave close to ideal (Bettelheim and Volman, 19 5 7). Finally the ‘B.E.T. ’ monolayer values (Brunauer, Emmett and Teller, 1938) have been also calculated. These data are given in Table I. The sorption experiment covers a different concentration range, 100-2 5 % crystallin. The strongly bound water as represented by the B.E.T. monolayer or by the sorption at low vapor pressure before ‘monolayer ’ completion occurred, or by the calculated isosteric heat, is conceptually different from the bound water represented by the non-freezable water content.
HYDRATION
PROPERTIES
OF
LENS
600
P/P*
FIG. 3. Water vapor sorption by crystallins as % (g water vapor per 100 g crystallin) as a function of relative vapor pressure of water at 37°C. OL (*-*), /I, (a--•), /?, (0-O) and y (m--m) (low molecular weight) crystallin. The small symbols represent data obtained volumetrically. The large symbols, those obtained gravimetrically. TABLE I
Water sorptiorl parameters
Sample
Percentage sorption at 5 torr 31 63 35 26
B.E.T. monolayer %
717
CRYSTALLINS
Isosteric heat at maximum kcal molm’
31 87 30 26
In spite of that, the order of water binding
4.9 9.8 6.3 4.1
capacity
among the crystallins remains the same. 4. Discussion
The definition of bound water depends on the method of measurements (Ali and Bettelheim, 1985). Therefore, it is reassuring that two different, albeit closely related measurements, volumetric and gravimetric water vapor sorption, essentially gave the same isotherms (Fig. 3). The bound water defined as strongly sorbed water from the vapor sorption isotherms is conceptually quite different from the bound water defined as the non-freezable water obtained from DSC and TGA measurements. Not only are these definitions
different but, by the nature of measurements. they were obtained at different concentration ranges when the extent of bound water was investigated as a function of crystallin concentration. In spite of these differences, we find that the different crystallin fractions have the same order in terms of water immobilization, fi, being the most efficient followed by CL.The p,, fraction binds water more strongly than the low molecular weight fraction (y) (Table I. Fig. 3). but in the thermal studies it immobilizes water to a somewhat lesser extent than y (Figs 1 and 2). However, both fi,, and y have less water binding capacities than ,4,., or Z. This water immobilization and binding may have to do with the openness or compactness of the quaternary structures. We know from X-ray diffraction studies (Lindley et al., 1985) that y-crystallins are compact and it was also proposed that the /I-crystallins have the same compact core with extended chains protruding (Slingsby. 1985: Slingsby et al.. 1988). Thus, is seems that in the more dilute concentration range of O-18% crystallin. the non-freezable water content of /J’, is highest, because it has an open quaternary structure. This can immobilize water to a greater extent than the layered or other proposed compact quaternary structures of a-crystallin (Siezen, Bindels and Hoenders, 1980: Tardieu et al.. 1986; Augusteyn and Koretz, 1987; Chiou and Azari. 1989). The compactness of y- and p,,-crystallins is derived from having lower aggregation in the case of ,4’,, and monomeric form in y. Thus, they bind and immobilize less water. The fact that, in the more concentrated range (SO-lOOo/, crystallin) of the vapor sorption studies. the y-crystallin has the least water binding capacity may indicate the strong self-aggregating tendencies of these monomeric crystallins. The ammonia gas accessibility (Bettelheim and Zigler, 1988) indicated preferential self-aggregation via hydrophobic domains and the crystallographic studies indicate that at least in the crystals, strong electrostatic interactions may also operate (Lindley et al.. 1985). Thus the present study of the low hydration tendency demonstrated by the low molecular weight (y) fraction is in agreement with previous structural and accessibility studies. Furthermore, y-crystallin is implicated in cold cataract Zigman and Lerman, 1965: Siezen et al., 1985) and in local separation in nuclear cytoplasm (Tanaka, Ishimoto and Chylack. 19 77 : Clark, Mengel and Benedek. 1980). This tendency is in agreement with the low water binding capacity of ?I-crystallin. Acknowledgments I would like to pay tribute to Dr Kinoshita, who with his pioneering studies on cataracts opened up new research fields and with his probing questions and interest, enticed us to look into diverse fields for answers. Thus, his influence on eye research was and is both extensive and intensive. This research was supported by a National Eye Institute grant. EY-0157 1.
718
References Ali. S. and Bettelheim, F. A. (1985). Non-freezing water in protein solutions. Co/bid Polymer Sci. 203. 396-8. Augusteyn. K. C. and Koretz. J. F. (1987). A possible structure for a-crystallin. FEBS. Lett. 222. I-5. Benedek, G. G. (1971). Theory of transparency of the eye. Appl. Optics 10, 459-73. Bettelheim, F. A. (1979). Syneresis and its possible role in cataractogenesis. Exp. Eye Res. 28, 189-97. Bettelheim, F. A. (1985). Physical basis of lens transparency. In The Oculnr Lens, Structure, Function and Pathology (Ed. Maisel. H.). Pp. 265-300. Marcel Dekker, Inc.: New York. Bettelheim, F. A., Castoro. J. A., White, 0. and Chylack, Jr.. L. T. ( 1986). Topographic correspondence between total and non-freezable water content and the appearance of cataract in human lenses. Cum. Eye Res. 5, 925-32. Bettelheim, F. A. and Paunovic, M. (19 79). Light scattering of normal human lens I. Application of random density and orientation fluctuation theory. Biophys. I. 26. 85-100. Bettelheim, F. A. and Siew, E. L. (1983). The effect of change in concentration upon lens turbidity as predicted by the random fluctuation theory. Biophys. 1. 41, 29-33. Bettelheim, F. A., Siew, E. L. and Chylack, Jr,, L. T. (1981). Studies on human cataracts III. Structural elements in nuclear cataracts and their contribution to the turbidity. Invest. Ophthalmol. Vis. Sci. 20. 348-54. Bettelheim, F. A. and Volman, D. (1957). Pectic-substanceswater II. Thermodynamics of water vapor sorption. I. Polymer Sci. 24, 445-54. Bettelheim. F. A. and Zigler, Jr., J. S. (1988). Preferential interaction among lens proteins as evidenced from accessibility of crystallins to ammonia gases. Exp. Eye Res. 47, 227-36. Bettelheim, F. A., Zigler, Jr., J. S. and Reddy, V. N. ( 198 7). Accessibility of low-molecular weight crystallins to ammonia and hydrogen chloride gases. Erp. Eye Res. 45. 271-9. Block, A. and Bettelheim, F. A. ( 19 70). Water vapor sorption by hyaluronic acid. Biochem. Biophys. Acta 210.29-75. Brunauer, S.. Emmett, P. H. and Teller, E. ( 1938 ). Adsorption of gases in multimolecular layers. 1. Am Chem. Sot. 60. 309-19. Castoro. J. A. and Bettelheim, F. A. (1987). Water gradients across bovine lenses. Exp. Eye Res. 45, 191-5. Chiou, S. H. and Azari, P. ( 1989). Physio-chemical characterization of x-crystallins from bovine lenses: Hydrodynamic and conformational studies. J. Protein Chem. 8. l-l 7. Clark, J., Mengel, I,. and Benedek, G. B. (1980). Scanning electron microscopy of opaque and transparent states in reversible calf lens cataracts. Ophthalmic Res. 12. 16-33. Delaye. M. and Tardieu, A. (1983). Short range order of crystallin proteins accounts for eye lens transparency. Nature 302, 415-7. Dvornik. D., Simard-Duquesne. N., Krami, M., Sestanj. K., Gabbay. K. H.. Kinoshita. J. H., Varma, S. D. and Merola. L. A. ( 1973). Polyol accumulation in galactosemic and diabetic rats: control by an aldose reductase inhibitor. Science 182, 1146-8. Harding, J. J. and Dilley. V. J. ( 19 76). Structural proteins of mammalian lens: A review with emphasis on changes
F.A.
BETTELHEIM
AND
N. POPDIMITROVA
in development, aging and cataract. h[~. I+~ ks. 22. l--73. Kinoshita. J. H. I 19 71). Mechanisms initiating c’ataract formation. Invrst. Ophthnlmol. 1 3. 71 3-24. Lahm. D.. Lee. I,. K. and Bettelheim. F. A. I 1985). Ape dependence of freezable and non-freezable water corltent of normal human lenses. /r?vrst. ~~phthr/n~o/. Vis. Ski. 26. 11 h2-5. Lindley. P. F.. Narebor, M. E.. Summers, I,. J. and Wistow. G. J. ( 1985). Structure of Lens Proteins. In The Ocular Lens Structure, Function and Pathology (Ed. Maisel. H.). Chapter 4. Pp. 123-67. M. Dekker: N.Y. Lorand. L.. Conrad. S. M. and Velasco. P. ‘I’. ( 198 5). Formation of a 5 5.000 weight cross-linked /i’-crystallin dimer in the Ca?+ treated ions. A model for cataract. Biochemistry 24, 152 5-3 1. Obazawa, H., Merola, I,. 0. and Kinoshita. J. H. ( 1974). The effect of xylose on the isolated lens. Invest Ophthalmol. 13, 204-9. Racz. P.. Tompa, K. and Pocsik. 1. ( 19 79). The state of water in normal and senile cataractous lenses studied by nuclear magnetic resonance. Exp. Eye Res. 28. 12 9- 3 5. Racz, P., Tompa. K., Pocsik. I. and Banki. P. ( 198 3). NMR spectroscopy of the ocular lens. Lens Res. 1, 199-206. Siew. E. L., Bettelheim, F. A.. Chylack. Jr.. L. T. and Tung. W. H. ( 1981). Studies on human cataracts 11. Correlation between the clinical description and the light scattering parameters of human cataracts. Invest. Ophthalmol. Vis. Sci. 20, 334-47. Siew. E. L., Opalecky, D. and Bettelheim. F. A. ( 1Y 8 1). Light scattering of normal human lens II. Age dependence of light scattering parameters. Exp. Eye Res. 33, 603-14. Siezen. R. J.. Bindels, J. and Hoenders. H. J. (1980). The quaternary structure of bovine a-crystallin: Effects of variations in alkaline pH. ionic strength, temperature and calcium ion concentration. Eur. 1. Biochem. 111, 43 5-44.
Siezen, K. J., Fish, M. K.. Slingsby. C. and Benedek, G. B. ( 198 5). Opacification of y-crystallin solutions from calf lens in relation to cold cataract formation. Proc. Nntl. Acad. Sci U.S.A. 82. 1701-5. Slingsby. C. ( 198 5). Structural variations in lens crystallins. Trends Biochem. Sri. 10. 28 1-4. Slingsby, C., Driessen, H. P. C.. Mahadevan, D.. Bax. B. and Blundell, T. L. (1988). Evolutionary and functional relationships between the basic and acidic /Gcrystallins. Exp. Eye Res. 46, 375-403. Tanaka, T., Ishimoto, C. and Chylack, Jr., T. I,. (1977). Phase separation of a protein water mixture in cold cataract in the young rat lens. Science 197, 1010-2. Tardieu. A., Laporte. D.. Licinio, P., Krop, B. and Delaye. M. (1986). Calf lens a-crystallin quaternary structure. A three-layer tetrahedral model. 1. Mol. Biol. 192. 71 1-4. Varma. S. D. and Kinoshita, J. H. ( 1974). Sorbitol pathway in diabetic and galactosemic rat lens. Biochim. Biophys. Acta 338, 632-40. Wang, X. and Bettelheim. F. A. ( 1988). Distribution of total and non-freezable water contents of galactosemic rat lenses. Cum. Eye Res. 7. 771-h. Zigler. Jr., J. S., Horwitz. J. and Kinoshita. J. ( 1980). Human /%crystallin I. Comparative studies on the /I,, /j, and ,I],crystallins. Exp. Eye Res. 31. 41-55. Zigman, S. and Lerman, S. (1965). Properties of a coldprecipitable protein fraction in the lens. Exp. Eye Res. 4. 24-30.
