The Molecular Chaperone α-Crystallin Inhibits UV-Induced Protein Aggregation

Exp. Eye Res. (1996) 62, 141–148

The Molecular Chaperone α-Crystallin Inhibits UV-Induced Protein Aggregation R A Y M O N D F. B O R K M A N*, G R A D Y K N I G H T    B E T T I E O B I School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, U.S.A. (Received Rochester 27 April 1995 and accepted in revised form 29 August 1995) Solutions of γ-crystallin, and various enzymes, at neutral pH and 24–26°C, became turbid upon exposure to UV radiation at 295 or 308 nm. SDS-PAGE analysis revealed interchain cross-linking and aggregate formation compared to dark control solutions as reported previously. When α-crystallin was added to the protein solutions in stoichiometric amounts, UV irradiation resulted in significantly less turbidity than in the absence of α-crystallin. For example, addition of 0±5 mg of α-crystallin to 0±5 mg of γ-crystallin in 1±0 ml solution yielded only 25 % of the turbidity seen in the absence of α-crystallin. Addition of 2.0 mg of α-crystallin resulted in 20 % of the turbidity. Given the molecular weights of α- and γ-crystallin (about 800 kDa and 20 kDa, respectively), a γ}α 1 : 1 weight ratio corresponds to a 40 : 1 molar ratio, and a γ}α 1 : 4 weight ratio corresponds to a 10 : 1 molar ratio. Hence, the molar ratio of α-crystallin needed to effectively protect γ-crystallin from photochemical opacification was γ}α ¯ n : 1, where n was in the range 10–40. In terms of subunits, this ratio is γ}α ¯ 1 : m, where m ¯ 1–4. Thus, each γ-crystallin molecule needs 1–4 α subunits for protection. Similar stoichiometries were observed for protection of the other proteins studied. The protection stems in part from screening of UV radiation by α-crystallin but more importantly from a chaperone effect analogous to that seen in thermal aggregation experiments. Key words : lens ; protein ; aggregation ; α-crystallin ; γ-crystallin ; chaperone ; UV ; opacity ; turbidity.

1. Introduction It has been found that certain proteins can function as ‘ molecular chaperones ’ (Ellis and van der Vies, 1991). These species prevent undesirable aggregation of denatured proteins (Schmidt and Buchner, 1992) and assist in their refolding to the native form (Buchner et al., 1991). The mechanism of protection involves binding of denatured substrate by the chaperone, for example, the chaperone GroEL binds denatured peptides within its central cavity (Braig et al., 1993). Horwitz (1992, 1993) reported that the structural protein, α-crystallin, isolated from bovine lens, can function as a chaperone in that it prevents thermal aggregation of a number of enzymes as well as the lens structural proteins β- and γ-crystallin. Thus, it appears that α-crystallin plays a multiple role in the lens, and in some other tissues (Kato et al., 1991), serving both as a structural unit and as a protective agent preventing undesirable aggregation of proteins which could lead to lens opacity. The mechanism by which α-crystallin protects proteins from aggregation is being investigated (Boyle et al., 1993 ; Takemoto, 1994), and model structures have been proposed (Tardieu et al., 1986 ; Augusteyn and Koretz, 1987 ; Wistow, 1993 ; Carver et al., 1994). A relevant piece of information is the measured stoichiometry of the protective interaction. In the case of thermal denaturation of α-}γ-crystallin mixtures, * For correspondence at : School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332-0400, U.S.A.

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Horwitz (1993) found this stoichiometry to be about α}γ ¯ 1 : 10–20 (mole}mole). γ-Crystallin is a monomer of molecular weight 20 kDa (Wistow et al., 1983). α-Crystallin is an oligomer composed of subunits—the αA and αB chains—each 20 kDa in # # size (van der Ouderaa et al., 1974). α-Crystallin particle sizes vary in the range 300–2000 kDa depending on conditions of temperature, pH and ionic strength (Thompson and Augusteyn, 1988). Thus, at room temperature and neutral pH the molecular weight of α-crystallin is about 800 kDa, while at the higher temperatures used in the thermal aggregation} opacification experiments of Horwitz (1993), the αcrystallin effective particle size was estimated to be about 400 kDa. (It is possible that other conformational changes occur in α-crystallin at the 48–66°C temperatures used in the thermal aggregation work, Das and Surewicz, 1995). Based on an α}γ mole ratio of 1 : 10 for protection at 66°C, it appears that each pair of subunits of the 400 kDa α-crystallin oligomer can protect one γ-crystallin molecule from aggregation. This suggests an open structure for αcrystallin, with most or all of the subunits accessible. The three-dimensional structure of α-crystallin is not known, but various models have been proposed. A three-layer model was first proposed by Tardieu et al. (1986). Augusteyn and Koretz (1987) suggested a micellar model. Wistow (1993) advanced a tetrameric arrangement of subunits in the structure. Most recently, Carver et al. (1994) submitted a model more akin to other known chaperones, i.e. one with a hole in the center, lined with hydrophobic residues, where # 1996 Academic Press Limited

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substrate binding is proposed to occur. The chaperone data seem to argue against any structure with a significant fraction of the αA and αB chains buried # # and hence inaccessible as binding sites. An open structure with most or all of the subunits accessible seems more likely. Our laboratory has been interested in the photochemical aggregation of lens proteins. We have observed that β- and γ-crystallins in neutral buffer solutions at room temperature undergo photochemical crosslinking (seen by SDS–PAGE) and subsequent solution opacification when exposed to UV radiation (Walker and Borkman, 1989 ; Li et al., 1990 ; Hott and Borkman, 1993). On the other hand, α-crystallin solutions did not become opaque upon exposure to UV radiation under the same conditions (Hott and Borkman, 1993). Thus, when the thermal opacification results of Horwitz (1992, 1993) appeared, we began to investigate the possible roll of αcrystallin in protecting γ-crystallin and other proteins from UV induced aggregation. The present paper reports photochemical opacification studies of mixtures of bovine α- and γcrystallin and α-crystallin with some other enzymes in neutral pH buffer solutions. It is important to note that our photochemical work was carried out at 24–26°C, where α-crystallin is more likely to retain its native tertiary and quaternary structures than in thermal aggregation experiments performed at higher temperatures, typically in the 45–70°C range. Also, since in our experiments both α-crystallin, and the substrate protein, absorb significant UV radiation, we considered an additional possibility not present in thermal experiments : namely, that protection of proteins from photo-aggregation might be the result of screening of the incident radiation by α-crystallin. 2. Materials and Methods Bovine α-crystallin was obtained from 3-month-old calf lenses (courtesy of Brown Packing Company, Gaffney, SC, U.S.A.) by size exclusion chromatography at 4°C on Sephadex G-200 according to the procedure originally reported by Bloemendal (1981) and used in our previous work (Hott and Borkman, 1993). Some samples of bovine α-crystallin were also obtained from Sigma, St. Louis, MO, U.S.A. No differences were found between this and the material which we prepared ourselves. Separation of γ-crystallin was done by the procedure of Bjork (1961) also used in our previous work. Proteins were lyophilized and stored at ®5°C until needed. The enzymes carbonic anhydrase, enolase, and aldolase were obtained from Sigma, St. Louis, MO, and were used without further purification. Stock solutions of proteins were prepared in 0±1  phosphate buffer at pH 7±4. Protein concentrations were in the range 0±1–10±0 mg ml−", and we employed 1±0 cm path quartz cuvettes containing 1±0 ml of solution, except that the low and high protein

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concentration studies at 0±1 mg ml−" and 10 mg ml−" used a 10 cm path length quartz cell and a 0±1 cm path length quartz cell, respectively, to maintain constant absorbance in the three solutions. Aliquots were allowed to come to room temperature for 1 hr prior to beginning UV irradiation experiments. All solutions were irradiated at an ambient temperature of 24–26°C. The absorbances of the protein solutions were measured, at 295, 308, 360 and 600 nm before, and at intervals during, UV irradiation (or during heating, as discussed below) with a Milton Roy UV-visible spectrophotometer. Some of the UV irradiation studies were done with a 1000 W xenon arc lamp with 10 cm water filter and a Bausch and Lomb high intensity grating monochromator. The monochromator was set to transmit 295 nm radiation with a spectral band pass of 10 nm. The UV output incident upon the sample cell under these conditions was 0±5 mW cm−# (5 W m−#) as measured with a Scientech Model 365 meter. Other UV irradiation studies were done with a Lumonics excimer laser model 520 using XeCl as the lasing medium to give an average power at 308 nm of 0±5 W in a 1 cm# cross-sectional beam. Thus, the laser output at 308 nm was about 1000 times greater than that from the xenon lamp–monochromator system at 295 nm, i.e. the output ratio was O }O ¯ 1000. $!) #*& However, since the absorbance of the protein solutions was greater at 295 than at 308 nm, the ratio of the radiation flux actually absorbed at the two wavelengths was much less than the ratio of incident intensities. For example, for a 1±0 mg ml−" solution of γ-crystallin in buffer the measured absorbance ratio was A }A ¯ 17. Thus, the ratio of absorption #*& $!) rates at the two wavelengths was I }I ¯ 1000}17 $!) #*& ¯ 60. This ratio is reflected in the fact that typical irradiation times to achieve opacity with the laser were on the order of minutes, while with the xenon lamp they were on the order of hours. Because of the relatively high intensity of the laser source, we were concerned about possible heating of the irradiated solutions. Checks of solution temperatures indicated less than 1°C rise during a typical 15 min laser irradiation. Thermal opacification studies were performed by placing protein solutions in a thermostated water bath maintained at a fixed temperature in the range 55–70°C for times of up to 10 min. Absorbance was monitored periodically during the heating period as described above. SDS–PAGE was done using Bio-Rad’s Mini-Protean II dual slab gel cell apparatus. The gels were made of 15 % polyacrylamide and the SDS content was 0±1 %. The gels were run at 200 V and stained with Coomassie blue. The sample buffer was : 50 % water, 12±5 % 0±5  Tris (pH 6±8), 10 % glycerol, 20 % of a 10 % (w}v) solution of SDS, 5 % β-mercaptoethanol and 2±5 % of a 0±05 % (w}v) solution of bromophenol

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blue. The protein samples to be analysed were first diluted 1 : 4 with sample buffer prior to application to the gel. Protein solutions which were partially insolubilized by UV irradiation were centrifuged for 15 min at 10 000 rpm. The insoluble material was dissolved in 100 µl of sample buffer, and the soluble material was diluted 1 : 4 with sample buffer.

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Figure 1 shows a plot of absorbance at 600 nm versus UV irradiation time at 295 nm and room temperature for : 1 mg ml−" γ-crystallin, and for the same solution with 1 mg of α-crystallin added. UV irradiation at room temperature caused the γcrystallin solution to become opaque, while the α–γ mixture remained transparent. Also shown in Fig. 1 is a plot of the hypothetical degree of opacification expected in a 1 mg ml−" γ- plus 1 mg ml−" α-crystallin solution based on screening of the UV intensity by αcrystallin according to the Beer–Lambert law I}Io ¯ 10−εlc where Io and I are the incident and transmitted light intensity, ε is the molar decadic absorption coefficient, l is the cell path length, and c is the molar concentration. We computed the radiation absorbed by α-crystallin at each depth and subtracted this from the total absorbance to give the radiation absorbed by γ-crystallin versus pathlength and integrated this over the total path, l. This is expected to result in a reduction in the number of photochemical events for γ-crystallin, and we refer to this phenomenon as the ‘ UV screening mechanism ’. In Fig. 1, one sees that the

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F. 2. Absorbance at 600 nm vs. time of irradiation at 295 nm for solutions of α- and γ-crystallin : (a) 10 cm path cell, 0±1 mg ml−" γ-crystallin in buffer, —D—, two runs are shown, and 0±1 mg ml−" γ- plus 0±1 mg ml−" α-crystallin in buffer, —E—, two runs are shown ; (b) 1±0 cm path cell, 1±0 mg ml−" γ-crystallin in buffer, —D—, two runs, 1±0 mg ml−" γ- plus 1±0 mg ml−" α-crystallin in buffer, —E—, two runs shown ; (c) 0±1 cm path cell, 10 mg ml−" γcrystallin in buffer, —D—, two runs, 10 mg ml−" γ- plus 10 mg ml−" α-crystallin, —E—, two runs shown. All irradiations at room temperature with cell path length indicated.

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F. 1. Absorbance at 600 nm vs. time of irradiation at 295 nm for solutions : 1±0 mg ml−" γ-crystallin in buffer, —D— ; 1±0 mg γ-crystallin plus 1±0 mg α-crystallin in 1±0 ml buffer, —E—. The broken line – – E – – represents the expected degree of opacity based on the UV screening mechanism alone (see text). All irradiations at room temperature in 1±0 cm path quartz cell.

screening mechanism can account for only about onethird of the total protection afforded by α-crystallin. Dark control and UV-irradiated solutions were examined by SDS–PAGE. UV-irradiated solutions were extensively cross-linked, with material in the 20– 100 kDa molecular weight range and above, as our lab (Li et al., 1990 ; Hott and Borkman, 1993) and others (Mandel et al., 1988) have reported previously.

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F. 3. Photo-opacification data for enzymes (1±0 mg ml−") and protection by addition of varying amounts of αcrystallin : 0±0 mg ml−", —D— ; 0±25 mg ml−", —*— ; 1±0 mg ml−", —V— ; and 2±0 mg ml−", —E—. In each case, solution absorbance at 600 nm is plotted against the time of UV-irradiation at room temperature and 308 nm. (A) Aldolase. (B) Carbonic anhydrase. (C) Enolase.

SDS–PAGE results for the dark controls showed only bands characteristic of the starting materials, i.e. 20 kDa peptides for both α- and γ-crystallin. UV

irradiated mixtures of α and γ-crystallin also showed cross-linking, but the data did not reveal whether these links involved α–α, γ–γ, α–γ or some mixture of all three. The data are not shown here since numerous previous publications have shown SDS–PAGE gels for UV irradiated crystallins as noted above. Experiments were done to assess the effect of protein concentration on the chaperone function. In Fig. 2 are UV opacification data for gamma crystallin in buffer solution at concentrations of 0±1, 1±0 and 10 mg ml−", measured in cells of path length 10, 1 and 0±1 cm, respectively. Since the UV absorbance of these three samples is the same under these conditions, the data reveal the true concentration dependence of photoopacification, independent of the rate of UV absorption. As seen in Fig. 2(A), (B) and (C), all three γ-crystallin solutions became opaque following UV irradiation at 295 nm. Also seen is the effect of adding a stoichiometric amount of α-crystallin to each solution. In each case, addition of α-crystallin significantly protected γ-crystallin from UV opacification. Two experimental runs are shown for each solution. There are some differences in the degree of opacification achieved in each solution, and in the apparent degree of protection afforded, but the important point is that significant protection occurs over the entire 100-fold range of protein concentrations. In Fig. 3 are data on UV-induced opacification of solutions of aldolase, carbonic anhydrase, and enolase. The irradiations were done with the excimer laser at a wavelength of 308 nm and room temperature. In each case, added α-crystallin is seen to prevent or reduce the degree of opacity produced in these solutions by exposure to UV radiation. For example, in Fig. 3(A), one sees that 0±25 mg ml−" of added α-crystallin afforded no protection against UV-induced opacification of aldolase, but that the level of protection was significant when 1–2 mg ml−" of alpha was added. With some variations, similar data were obtained for the other enzymes, seen in Fig. 3. Thus, the protective or chaperone effect of α-crystallin was not limited to the lens protein γ-crystallin but extended to other proteins too. For comparison purposes, we plot in Fig. 4 results of thermal opacification experiments on the same enzymes, with and without added α-crystallin. The data for aldolase in Fig. 4(A) indicate that heating a 1±0 mg ml−" solution to 60°C resulted in opacification, but that such opacification was largely prevented by addition of as little as 0±25 mg ml−" of α-crystallin and completely prevented by addition of 1±0 mg ml−". Analogous results were obtained for carbonic anhydrase heated to 70°C except that more αcrystallin was needed to afford complete protection. In the case of enolase at 55°C, protection was significant, but incomplete, even at 2 mg ml−" of α-crystallin, as seen in Fig. 4. Thermal opacification data were also obtained for γ-crystallin (data not shown). As an example,

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F. 5. Photo-opacification data for 0±5 mg ml−" γ-crystallin with varying amounts of α-crystallin added : 0±0 mg ml−", —D— ; 0±10 mg ml−", —*— ; 0±25 mg ml−", —V— ; 1±0 mg ml−", —+— ; 2±0 mg ml−", —E—. Solutions irradiated at 308 nm, room temperature.

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F. 4. Thermal-opacification data for enzymes (1±0 mg ml−") and protection by addition of varying amounts of αcrystallin : 0±0 mg ml−", —D— ; 0±25 mg ml−", —*— ; 1±0 mg ml−", —V— ; and 2±0 mg ml−", —E—. In each case, solution absorbance at 600 nm is plotted against time at elevated temperature. (A) Aldolase, 60°C. (B) Carbonic anhydrase, 70°C. (C) Enolase, 55°C.

0±4 mg ml−" solutions of γ-crystallin, initially at room temperature, and containing varying amounts of αcrystallin were immersed into a 63°C water bath and the absorbance was monitored at 600 nm. The results showed that, in the absence of α-crystallin, solution opacification was complete within 10–15 min. When 0±4 mg of α-crystallin was added, the solution remained clear upon immersion in the 63°C bath. Thermally aggregated solutions were examined by SDS–PAGE. The heated solutions contained only the 20 kDa starting peptides of α- and γ-crystallin. Thus, in contrast, UV treatment of γ-crystallin produced high molecular weight aggregates which survived treatment with the detergent SDS as we and others have reported previously (Mandel et al., 1988 ; Hott and Borkman, 1993) but thermal aggregates of γcrystallin did not survive SDS treatment. Interestingly, both types of aggregation were prevented by addition of α-crystallin. In Fig. 5 are photochemical opacification data for 0±5 mg ml−" γ-crystallin solutions containing varying concentrations of added α-crystallin. Higher levels of α-crystallin are seen to afford greater protection than lower concentrations. In Fig. 6, these results are plotted so as to display the dependence of γ-crystallin solution opacity on α-crystallin concentration. Examining the data in Fig. 6 shows that a 0±5 mg ml−" solution of γ-crystallin was 75 % protected from UVinduced opacification by an α-crystallin concentration of 0±5 mg ml−" and 80 % protected by 2±0 mg ml−" of α-crystallin. Thus, 90 % of the limiting protection was afforded by 0±5 mg ml−" of α-crystallin. Weight ratios in the observed protection range of γ}α ¯ 1 : 1–4 correspond to molar ratios of γ}α ¯ n : 1 where n ¯ 10–40. Since monomeric γ-crystallin has molecular weight 20 kDa, and oligomeric α-crystallin is com-

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posed of 20 kDa subunit peptides, the data suggest that each α-crystallin subunit can protect m γcrystallin molecules from aggregation, i.e. γ}α ¯ m : 1 where m ¯ 0±25–1±0. Similar stoichiometries were observed for protection of other proteins from UVaggregation by α-crystallin (Fig. 3). An experiment was performed to test whether protein solutions which had been photoaggregated could be deaggregated by subsequent addition of αcrystallin. A 0±5 mg ml−" γ-crystallin in buffer solution was irradiated at 308 nm with the excimer laser source for 15 min. The solution became opaque and solid precipitate formed in a similar fashion to that shown in Fig. 5, with an absorbance at 600 nm of about 0±45. α-Crystallin was then added, to a concentration of 2 mg ml−", and the absorption at 600 nm, as well as the visual appearance of the sample, were monitored for 1 hr. The solution remained visually opaque, and the absorbance at 600 nm remained constant at 0±45³0±05. The solution was then warmed to 40°C and incubated for an additional hour. There was no evidence for deaggregation, and absorbance remained constant during this period.

4. Discussion The data in Figs 3 and 4 show that α-crystallin can protect proteins from both thermal and UV-induced protein opacification}aggregation. The thermal case has been studied by Horwitz (1992, 1993), by Wang and Spector (1995), and ourselves, and our UVinduced opacity data can be compared with results of Raman and Rao (1994). The degree of protection afforded by α-crystallin against thermal insult (Horwitz, 1993, found a molar ratio of γ}α ¯ 10 : 1 at

elevated temperatures) and our data on UV-induced aggregation at 24–26°C which gave γ}α ¯ 10–40 : 1 are seen to be of comparable magnitude. Wang and Spector (1994) found a subunit ratio of γ}α ¯ 1 : 1 for binding of partially denatured gamma, and they obtained chromatographic evidence that not all of the γ-crystallin molecules are bound to α-crystallin. Raman and Rao (1994) found that α-crystallin afforded increasing protection against UV aggregation of γ-crystallin with increasing temperature, particularly above 30°C, while all of our UV irradiation data were taken at 24–26°C. There is an apparent discrepancy between the two data sets in that we found significant protection against UV aggregation even at room temperature while Raman and Rao only began to see protection above 30°C. But, examining the data in Fig. 2 of Raman and Rao (1994), one sees that in fact their percentage protection does not go to zero for temperatures of 10–30°C but hovers in the range about 5–10 % of the maximum high temperature value. Thus, it is not correct to think that the chaperone function is controlled by an ‘ on–off switch ’ at 30°C, but rather than there is an increase in chaperone activity with increasing temperature. We agree that the α-crystallin chaperone function may be partially temperature activated, but we would not agree that there is no activity whatsoever below 30°C. This conclusion agrees with the findings of Farahbakhsh et al. (1995) who found that α-crystallin protected insulin from chemically induced aggregation at 25°C. A second important point is that Raman and Rao took pains to strictly maintain their stock solutions of α-crystallin at 4°C or less prior to beginning the UV irradiation experiments, while our solutions were equilibrated at 25°C for up to 1 hr prior to UV irradiation. Thus, our α-crystallin could have undergone some partial denaturation (chaperone activation ?) during this time while Ramon and Rao’s material did not. α-Crystallin absorbs UV radiation at the same wavelengths as the substrate proteins. This is an additional factor to be considered, which is not present in thermal experiments, and may contribute to the protection phenomenon. We propose that the protection mechanism in UV aggregation is the sum of a screening effect plus a binding effect analogous to that in thermal experiments (Horwitz, 1992, 1993). Both protection mechanisms are operative, as indicated by the data in Fig. 1, which shows that UV screening can only account for about one-third of the protection. An important conclusion that can be drawn from the UV chaperone results is that α-crystallin is able to bind the intermediates which occur in UV-irradiated proteins (free radicals ?) as well as the intermediates which occur in heat treated proteins (random coil peptides ?). The notion that the reactive intermediates are different in UV versus thermal aggregation experiments is supported by our SDS–PAGE results which showed that UV radiation led to covalently

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linked aggregates which were not dissociated by reducing SDS while heating led to aggregates which were dissociated under the same conditions. Thus, it seems clear that different reaction intermediates are involved in UV versus heat aggregation but that αcrystallin can bind either type. Comparing the ability of α-crystallin to protect one protein versus another reveals some differences amongst the proteins. Horwitz (1993) noted that the lens protein γ-crystallin was slightly more effectively protected than some other enzymes which he studied. A conclusion that can be drawn from comparison of the UV and thermal opacification data stems from the fact that the former were all performed at room temperature, while the latter were done at elevated temperatures in the range 50–70°C. In the thermal chaperone experiments, the α-crystallin was heated above room temperature. The question arises as to whether this heating is necessary to activate the chaperone function. The UV experiments were done at room temperature and thus the α-crystallin was not exposed to elevated temperatures, yet it still functioned as an effective chaperone. Thus thermal activation may enhance the chaperone function but does not seem to be required in order to have minimal protection. The same conclusion was reached in the chemical denaturation studies of Farahbakhsh et al. (1995) on insulin at 25°C. A related point is that the quaternary structure of α-crystallin is known to be temperature sensitive. The macromolecule has a molecular weight of about 800 kDa at 25°C (neutral pH and physiological ionic strength). At the elevated temperatures used in the thermal denaturation work, Horwitz (1993) estimated the particle size at 360 kDa. Considering the combined UV and thermal aggregation data, one sees differences in α-crystallin’s effectiveness as a chaperone at 800 kDa and at 360 kDa. For example, aldolase in Figs 3(A) and 4(A) is seen to be much better protected from thermal insult than UV insult at an α-crystallin concentration of 0±25 mg ml−". On the other hand, the observed protection stoichiometry for γ-crystallin, was α}γ ¯ 1 : n with n ¯ 10–40 regardless of whether α-crystallin was present in the 800 kDa at 25°C or the 360 kDa form at elevated temperature. Thus, enhanced chaperone activity may be present at elevated temperatures but activity persists even at room temperature. Most of our UV induced opacification experiments were done in the concentration range of a few milligrams per milliliter of both α-crystallin and substrate protein. The results obtained at lower (0±1 mg ml−") and higher (10 mg ml−") concentrations did not differ qualitatively from those at intermediate concentrations. There are some variations in the apparent degree of protection by α-crystallin at the three concentrations. The important point is that significant protection was observed throughout the 0±1–10 mg ml−" concentration range. It should be pointed out that our highest concentration,

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10 mg ml−", is an order of magnitude less than that present in the intact ocular lens. The question of whether a chaperone effect operates in the intact lens has been addressed by Boyle and Takemoto (1994).

Acknowledgements This work was supported in part by NIH Grant EY-06800 to RFB from the National Eye Institute (U.S.A.). BO was an NSF REU Research Participant from the University of West Florida, Pensacola, FL, U.S.A., Summer, 1993.

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