Interaction of 1,1'-Bi(4-anilino)naphthalene-5,5'-Disulfonic Acid with alpha -Crystallin*

K. Krishna SharmaDagger §, Harjeet KaurDagger , G. Suresh KumarDagger , and Kathryn KesterDagger

From the Dagger  Mason Eye Institute, Department of Ophthalmology and the § Department of Biochemistry, University of Missouri, Columbia, Missouri 65212

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The hydrophobic sites in alpha -crystallin were evaluated using a fluorescent probe 1,1'-bi(4-anilino)naphthalenesulfonic acid (bis-ANS). Approximately one binding site/subunit of alpha -crystallin at 25 °C was estimated by equilibrium binding and Scatchard analysis (Kd = 1.1 µM). Based on fluorescence titration, the dissociation constant was 0.95 µM. The number of bis-ANS binding sites nearly doubled upon heat treatment of the protein at 60 °C. Likewise, the exposure of alpha -crystallin to 2-3 M urea resulted in increased binding of bis-ANS. Above 3 M urea there was a rapid loss in the fluorescence indicating the loss of interaction between bis-ANS and protein. The alpha -crystallin refolded from 6 M urea showed tryptophan fluorescence emission similar to the native alpha -crystallin. However, the refolded alpha -crystallin showed a 60% increase in bis-ANS binding, suggesting distinct changes on the protein surface resulting from exposure to urea similar to the changes occurring due to heat treatment. The fluorescence of tryptophan in native alpha -crystallin was quenched by the addition of bis-ANS. The quenching was inversely related to the amount of bis-ANS bound to alpha -crystallin. Additionally, the binding of bis-ANS reduced the chaperone-like activity of the protein. Photolysis of bis-ANS-alpha -crystallin complex resulted in incorporation of the probe to both A- and B-subunits, indicating that both subunits in native alpha -crystallin contribute to the surface hydrophobicity of the protein.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

alpha , beta -, and gamma -crystallins constitute the major portion of the eye lens fiber cells (1). Among the crystallins, alpha -crystallin is the most abundant protein, existing as a polydisperse aggregate with the average molecular mass of 800 kDa (2). alpha -Crystallin is made up of two types of subunits, designated alpha A and alpha B with molecular masses 19,832 and 20,079 kDa, respectively (2). The sequences of the subunits of alpha -crystallin have high homology to small heat shock proteins (3, 4). alpha -Crystallin subunits, once thought to be lens-specific, are now widely known to be present in other tissues as well (5-8), and increased expression of alpha B-crystallin has been documented in some neurological disorders (6, 9, 10).

Recently, the ability of native alpha -crystallin to suppress the aggregation of heat-denatured (11-26), UV-irradiated (26, 27), and chemically denatured (28) proteins and enzymes has been demonstrated. Complex formation between alpha -crystallin and denatured proteins and enzymes or beta - and gamma -crystallins has been demonstrated (14, 18). On the basis of these in vitro data, it has been proposed that alpha -crystallin acts as a chaperone in vivo to maintain the lens clarity and that alpha -crystallin loses this ability during aging. Consistent with this hypothesis, a decreased chaperone-like activity has been observed for the alpha -crystallin present in high molecular mass aggregates from aged bovine and human lens (29, 30).

It has been proposed that surface hydrophobic sites in the native alpha -crystallin aggregate are involved in binding of target proteins to alpha -crystallin during chaperone-like activity display (17). A direct correlation between the extent of alpha -crystallin hydrophobicity and chaperone-like activity has been demonstrated (31-34). Liang and co-workers (35) in their recent study used recombinant alpha A- and alpha B-homopolymers and reported that the relative fluorescence enhancement of ANS1 is greater with alpha B compared with alpha A and concluded that alpha B has higher hydrophobicity. However, so far the amino acid sequences that contribute to the hydrophobic site(s) have not been identified. In a recent report, Smulders and de Jong (36) described that the N-terminal domain of recombinant murine alpha B-crystallin binds hydrophobic probe bis-ANS. We have recently reported that amino acid residues 57-69 and 93-107 of alpha B-crystallins interact with heat-denaturing alcohol dehydrogenase (37). Liang and Li (38) reported that there are about 40 ANS binding sites/native alpha -crystallin. Stevens and Augusteyn (39) have disputed the study of Liang and Li and reported that there is one ANS binding site/24 subunits of alpha -crystallins. It is rather difficult to explain the stoichiometry of ANS binding to alpha -crystallin in view of the proposed complex but ordered structure for alpha -crystallin (2).

In the present study we have determined the binding of bis-ANS to alpha -crystallin by equilibrium dialysis. The data presented here show the binding of bis-ANS to both A- and B-subunits of alpha -crystallin and transfer of the energy from protein tryptophan to the bound fluorophore. Furthermore, we show that prior binding of bis-ANS to alpha -crystallin can affect the chaperone-like activity.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- bis-ANS was obtained from Molecular Probes, Inc. (Junction City, OR). The stock solutions of bis-ANS were prepared in 95% alcohol, and the concentration was determined by absorbance at 385 nm using an extinction coefficient, epsilon  385 = 16,790 cm-1 M-1 (40). Ultra pure urea was purchased from U. S. Biochemical Corp. Yeast alcohol dehydrogenase (ADH) was obtained from Sigma. All other chemicals were of the highest grade commercially available.

Preparation of alpha -Crystallin-- alpha -Crystallin was isolated from young bovine lens cortex by gel filtration on Sephadex G-200 and ion exchange chromatography on trimethylaminoethyl-fractogel column (EM-Separations) as described earlier (21). The alpha -crystallin thus obtained was >99% pure as judged by SDS-PAGE and used in this study.

Equilibrium Binding of bis-ANS to alpha -Crystallin-- Bovine alpha -crystallin (0.3 µM) was incubated with 0-40 µM bis-ANS in 50 mM sodium phosphate buffer, pH 7.0, containing 100 mM NaCl (buffer A) for 1 h at 25 °C. The entire sample was then subjected to equilibrium dialysis in microdialysis cells (Technilab Instruments Inc.) using 1.2-ml volumes of alpha -crystallin and bis-ANS. Following equilibration for 24 h with stirring, the concentration of bis-ANS in each chamber was measured by its absorbance at 385 nm. The number of bis-ANS binding sites/alpha -crystallin was determined by Scatchard analysis (41).

Fluorescence Titration of bis-ANS Binding to alpha -Crystallin-- Fluorescence measurements were made on a Perkin-Elmer Spectrofluorimeter model 650-40 with a 3600 data station. The concentration of protein used was approximately 0.125 µM. The excitation and emission slit width were set to 4 nm. Samples with bis-ANS were excited at 390 nm, and the emission was measured at 490 nm in a cuvette with a 1-cm path length or recorded between 400 and 600 nm. Briefly, bis-ANS at several fixed concentrations was mixed with alpha -crystallin, and the fluorescence was measured. All the measurements were made at 25 °C. The fluorescence intensities of the samples were corrected for the absorption of the dye by the relation (42), Fcorr = Fobs antilog (ODex + ODem/2), where ODex and ODem are the optical densities at excitation and emission wavelengths, respectively. To see the effect of urea on bis-ANS binding to alpha -crystallin, a known amount of protein was mixed with 0-6 M urea in buffer A. After 14 h at 5 °C, bis-ANS was added, and the fluorescence spectra was recorded as above. Blanks without protein were prepared in same buffer.

For energy transfer experiments, an excitation wavelength of 295 nm was used to reduce the involvement of tyrosine fluorescence from the protein. The efficiency of energy transfer (EET) (43) from tryptophan to bis-ANS was calculated from the formula, EET = 1 - (Qt/Qo), where Qt and Qo are the relative fluorescence intensities of energy donor (tryptophan) in the presence and the absence of energy acceptor (bis-ANS), respectively. The quenching of tryptophan fluorescence upon the addition of bis-ANS was analyzed according to the equation Fo - F = (Fo - Fi- Kd app(Fo - F)/[bis-ANS] (44), where Fo and F denote the observed fluorescence in the absence and the presence of the bis-ANS, respectively, and Fi is the fluorescence at infinite concentration of bis-ANS. A plot of Fo - F versus (Fo - F)/[bis-ANS] yields a straight line whose slope equals Kd app of bis-ANS.

Photoincorporation of bis-ANS into alpha -Crystallin-- alpha -Crystallin (1 mg) was mixed with 100 µg of bis-ANS in 1 ml of buffer A, and the excess bis-ANS was removed by dialysis using buffer A. After dialysis the sample was taken in a glass Petri dish. The Petri dish was kept on ice, and a hand-held UV lamp (UVL-56 from Ultra-violet Products, Inc., San Gabriel, CA) was placed approximately 2 cm above the sample (45). The sample was illuminated for 10 min with constant stirring. The sample was analyzed by HPLC and SDS-PAGE after photolysis.

Separation of bis-ANS-labeled alpha A- and alpha B-Crystallin-- The alpha -crystallin-bis-ANS complex was treated with 5 mM dithiothreitol for 2 h and filtered. The alpha A- and alpha B-subunits were separated from one another by HPLC using a C18 column (Vydac C18, 218TP1010 from The Separation Group, Hesparia, CA) and linear gradient formed between 0.065% trifluoroacetic acid in water and 0.065% trifluoroacetic acid in acetonitrile. The elution was monitored by absorbance at 280 nm. All the fractions were tested for fluorescence (excitation, 350 nm; emission, 450 nm) in a Perkin-Elmer Spectrofluorimeter. The identities of alpha A- and alpha B-subunit peaks were confirmed by SDS-PAGE and pooled separately. The fluorescence spectrum of both the alpha A- and alpha B-crystallin fractions was obtained (excitation, 390 nm).

Thermal Denaturation and Light Scattering Assay-- The capacity of the bis-ANS treated alpha -crystallin to protect against heat-induced aggregation of ADH was determined according to the procedure described earlier (19). Briefly, 400 µg of ADH was heated in 50 mM PO4, pH 7.0, and 0.1 M NaCl (buffer A) in the presence or the absence of different amounts of the bis-ANS-alpha -crystallin in a final volume of 1.0 ml. bis-ANS-alpha -crystallin was prepared by saturating bis-ANS binding sites in alpha -crystallin at 48 °C and removing excess bis-ANS by dialysis. The aggregation of proteins at the specified temperature was followed by recording the increase in light scattering as a function of time in a Perkin-Elmer Lambda 3 spectrophotometer equipped with multicell transporter attached to a circulating water bath.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Interaction of bis-ANS with alpha -Crystallin-- bis-ANS is an environment-sensitive probe that shows an emission maximum at 533 nm in aqueous medium (46). When bis-ANS binds to a hydrophobic protein such as alpha -crystallin, its fluorescence intensity increases severalfold, and the emission maxima is blue shifted (32). Titration of alpha -crystallin (0.125 µM) with bis-ANS (0-10 µM) gave a hyperbolic plot as shown in Fig. 1, suggesting the saturation of bis-ANS binding sites in alpha -crystallin. The titration data, when analyzed by a double reciprocal plot (47), gave a Kd value of 0.95 µM for the binding of bis-ANS to alpha -crystallin.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 1.   Binding of alpha -crystallin at different concentrations of bis-ANS. alpha -Crystallin was at 0.125 µM. The increase in bis-ANS fluorescence was monitored at a fixed wavelength of 490 nm. The excitation wavelength was 390 nm. The maximum dye/protein ratio for complex formation was approximately 50.

The bis-ANS binding to alpha -crystallin was also determined by equilibrium dialysis method. alpha -Crystallin was dialyzed against bis-ANS in buffer A at 4 °C for 24 h following a initial incubation for 1 h at 25 °C. After the dialysis the amount of bis-ANS bound to alpha -crystallin was estimated on the basis of 385 nm absorption (40) of bis-ANS-alpha -crystallin complex. Scatchard analysis (41) of bis-ANS bound to different concentrations of alpha -crystallin is shown in Fig. 2. The Kd and n values calculated from the graph were 1.15 µM and 40, respectively. An average molecular mass of 800 kDa for alpha -crystallin (2) was used in these calculations.


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 2.   Scatchard plot for the binding of bis-ANS to alpha -crystallin. The data of bis-ANS binding to alpha -crystallin (0.27 µM) are plotted according to the Scatchard equation b/f = 1/Kd(t - b), where b is the concentration of bis-ANS bound to alpha -crystallin (calculated from absorption at 385 nm as described under methods), t is the concentration of bis-ANS bound at saturation of the sites, and f is the concentration of free bis-ANS.

Effect of Heat Treatment on bis-ANS Binding to alpha -Crystallin-- Earlier reports have shown that interaction of ANS or bis-ANS with alpha -crystallin is temperature-dependent (31-34). When alpha -crystallin was incubated with excess of bis-ANS at 48 or 70 °C for 1 h and dialyzed to remove the unbound dye, considerably higher amounts of bis-ANS remained bound to alpha -crystallin compared with bis-ANS-alpha -crystallin incubated at 25 °C. alpha -Crystallin incubated at 48 °C was able to bind 80 ± 4 mol of bis-ANS, whereas prior heat treatment of alpha -crystallin at 70 °C resulted in binding of 110 ± 6 mol of bis-ANS. This translated to 2 and 2.7 binding sites/subunit of alpha -crystallin at 48 and 70 °C, respectively.

Effect of Urea on bis-ANS Binding to alpha -Crystallin-- The effect of varying concentration of urea on bis-ANS binding to alpha -crystallin is shown in Fig. 3. Maximum dye binding to alpha -crystallin was observed in the presence of 3.0 M urea. The increase in bis-ANS fluorescence at low urea concentrations and the sharp decrease in the fluorescence above 3 M urea suggest an initial exposure of the buried binding sites at low urea concentration and the destruction of protein quaternary, tertiary, and secondary structures at higher urea concentration. The bis-ANS emission maximum also red shifted in samples with urea (data not shown). Fig. 3 also shows the change in tryptophan emission maximum at various concentrations of urea used. The graph depicts a partial exposure of buried tryptophan fluorescence (maximum emission, 346 nm) at 2.5 M urea concentration, which also showed maximal bis-ANS fluorescence. In another experiment, alpha -crystallin that had been denatured and unfolded in 6 M urea was allowed to refold and renature by dialysis and tested for bis-ANS binding by equilibrium dialysis method at 25 °C. The urea-denatured and renatured alpha -crystallin and the native alpha -crystallin, however, showed similar tryptophan emission maxima at 336 nm (data not shown). However, the alpha -crystallin exposed to urea was found to bind 60% more bis-ANS after refolding compared with the native alpha -crystallin (Fig. 4).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 3.   Intrinsic fluorescence and fluorescence intensity of bis-ANS bound to alpha -crystallin as a function of urea concentration. To determine the effect of urea on bis-ANS binding, individual samples at the indicated urea concentrations were prepared as described under "Experimental Procedures" with a protein concentration of 0.1 mg and a bis-ANS concentration of 12 µM. To determine the effect of urea on tryptophan emission maximum, samples prepared in different concentrations of urea without bis-ANS were used. bullet , bis-ANS fluorescence; black-square, tryptophan emission maximum.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4.   bis-ANS interaction with native and urea-denatured and refolded alpha -crystallin. Experimental details are under "Experimental Procedures." Broken line, native alpha -crystallin; solid line, refolded alpha -crystallin.

Energy Transfer from Tryptophan to Bound bis-ANS-- Energy transfer was detected from the overlap of the tryptophan fluorescence emission spectrum of alpha -crystallin and excitation spectrum of bis-ANS. The emission spectra of alpha -crystallin and the alpha -crystallin-bis-ANS complex when excited at 295 nm are shown in Figs. 5 and 6. In the absence of bis-ANS, the fluorescence was emitted maximally at 336 nm due to the excitation of tryptophan residues in alpha -crystallin. Upon addition of bis-ANS to alpha -crystallin there was a decrease in the tryptophan fluorescence at 336 nm concurrent with an increase in the fluorescence at 488 nm. Because free bis-ANS does not fluoresce when excited at 295 nm, the 488 nm band represents emission due to tryptophan-excited bis-ANS bound to alpha -crystallin. The isoemmissive point was 420 nm. A Kd value of 1.4 µM was obtained for the binding of bis-ANS by the analysis of tryptophan quenching data (44). This value is not significantly different from the Kd value obtained by equilibrium dialysis method. A similar value was obtained when the tryptophan quenching data were analyzed by a modified Stern-Volmer method (48, 49). The maximum transfer efficiency of 0.9 was obtained when the quenching data were analyzed by the method of Wallach et al. (50). The transfer efficiency was also calculated by the method described by Stryer (43) and found to be 0.83.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 5.   Energy transfer between the tryptophan residues and bound bis-ANS in alpha -crystallin. Excitation wavelength was 295 nm. Protein concentration was 1 mg/ml, and the added bis-ANS concentrations were 0 (trace 1), 2.2 (trace 2), 4.4 (trace 3), 6.6 (trace 4), 8.8 (trace 5), 11 (trace 6), and 13.2 µM (trace 7).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 6.   Concentration dependence of energy transfer between tryptophan and bound bis-ANS. alpha -Crystallin was excited at 295 nm, and emission was measured at 338 and 490 nm. bis-ANS at 150 µM was added in 15-µl aliquots, and emission was measured after 30 s. Measured fluorescence intensities were corrected for the absorption of bis-ANS at 295, 338, and 490 nm, respectively. black-square, 338 nm fluorescence; bullet , 490 nm fluorescence.

Effect of bis-ANS Binding on Chaperone-like Activity of alpha -Crystallin-- The effect of prior binding of bis-ANS to alpha -crystallin on its chaperone-like activity is shown in Fig. 7. Only a marginal decrease in the alpha -crystallin chaperone-like activity was observed when tested with ADH.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of bis-ANS binding on the chaperone-like activity of alpha -crystallin. The assays were done as described under "Experimental Procedures" using alpha -crystallin-bis-ANS and ADH. black-square, ADH; bullet , ADH + 25 µg of alpha -crystallin; black-triangle, ADH + 25 µg of bis-ANS-alpha -crystallin.

bis-ANS Incorporation to alpha -Crystallin-- It has been shown that bis-ANS can be incorporated to specific hydrophobic sites in proteins by UV activation and that the dye incorporated into the proteins remains sensitive to the polarity of its general environment (45). Furthermore, it has been shown that bis-ANS binding sequences in molecular chaperones can be identified by photocross-linking of the dye to the protein, peptide mapping, and sequencing (45). During this study bis-ANS was initially allowed to bind to alpha -crystallin by the addition of saturating amounts of the probe. The excess probe was removed by dialysis. The alpha -crystallin-bis-ANS complex was photolyzed by UV-A light (366 nm). Fig. 8 shows the SDS-PAGE of the bis-ANS-alpha -crystallin complex subjected to a 10-min photolysis. The fluorescence seen in the 20-kDa region (Fig. 8, lane 3, left panel) was due to the covalently bound bis-ANS. Lane 2 of Fig. 8 contains the unphotolyzed alpha -crystallin-bis-ANS complex. The alpha A- and alpha B-subunits of photolyzed alpha -crystallin-bis-ANS complex were separated by HPLC. SDS-PAGE of alpha A and alpha B is shown in Fig. 8 (lanes 4 and 5). Both lanes show fluorescence in alpha A- and alpha B-crystallin protein band region. These data suggest that both alpha A- and alpha B-subunits may be contributing to the bis-ANS binding sites in alpha -crystallin.


View larger version (44K):
[in this window]
[in a new window]
 
Fig. 8.   Photoincorporation of bis-ANS to alpha -crystallin as analyzed by SDS-PAGE. The experimental details are under "Experimental Procedures." Lanes 1, molecular mass standard; lanes 2, bis-ANS + alpha -crystallin without photolysis; lanes 3, bis-ANS + alpha -crystallin after photolysis; lanes 4 and 5, alpha A and alpha B isolated from bis-ANS-labeled alpha -crystallin by HPLC. Left panel, fluorescence; right panel, Coomassie Blue R-250 stain.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Although the presence of surface hydrophobic sites on alpha -crystallin has been known for a number of years (51), only recently has considerable interest been shown in the hydrophobic sites within alpha -crystallins because these sites have been implicated in chaperone-like function of the protein (17, 31-36, 38, 39). There is, however, disagreement with respect to the number of hydrophobic sites available within alpha -crystallin as determined by the environment-sensitive probe ANS (38, 39). We used bis-ANS instead of ANS to determine the nature of hydrophobic sites in alpha -crystallin. Consistent with the report published earlier (32), our data show that alpha -crystallin has hydrophobic sites capable of binding environment-sensitive probe bis-ANS. bis-ANS binds to alpha -crystallin very tenaciously. Unlike ANS-alpha -crystallin, dialysis of bis-ANS-alpha -crystallin for 2 days in buffer A did not dissociate the protein bound bis-ANS. We determined the affinity of bis-ANS to alpha -crystallin by three methods. Although the fluorescence titration method gave a Kd of 0.95 µM (Fig. 1), the equilibrium dialysis method and the tryptophan quenching studies gave Kd and Kd app values of 1.15 µM (Fig. 2) and 1.4 µM (Fig. 6), respectively. The difference in dissociation constants determined by the three methods was not significant. These values are five to eight times lower than the Kd value for ANS binding to alpha -crystallin reported recently (39).

We determined the number of bis-ANS binding sites/subunit of alpha -crystallin by equilibrium dialysis and measuring the absorbance of bound bis-ANS at 385 nm rather than the fluorescence titration method for the following reasons. The titration method is likely to give incorrect values if all the binding sites are not homogenous. The quantum yield of the dye bound at different sites in subunits may vary significantly. It has also been shown recently that alpha A- and alpha B-crystallins have different ANS binding characteristics (35). From earlier studies we know that there is no constant stoichiometry between the two types of subunits (2). Therefore if we do not know the contribution of each type of binding site to the total sites, a reliable estimate of the total binding sites cannot be made by fluorescence titration studies. In view of this an estimate of binding sites determined earlier by fluorescence titration is likely to include significant error. The equilibrium dialysis method we employed involves the direct estimation of the bound dye on the basis of molar absorbance. The absorbance of bis-ANS is not altered upon binding to proteins. Our estimate of one bis-ANS binding site/alpha -crystallin subunit at 25 °C is much higher than one ANS binding site/24 subunits estimated by titration method (39). The estimation of one binding site/24 subunits of alpha -crystallin cannot be explained by any of the proposed structural models (2) for the protein. The increase in the bis-ANS binding sites at elevated temperature, the earlier demonstration that alpha -crystallin displays higher chaperone-like activity if exposed to elevated temperatures (32-34), and the estimated stoichiometry of interactions between the target protein and alpha -crystallin during chaperoning (18) suggest that one to three hydrophobic sites may be involved in binding a target protein.

Although we could not quantify the extent of A- and B-subunit contribution to the total bis-ANS binding sites on alpha -crystallin, the photocross-linking experiments (Fig. 8) show that both alpha A- and alpha B-subunits in alpha -crystallin bind bis-ANS. By cross-linking studies we have recently shown that both A- and B-subunits of alpha -crystallin participate in chaperone-like activity display (37). As has been reported for rat alpha B-crystallin (36), bovine alpha -crystallin also showed a decrease in chaperone-like activity after complexing with bis-ANS. Although only a partial loss in chaperone-like activity of alpha -crystallin was observed when it was complexed with bis-ANS (Fig. 7), the data support the hypothesis that hydrophobic sites in alpha A and alpha B are involved in chaperone activity. The high residual chaperone-like activity observed for bis-ANS-alpha -crystallin may be due to the retention of the hydrophobic nature of the site subsequent to the binding of the probe. We have also observed that glycated alpha -crystallin binds less ANS and displays lower chaperone-like activity. The residual chaperone-like activity of the glycated alpha -crystallin was directly proportional to the residual ANS binding.2 These data suggest the involvement of same site/amino acid residues in alpha -crystallin during ANS binding, glycation, and chaperone-like activity.

The binding of ANS or bis-ANS to alpha -crystallin also increases after heat treatment (31-34), partial unfolding by urea (Fig. 3), or urea denaturation and refolding (Fig. 4). Although earlier studies have demonstrated that increased bis-ANS or ANS fluorescence is seen with alpha -crystallin at 25 °C that has been exposed once to higher temperature (32-34), there are no reports on the number of newly formed hydrophobic sites. Our data show that approximately 40 new bis-ANS binding sites are formed per alpha -crystallin molecule when it is heated to 48 °C. Retention of the additional bis-ANS sites by alpha -crystallin exposed to at higher temperatures (48 and 72 °C) points to the inability of alpha -crystallin to regain its native structure after cooling. It is unlikely that in our experiments bis-ANS was trapped in alpha -crystallin at higher temperature because experiments where bis-ANS was added after cooling alpha -crystallin also showed increased bis-ANS binding. The increased binding of bis-ANS to urea-denatured and renatured alpha -crystallin suggests that although the refolded alpha -crystallin displays intrinsic fluorescence similar to the native protein as reported earlier (52), there is a measurable difference with respect to hydrophobic sites between the native protein and renatured protein (Fig. 4). Therefore caution should be exercised in interpreting the recombinant alpha -crystallin chaperone-like activity data if the protein isolation step involves urea.

The increased binding of bis-ANS to structurally perturbed alpha -crystallin reported here is similar to that observed with molecular chaperone GroEL (49, 53, 54), but the number of bis-ANS that can bind to native or partially unfolded alpha -crystallin is greater. Although the oligomeric GroEL (800 kDa), binds one or two bis-ANS molecules in its native form and about 14 bis-ANS molecules in the presence of ~2.5 M urea (54), we estimated that 40 and 65 bis-ANS molecules bind to alpha -crystallin in its native form and in the presence of 2.5 M urea, respectively. The high number of available surface hydrophobic sites are probably responsible for the increased capacity of alpha -crystallin to bind denaturing proteins compared with GroEL, which is believed to bind one protein at a time (55).

The alpha -crystallin A-subunit has one Trp (Trp-9), whereas the B-subunit has two tryptophans (Trp-9 and 60) (2). The fluorescence emission studies have shown that two tryptophans near the N terminus are relatively buried and the other Trp is near the surface (56). The fluorescence quenching studies reported here suggest that the bis-ANS binding sites are relatively closer to the Trp. During our studies to identify the alpha -crystallin amino acid sequences involved in chaperone-like function, we observed that one of the binding sequences, APSWIDTGLSEMR (37), contained the Trp-60 in alpha B-crystallin. The sequence is also a hydrophobic sequence predicted by hydropathy plot and deuterium exchange studies (57). Therefore we hypothesize that Trp-60 of alpha B-crystallin forms the part of chaperone binding site as well as bis-ANS binding site. While this work was in progress, Smulders and de Jong (36) reported that the bis-ANS binding site is on the N-terminal domain of the recombinant rat alpha B-crystallin. However, it is not known whether the bis-ANS binding to rat alpha B-crystallin was influenced by the urea used during the isolation of the recombinant protein.

It remains to be determined which amino acid residues form the bis-ANS binding site(s) in native, heat-treated, and refolded alpha -crystallin. Once the chaperone binding sites are identified in both A- and B-subunits of alpha -crystallin, the extent of involvement of bis-ANS binding sites in chaperoning can be determined.

    ACKNOWLEDGEMENT

We thank Dr. B. J. Ortwerth for helpful discussions on this project and critically reading the manuscript.

    FOOTNOTES

* This work was supported in part by a grant-in-aid from the Fight For Sight Research Division of Prevent Blindness America and Research to Prevent Blindness, Inc.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Mason Eye Inst., University of Missouri, Columbia, One Hospital Drive, Columbia, MO 65212. Fax: 573-884-4100; E-mail: opthks{at}showme.missouri.edu.

1 The abbreviations used are: ANS, 1-anilinonaphthalene-8-sulfonic acid; bis-ANS, 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid; ADH, alcohol dehydrogenase; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography.

2 K. K. Sharma, unpublished data.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Bloemendal, H. (1981) in Molecular and Cellular Biology of the Eye Lens (Bloemendal, H., ed), pp. 1-47, John Wiley & Sons, New York
  2. Groenen, P. J. T. A., Merck, K. B., de Jong, W. W., and Bloemendal, H. (1994) Eur. J. Biochem. 225, 1-19[Abstract]
  3. Ingolia, T. D., and Craig, E. A. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2360-2364[Abstract]
  4. Sax, C. M., and Piatigorsky, J. (1994) Adv. Enzymol. Relat. Areas Mol. Biol. 69, 155-201[Medline] [Order article via Infotrieve]
  5. Bhat, S. P., and Nagineni, C. N. (1989) Biochem. Biophys. Res. Commun. 158, 319-325[Medline] [Order article via Infotrieve]
  6. Iwaki, T., Kume-Iwaki, T., Liem, R. K. H., and Goldman, J. E. (1989) Cell 57, 71-78[Medline] [Order article via Infotrieve]
  7. Kato, K., Shinohara, H., Kurobe, N., Goto, S., Inaguma, Y., and Ohshima, K. (1991) Biochim. Biophys. Acta 1080, 173-180[Medline] [Order article via Infotrieve]
  8. Srinivasan, A. N., Nagineni, C. N., and Bhat, S. P. (1992) J. Biol. Chem. 267, 23337-23341[Abstract/Free Full Text]
  9. Iwaki, T., Wisniewski, T., Iwaki, A., Corbin, E., Tomokane, N., Tateishi, J., and Goldman, J. E. (1992) Am. J. Pathol. 140, 345-356[Abstract]
  10. Murano, S., Thweatt, R., Shmookler Reis, R. J., Jones, R. A., Moerman, E. J., and Goldstein, S. (1992) Mol. Cell. Biol. 11, 3905-3914
  11. Horwitz, J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10449-10453[Abstract]
  12. Horwitz, J., Emmons, T., and Takemoto, L. (1992) Curr. Eye Res. 11, 817-822[Medline] [Order article via Infotrieve]
  13. Jacob, U., Gaestel, M., Engel, K., and Buchner, J. (1993) J. Biol. Chem. 268, 1517-1520[Abstract/Free Full Text]
  14. Rao, P. V., Horwitz, J., and Zigler, J. S., Jr. (1993) Biochem. Biophys. Res. Commun. 190, 786-793[CrossRef][Medline] [Order article via Infotrieve]
  15. Merk, K. B., Groenen, p. J. T. A., Voorter, C. E. M., de Haard-Hoekman, W. A., Horwitz, J., Bloemendal, H., and de Jong, W. W. (1993) J. Biol. Chem. 268, 1046-1052[Abstract/Free Full Text]
  16. Ganea, E., and Harding, J. J. (1995) Eur. J. Biochem. 231, 181-185[Abstract]
  17. Raman, B., and Rao, Ch. M (1994) J. Biol. Chem. 269, 27264-27268[Abstract/Free Full Text]
  18. Wang, K., and Spector, A. (1994) J. Biol. Chem. 269, 13601-13608[Abstract/Free Full Text]
  19. Sharma, K. K., and Ortwerth, B. J. (1995) Exp. Eye Res. 61, 413-421[Medline] [Order article via Infotrieve]
  20. Smulders, R. H. P. H., Merh, K. B., Aendekerk, J., Horwitz, J., Takemoto, L., Slingsby, C., Bloemendal, H., and de Jong, W. W. (1995) Eur. J. Biochem. 232, 834-838[Abstract]
  21. Das, K. P., Petrash, J. M., and Surewicz, W. K. (1996) J. Biol. Chem. 271, 10449-10452[Abstract/Free Full Text]
  22. Plater, M. L., Goode, D., and Crabbe, J. M. (1996) J. Biol. Chem. 271, 28558-28566[Abstract/Free Full Text]
  23. Carver, J. A., Nicholls, K. A., Aquilina, J. A., and Truscott, R. J. W. (1996) Exp. Eye Res. 63, 639-647[CrossRef][Medline] [Order article via Infotrieve]
  24. Smulders, R. H. P. H., Carver, J. A., Lindner, R. A., van Boekel, M. A. M., Bloemendal, H., and de Jong, W. W. (1996) J. Biol. Chem. 271, 29060-29066[Abstract/Free Full Text]
  25. Clark, J. I., and Haung, Q. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 15185-15189[Abstract/Free Full Text]
  26. Andley, U. P., Mathur, S., Griest, T. A., and Petrash, J. M. (1996) J. Biol. Chem. 271, 31973-31980[Abstract/Free Full Text]
  27. Borkman, R. F., Knight, G., and Obi, B. (1996) Exp. Eye Res. 62, 141-148[CrossRef][Medline] [Order article via Infotrieve]
  28. Farahbakhsh, Z. T., Huang, Q.-L,., Ding, L.-L., Altenbach, C., Steinhoff, H.-J., Horwitz, J., and Hubbell, W. L. (1995) Biochemistry 34, 509-517[Medline] [Order article via Infotrieve]
  29. Takemoto, L., and Boyle, D. (1994) Curr. Eye Res. 13, 35-44[Medline] [Order article via Infotrieve]
  30. Rao, P. V., Haung, Q.-L., Horwitz, J., and Zigler, J. S., Jr. (1995) Biochem. Biophys. Acta 1245, 439-447[Medline] [Order article via Infotrieve]
  31. Raman, B., Ramakrishna, T., and Rao, Ch. M (1995) FEBS Lett. 365, 133-136[CrossRef][Medline] [Order article via Infotrieve]
  32. Das, K. P., and Surewicz, W. K. (1995) FEBS Lett. 369, 321-325[CrossRef][Medline] [Order article via Infotrieve]
  33. Das, B. K., Liang, J. J., and Chakrabarti, B. (1997) Curr. Eye Res. 16(4), 303-309
  34. Lee, J.-S., Lio, J.-H., Wu, S.-H., and Chiou, S.-H. (1997) J. Protein Chem. 16, 283-289[Medline] [Order article via Infotrieve]
  35. Sun, T.-X., Das, B. P., and Liang, J. N. (1997) J. Biol. Chem. 272, 6220-6225[Abstract/Free Full Text]
  36. Smulders, R. H. P. H., and de Jong, W. W. (1997) FEBS Lett. 409, 101-104[CrossRef][Medline] [Order article via Infotrieve]
  37. Sharma, K. K., Kaur, H., and Kester, K. (1997) Biochem. Biophys. Res. Commun. 239, 217-222[CrossRef][Medline] [Order article via Infotrieve]
  38. Liang, J. N., and Li, X.-Y. (1991) Exp. Eye Res. 53, 61-66[Medline] [Order article via Infotrieve]
  39. Stevens, A., and Augusteyn, R. C. (1997) Eur. J. Biochem. 243, 792-797[Abstract]
  40. Sudhakar, K., and Fay, P. J. (1996) J. Biol. Chem. 271, 23015-23021[Abstract/Free Full Text]
  41. Scatchard, G. (1949) Ann. N. Y. Acad. Sci. 51, 660-672
  42. Lakowicz, J. R. (1983) Principles of Fluorescence Spectroscopy, Plenum Publishing Corp., New York
  43. Stryer, L. (1978) Annu. Rev. Biochem. 47, 819-846[CrossRef][Medline] [Order article via Infotrieve]
  44. Kollmann-kock, A., and Egger, H. (1989) Eur. J. Biochem. 185, 441-447[Abstract]
  45. Seale, J. W., Martinez, J. L., and Horowitz, P. M. (1995) Biochemistry 34, 7443-7449[Medline] [Order article via Infotrieve]
  46. Shi, L., Palleros, D. R., and Fink, A. L. (1994) Biochemistry 33, 7536-7546[Medline] [Order article via Infotrieve]
  47. Wang, J. L., and Edelman, G. M. (1971) J. Biol. Chem. 246, 1185-1191[Abstract/Free Full Text]
  48. Eftnik, M. R., and Ghiron, C. A. (1981) Anal. Biochem. 114, 199-227[Medline] [Order article via Infotrieve]
  49. Prasad, A. R., Ludeuena, R. F., and Horowitz, P. M. (1986) Biochemistry 25, 3536-3540[Medline] [Order article via Infotrieve]
  50. Wallach, D. F. H., Ferber, E., Selin, D., Weidekamm, E., and Fischer, H. (1970) Biochim. Biophys. Acta 203, 67-76[Medline] [Order article via Infotrieve]
  51. Andley, U. P., Liang, J. N., and Chakrabarti, B. (1982) Biochemistry 21, 1853-1858[Medline] [Order article via Infotrieve]
  52. Raman, R., Ramakrishna, T., and Rao, Ch. M (1995) J. Biol. Chem. 270, 19888-19892[Abstract/Free Full Text]
  53. Gorovits, B. M., Seale, J. W., and Horowitz, P. M. (1995) Biochemistry 34, 13928-13933[Medline] [Order article via Infotrieve]
  54. Horowitz, P. M., Hua, S., and Gibbons, D. L. (1995) J. Biol. Chem. 270, 1535-1542[Abstract/Free Full Text]
  55. Hartl, F. U. (1996) Nature 381, 571-579[CrossRef][Medline] [Order article via Infotrieve]
  56. Augusteyn, R. C., Ghiggino, K. P., and Putilina, T. (1993) Biochim. Biophys. Acta 1162, 61-71[Medline] [Order article via Infotrieve]
  57. Smith, J. B., Liu, Y., and Smith, D. L. (1996) Exp. Eye Res. 63, 125-128[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.