Exp. Eye Res. (1992) 54, 719-724

Spectroscopic

Studies

on the Interaction with Crystallins

JACK Howe

J. N. LIANG”

AND

of Calf

XIAO-YAN

Lens Membranes

LI

Laboratory of Ophthalmology, Massachusetts Eye and Ear Infirmary, Department Ophthalmology, Harvard Medical School, 243 Charles Street, Boston, MA, U.S.A.

(Received

Bethesda

5 February

7991 and accepted

in revised form 25 June

of

1991)

The interaction of crystallins with lens membranes and liposomes was studied by fluorescence and circular dichroism (CD) measurements. Two extrinsic fluorescence probes ANS (l-anilino-naphthalene8-sulfonic acid) and DPH (1.6-diphenyl, 1,3,5-hexatriene) were used to detect the binding and to explore the binding site. The ANS fluorescence intensity is greater in membranes than in liposomes, but is reverse for DPH. Among a, @and y-crystallins, only a,-crystallin decreased the ANS fluorescence intensity in membranes, indicating a binding between membranes and a,-crystallin. The binding site appears to be at the polar-apolar interface in membrane protein (hUP26) and a,-crystallin. Fluorescence polarization measurements show that lipid bilayer becomes less mobile with a,-crystallin binding. The change in the near UV CD due to the binding also indicates a decreased freedom of rotation of aromatic amino acid residues either in MIP26 or in a-crystallin. Key words: crystallin ; membrane ; liposome : fluorescence : circular dichroism.

1. Introduction The interaction between lens membranes and crystallin has been thought important in the formation of cataracts (Spector, 1984; Alcala and Maisel, 1985). Recent studies showed that only a-crystallin isolated from the calf cortex bound to the membranes (Ifeanyi and Takemoto, 1989; Mulders et al., 1989). An earlier study suggested that the binding arose from surface-seeking segments of aA and MIP26 (Mulders et al., 1985). It is still unknown, however, what specific sites of membranes and a-crystallin are involved in the binding. Since membrane proteins are very hydrophobic and are mostly buried inside the bilayer, it is logical to assume that the binding sites are at the polar-apolar interface of MIP26. In the present study, we have used two fluorescent probes, one anionic ANS and one non-ionic DPH, to study the interaction between a-crystallin and membranes. Both probes have been extensively used in the study of

membranes (Lents, 1988; Liang, Rossi and Andley, 1989). ANS binds to the polar-apolar interface, while DPH binds to the interior of the bilayer. The effect of binding on membranes or a-crystallin was studied by polarization and CD measurements. 2. Materials

alkali treatment (0.1 M NaOH) (Russell, Robison and Kinoshita, 1981). The age of the calf was about 4 weeks and the cow 4-6 years. These preparations yield membranes consisting of mainly MIP26, as shown by SDS-PAGE (Liang et al., 1989). Lipids were extracted from calf lenses by chloro-

form/methanol (2: 1 by volume) as described by Folch, Lees and Sloane-Stanley (19 5 7). Briefly, decapsulated lenses were homogenized in water (0.5 ml per lens) and extracted with chloroform/methanol. After filtering, the liquid extract was washed with 0.1 M KC1 and was allowed to separate thoroughly. The chloroform phase was recovered and dried with a stream of nitrogen. The phospholipids obtained were used in the present study without further separation into individual lipids. Liposomes were made by a mild sonication of lipid suspension. isolation of Lens Crystallins

Crystallins were prepared from calf lens cortical and nuclear extracts by gel filtration on a superose 6 column (FPLC System, Pharmacia-LKB) (Liang and Li, 199 la). Both cortical and nuclear a-crystallins were used (labeled as a,- and a,-crystallin). For p-&-) and y-crystallin, only cortical samples were used.

and Methods

isolation of Membranes and Liposomes Membranes were isolated from bovine lenses, using successive washing by 7 M urea and then by a mild * For correspondence at: Longwood Medical Research Center. Brigham and Women’s Hospital, 221 Longwood Ave. Boston, MA 02115. U.S.A. 00144835/92/050719+06

$03.00/O

FluorescenceMeasurements

ANS and DPH were purchased from Molecular Probes (Junction City, OR). Fifteen microliters of ANS or DPH stock solutions (l-10 x 10e3M) was added to 3 ml of membrane solutions (0.05 M Tris buffer, pH 7.6 ; protein concentration, 0.02 mg ml-‘. or mem-

brane solutions containing

crystallins (S-20 lug). The

0 1992 AcademicPressLimited

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500 Wavelength

(nm)

FIG. 1. ANS Buorescence spectra in (A) calf lens membranes and (B) liposomes. Membranes and liposomes suspended in 0.05 M Tris buffer, pH 7.6, were adjusted to have an optical density (0.1-0.2) at 400 nm. ANS concentration is 5 x 10m5M. The excitation wavelength is 380 nm and slit widths are 5 x 8 nm. All fluorescence intensities are in an arbitrary unit.

mixtures of membranes and crystallin were incubated at room temperature for 30 min before adding dye solution. In the measurements of liposomes, a suspension was made having the same optical density at 400 nm with membrane suspension. Fluorescence measurements were made in a PerkinElmer MP66 Fluorometer, with a corrected excitation and emission mode (Liang, 1990). ANS and DPH emission spectra were obtained by exciting at 3 80 and 360 nm, respectively. Both 90” and front surface detection were used, but no appreciable difference in relative intensity was observed. Polarization Measurements Polarization measurements of DPH in membranes were described previously (Liang et al., 1989). The calculated anisotropy (r) values were converted into rotational diffusion parameters (r,/r1)-l, where r,, is intrinsic anisotropy and is 0.362. The rotational relaxation time (p) is related to the rotational diffusion parameter by Perrin equation (Weber, 1953 ; Lakowicz, 1981), (r,/r- 1)-l = p/37, where 7 is the lifetime of the probe and has a value of 6.7 nsec for DPH in the calf membranes (Liang et al., 1989). CD Measurements CD measurements were made in an Aviv Circular Dichroism Spectrometer, model 60 DS (Liang and Rossi, 1989). The reported CD spectra are the average of five scans, smoothed by polynomial curve fitting. The system for correction due to differential scattering consistsof an end-window photomultiplier tube with a variable aperture diaphragm (342 mm diameter), mounted on a movable carriage, whose position can be varied from 18 to 46 cm from the sample cuvette. The reported CD spectra of membranes were obtained with an acceptance half-angle of 8”. The CD data were expressed as specific ellipticity in millidegrees. The CD change due to the binding of ol-crystallin

400

420

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480

500

520

540

inml

FIG. 2. ANS fluorescencespectrain (A) membranesonly,

(B) mixture of membranesand a,-crystallin, and (C) x~crystallin only. Samplesare in 0.05 M Tris buffer, pH 7.6. Protein concentrationsof membranesand cc,-crystallinare 20 pg ml-’ and 3 pugml-‘, respectively. Conditions of measurements were the sameasin Fig. 1. was measured with a mixing cell, which has two compartments each with 5 mm path length. CD was measured before and after mixing the two components. 3. Results ANS Fluorescence ANS fluorescence displayed a much greater intensity in membranes than in liposomes,as shown in Fig. 1. A decrease in ANS fluorescence intensity was shown only in the mixture of a,-crystallin membranes (Fig. 2). The addition of a,-crystallin and /3- or y-crystallin increased the ANS fluorescence intensity (Fig. 3, only

CALF

LENS

CRYSTALLINS

721

1

. (400

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480

Wavelength

1

500

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(nm)

400

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FIG. 3. ANS fluorescencespectrain (A) membranesonly,

460

Wavelength

(B) mixture of membranesand a,-crystallin, and (C) a,crystallin. Conditionsof measurements were the sameasin Fig. 2.

480

500

[nm)

FIG. 5. DPH fluorescencespectrain (A) membraneonly.

(B) mixture of membraneand a,-crystallin, and (C) a,crystallin only. Samplesare in 0.05 M Tris buffer, pH 76. Concentrationsof protein are the sameas in Fig. 2 and conditionsof measurements are the sameas in Fig. 4.

a,-crystallin data are shown). The decrease of ANS

fluorescence intensity was saturable : addition of more cz,-crystallin increased ANS fluorescence intensity because of the presence of an excess of ol,-crystallin. The small increase in intensity with addition of a,crystallin is due to ANS binding with a,-crystallin. The liposome-cr,-crystallin system did not show a decrease in ANS fluorescence intensity as did membrane-a,-crystallin. ANS spectra are very similar to those of a,-crystallin in membranes (data not shown). It appears that a,-crystallin or any other crystallins did not bind to liposomes.

crystallin data are shown). The magnitude of the

increase is equal to the intensity

of DPH in CI-

crystallins. Polarizution

The rotational relaxation time of calf lens membranes is 5 7 f 4 nsec, increased to 69 -f.2 nsec in the presence of a,-crystallin (P < OOOOl), but remained the same, 59 f 5 nsec in the presence of a,-crystallin (P > 0.5). For the purpose of comparison, cow membranes were also measured and had a value of

DPH Fluorescence

73 f 4 nsec, which is significantly calf membranes (P < 0.001).

DPH displayed a far greater fluorescence intensity in liposomes than in membranes, a complete reversal shown by ANS (Fig. 4). Unlike ANS, DPH fluorescence

Circular Dichroism CD spectra of the calf lens membrane protein and a,,-

intensity increased in both membranes and liposomes with addition of all crystallins (Figs 5 and 6. only a-

I

380

I

crystallin are shown in Fig. 7. CD of the membrane

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higher than that of

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(nm)

FIG. 4. DPH fluorescencespectra in (A) calf lens membraneand (B) liposomes.DPH concentration is 5 x 1O-5M. The excitation wavelength is 360 nm and slit widths are 5 x 2.5 nm. Other conditionswere the sameasin Fig. 1.

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ment than fluorescence, concentrations

of both membranes and a-crystallin have to be higher in the CD measurement than in the fluorescence measurement. No change was observed for cc,,-crystallin-membrane system (data not shown). 4. Discussion

Wavelength

(nm)

FIG. 6. DPH tluorescence spectra in (A) membrane only, (B) mixture of membrane and a,-crystallin, and (C) LX,,crystallin. Conditions are the same as in Fig. 5.

protein in trifluoroethanol (TFE) is included for comparison, in which membrane proteins are soluble. The CD spectrum of membrane protein in TFE is very similar to that reported by Horwitz and Bok (1987). Membrane proteins in Tris buffer lost the structured CD as shown in TFE, apparently due to a differential scattering contribution. The geometry of the photomultiplier could not eliminate all of this contribution. However, as shown in Fig. 7, the CD intensity is proportional to membrane protein concentration and the differential scattering does not contribute any additional band. Figure 8 shows the CD change due to the binding of a,-crystallin with the membranes. The change is small (CD becomes more negative), but is always reproducible. Because of a lesser sensitivity of CD measure-

The difference in the relative fluorescence intensity of ANS and DPH in membranes and liposomes indicates that the size of the binding sites is not the same. The circularly shaped liposomes are apparently smaller in size and have less polar-apolar interfaces for ANS binding. The non-ionic DPH, however, can penetrate into the hydrophobic interior. Since the amounts of lipids in membranes and liposomes in these measurements were not determined, the greater DPH intensity in liposomes than in membranes (Fig. 2) may indicate that there are more lipids in liposomes, rather than that liposomes have more effective DPH binding. The present results show that only a,,-crystallin interacts with membranes; all other crystallins (either a,-crystallin or /3- and y-crystallin) do not show this property. Apparently, the membrane binding occurs at a specific site in a,,-crystallin, and this binding site is lost when the protein becomes old (a,-crystallin). Previous reports indicated that ANS binding sites decreased from a,-crystallin to a,-crystallin (Liang and Li, 1991a). The lost ANS binding sites are believed to be the same sites for membrane binding. Our data indicate that liposomes do not bind to SI,.crystallin, or any other crystallins, which may indicate that binding requires membrane proteins (mainly MIP26). Furthermore, since only ANS binding is affected by a,-crystallin-membrane interaction, the binding site in membranes is most probably at the polar-apolar interface of MIP26. The requirement of MIP26 for a-crystallin binding has also been reported

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360 Wavelength

360

400

(nm)

FIG. 7. Near UV CD of calf lens membrane and a,-crystallin: (A) a,-crystallin in 0.05 M Tris buffer, pH 7.6 (0.50 mg ml-l); (B) membrane in Tris buffer (0.04 mg ml-l) ; (C) membrane in Tris buffer (0.09 mg ml-‘); and (D) membrane in trifluoroethanol (0.25 mg ml-‘). All near UV CD was measured with a cell of 10 mm path length.

CALF

LENS

CRYSTALLINS

723

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340

360

360

4c

(nm)

FIG. 8. Near UV CDchangedue to the interaction betweencalf lensmembraneand a,-crystallin : (A) beforemixing ; and (B) after mixing. Samplesare in 0.05 M Tris buffer, pH 7.6. Protein concentrations are 0.09 mg mill and 0.50 mg ml-l for membraneand a,-crystallin, respectively.

by Mulders et al. (1985). The other possibility is that ANS binding measurement may not be able to detect the a,-crystallin interaction with liposomes,since ANS fluorescence intensity in liposomesis rather weak, as compared to membranes (Fig. 1). The increase of rotational relaxation time of membranes upon binding of a,-crystallin indicated a slower lipid lateral diffusion rate. The effect appears small when compared with the age-related increase of relaxation time of cow membranes. The increase of CD due to the interaction between membranes and 01,crystallin is small, but is consistent with the results of polarization measurements. The increase of CD indicates that the interaction decreases the freedom of rotation of aromatic amino acid residues either in MIP26 or in ol-crystallin (Cantor and Timasheff, 1982). The observed binding of a,-crystallin to membranes is probably not a major event in the aging process, rather the changes in the bound a-crystallin or other components are more important. It would be logical to argue that aged proteins are more susceptible to this type of interaction, because of post-translational modifications and partial protein unfolding. The observation that old human lens membranes, especially cataractous and diabetic ones, have a large amount of bound crystallins, showed that the in vivo interaction is different from the in vitro one (Garner and Spector, 1980; Liang and Li, 1991b). The bound crystallins have been modified and exhibit the characteristic blue fluorescence of the brown pigments (Liang and Li, 1991b). The binding between crystallin and membranes apparently requires a prior action by an oxidative stress, which modifies either membrane components or crystallins or both. It would be of great interest to find out whether normal membranes and crystallins under a stress, either UV irradiation or advanced glycation, will bind together as observed in human lenses.

Acknowledgement Supportedby a grant from the National Institutes of Health (EYO5803).

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724 Liang, J. N., Rossi, M. and Andley. U. P. (1989). Fluorescence studies on the age-related changes in bovine and human lens membrane structure. Curr. Eye Res. 8. 293-8. Mulders, J. W. M.. Stokkermans. J., Leunissen, J. A. M., Benedetti, E. L., Bloemendal, H. and De Jong. W. W. (1985). Interaction of a-crystallin with lens plasma membranes. Eur. I. Biochem. 152, 721-8. Mulders, J. W. M., Wojcik. E.. Bloemendal, H. and De Jong. W. W. (1989). Loss of high-affinity membrane binding

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of bovine nuclear a-crystallin. Erp. Q/r Res. 49. 149-52. Russell, P., Robison. W. G. Jr and Kinoshita, J. H. ( 19X 1). A new method for rapid isolation of the intrinsic membrane proteins from lens. Exp. Eye Rrs. 32, 5 1 l-h. Spector, A. (1984). The search for a solution to senile cataracts. Invest. Ophthalmol. Vis. Sci. 25, 130-46. Weber. G. (1953). Rotational Brownian motion and polarization of the fluorescence of protein solutions. Adv. Protein Chem. 8, 415-59.