Exp. Eye Res. (1996) 63, 407–410

Binding Capacity of α-Crystallin to Bovine Lens Lipids

DOUGLAS BORCHMAN* DAXIN TANG

Department of Ophthalmology and Visual Sciences, University of Louisville, Louisville, KY 40202, U.S.A.

(Received Rochester 11 December 1995 and accepted in revised form 29 March 1996)

Three experiments were performed to determine the α-crystallin binding capacity of bovine lens lipidvesicles. In one experiment lipid was kept constant (2±5 mg ml−") and the α-crystallin concentration waschanged (0±5 to 3±0 mg ml−"). In another experiment, α-crystallin was kept constant (1 mg ml−") and theconcentration of lipid was varied (0±25 to 3 mg ml−"). We calculated the binding capacity of the lipid tobe 0±33³0±05 (..) mg α-crystallin (mg lens lipid)−". This was confirmed by changes in the anisotropyand fluorescent intensity of a probe that partitions at the headgroup region of the lipid bilayer. Near0±33 mg α-crystallin (mg lens lipid)−" the fluorescence intensity and anisotropy of the probe increases andplateaus which indicates that concomitant with α-crystallin binding, water is excluded from the headgroup region of the bilayer and the headgroup region becomes less mobile. It is possible that α-crystallinbinding could protect and stabilize the lipid bilayer and decrease membrane permeability.

# 1996 Academic Press LimitedKey words : lens ; α-crystallin ; lipids ; binding.

1. Introduction

Human lens alpha crystallin concentration may be as

high as 40% of the total protein and is the major

extrinsic protein of lens membranes (Bloemendal et al.,

1972; Chandrasekher and Cenedella, 1995; Fleschner

and Cenedella, 1992). When solubilized, α-crystallin

can function as a molecular chaperone that inhibits

the heat-induced aggregation of other crystallins and

proteins (Horwitz, 1992). Alpha-crystallin–lens mem-

brane binding has been the focus of a number of recent

studies (Cenedella and Chandrasekher, 1993; Ifeani

and Takemoto, 1989, 1990a, 1990b, 1991a, 1991b;

Mulders et al., 1985, 1989; Liang and Li, 1992;

Ramaekers, Versteegen and Bloemendal, 1980; Zhang

and Augusteyn, 1994). Mulders et al. (1985) showed

that association of α-crystallin with lens membranes

was temperature, pH and time dependent. The

interaction of α-crystallin with bovine lens membranes

is age dependent (Ifeani and Takemoto, 1989) and

lipid alone may be all that is necessary for α-

crystallin–membrane binding since α-crystallin may

bind to pure phospholipid vesicles (Ifeani and

Takemoto, 1991a). When bound α-crystallin

immobiles lipids (Liang and Li, 1992), but does not

alter the lipid hydrocarbon chain structure (Sato et al.,

1996). In semiquantitative experiments it has been

demonstrated that α-crystallin may bind to lens lipids

at a capacity five times higher than that of vesicles

made from phosphatidylcholine (Sato et al., 1996).

The purpose of this study was to determine the binding

capacity of bovine α-crystallin to bovine lens lipids.

* For correspondence at : Department of Ophthalmology andVisual Sciences, 301 E. Muhammad Ali Blvd., Louisville, KY 40202,U.S.A.

2. Materials and Methods

All reagents and bovine α-crystallin were purchased

from the Sigma Chemical Co. (St. Louis, MO, U.S.A.)

except where indicated. Bovine lens lipids were

extracted from eyes obtained fresh from a slaugh-

terhouse.

N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexa-

decanoyl-sn-glycero-3phosphoethanolamine, triethyl-

ammonium salt (NBD-PE) was purchased from

Molecular Probes (Eugene, OR, U.S.A.). A monophasic

methanolic extraction followed by a hexane}isopropanol purification was used to extract lipid from

60 bovine lenses (Sato et al., 1996).

Three experiments were performed to determine

the α-crystallin binding capacity of bovine lens

lipid vesicles. Binding was determined at 36°C in a

5m [4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic

acid], buffer, pH¯7.5, (Hepes), bubbled with argon

gas for 10 min to remove oxygen.

Experiment 1

Lipid was kept constant (2±5 mg ml−") and the α-

crystallin concentration was changed (0±5 to 3±0 mg

ml−").

Experiment 2

Alpha-crystallin was kept constant (1 mg ml−") and

the concentration of lipid was varied (0±25 to 3 mg

ml−").

The following protocol was followed for experiments

1 and 2: stock bovine lipid in methanol (1 mg ml−")

was added to 1 ml capacity ultracentrifuge tubes. The

samples were frozen in liquid nitrogen and freeze dried

0014–4835}96}100407­04 $25.00}0 # 1996 Academic Press Limited

408 D. BORCHMAN AND D. TANG

in a lyophilizer for 20 min to remove methanol. Hepes

buffer then stock α-crystallin (10 mg ml−") was then

added. The centrifuge tubes were sealed in an

atmosphere of argon mixed and sonicated in a bath

sonicator for 15 min then allowed to equilibrate for

12 h at 36°C with gentle shaking. To remove lipid and

bound α-crystallin from solution, the samples were

centrifuged at 170000 g for 1±5 h at 36°C. Alpha

crystallin in the supernatant was determined by

measuring the optical density at 280 nm. The ex-

tinction coefficient at 280 nm for the α-crystallin was

calculated to be 0±645 O.D. mg ml−"³0±015 (...)

n¯28. The absorbance of the supernatant was

corrected for a small absorbance due to chromagens

from the lipid which had an extinction coefficient of

0±125 O.D. mg−" ml−"³0±002 (...), n¯17. Protein

concentration was also quantified using the Peterson

assay (Peterson, 1977) with identical results.

Controls

To test whether sonication influenced binding,

0±5 mg ml−" of α-crystallin and lipid were prepared as

described above, but in one set of samples the lipid was

sonicated in the absence of α-crystallin, and then

mixed with the α-crystallin, equilibrated and the

binding determined.

To determine if α-crystallin became insolubilized

during the prolonged incubation period, nine 1 mg

ml−" α-crystallin solutions were prepared. Three

samples were equilibrated at 36°C for 12 hr, three

were equilibrated at 25°C and three samples were

prepared within 1 hr of protein measurement.

Experiment 3

The fluorophore, NBD-PE, that partitions near the

phospholipid head group region was mixed with

bovine lipid to indirectly measure the maximum

binding capacity of α-crystallin to bovine lipid. When

α-crystallin binds to the lipid membrane, the ani-

sotropy (1}wobble) of the probe and the fluorescence

would be expected to increase due to the immobility of

the probe and the exclusion of water, respectively. The

fluorescent probe, NBD-PE, was mixed with bovine

lipid in chloroform at a weight ratio between 0±005

and 0±02 to 1 lipid. Samples were prepared as described

in the protocol for Experiment 2 except sonication was

not used and a buffer solution consisting of 10 m

Tris(hydroxymethyl)aminomethane hydrochloride

(Tris–HCl), pH 7.5, 0±1 m ethylenediamine-

tetraacetic acid (EDTA) and 50 m KCl was used to

eliminate the possibility of interference due to calcium–

fluorophore interactions.

Fluorescence Measurements

Anisotropy and intensity measurements were per-

formed on an ISS PC1 photon counting spectro-

fluorometer (Champagne, IL, U.S.A.) with a

polarization accessory unit. The excitation and

emission wavelengths used were 460 and 540 nm,

respectively, for the NBD-PE probe to detect en-

vironmental and structural changes near the bilayer

surface. Fluorescence anisotropy, r, was calculated by

r¯ (Ill®gIv)}(I

ll­2gI

l) in which g¯ Iv}I

ll(1)

The fluorescence intensity was measured as the ratio

of the sample detector signal and the reference detector

signal.

3. Results

The plot of α-crystallin bound per lipid verses 1}α-

crystallin free (Fig. 1) was used to estimate the

maximum binding capacity of α-crystallin to bovine

lipid by extrapolating the linear curve through our

data to the y axis. The maximal binding capacity of

bovine lens lipid to α-crystallin was estimated from the

data in Fig. 1 to be 0±34³0±4 (..) mg α-crystallin

(mg bovine lens lipid)−".

The binding capacity of α-crystallin to bovine lens

lipids was determined by varying the concentration of

bovine lens lipid and by keeping the concentration of

α-crystallin constant and above the saturation con-

centration. From the data obtained using this meth-

odology, we calculated the binding capacity of the lipid

to be 0±33³0±05 (..) mg α-crystallin (mg lens

lipid)−", n¯5. The capacity calculated by both

methods was almost identical.

Samples prepared with and without sonication gave

similar binding values (³6%) which indicates

sonication did not influence the binding capacity of α-

crystallin to the bovine lipids. The sonication step was

eliminated in the fluorescence measurement protocol.

3

0.4

1/α-Crystallin free (ml mg–1)

α-C

ryst

alli

n b

oun

d/li

pid

(g/g

)

0.3

0.2

0.1

1 20

F. 1. Binding of bovine lens α-crystallin to bovine lipids,36°C. The concentration of lipid was kept constant.

α-CRYSTALLIN–LIPID BINDING 409

1.0

0.10

Alpha-crystallin/lens lipid (w/w)

An

isot

ropy

0.09

0.08

0.07

0.2 0.40.0

(B)

0.06

0.050.80.6

1.0

1.20

Flu

ores

cen

ce

1.16

1.12

1.08

0.2 0.40.0

(A)

1.04

1.000.80.6

1.18

1.14

1.10

1.06

1.02

F. 2. Change in the (A) fluorescence intensity and(B) anisotropy of the head group lipid probe, NBD-PE, withα-crystallin binding. An increase in fluorescence indicatesexclusion of water. An increase in anisotropy indicates adecrease in the mobility of the probe.

Less than 1% of the α-crystallin became insoluble

after incubation in an atmosphere of argon after a

12 hr incubation at either 21 or 36°C. An incubation

period of over 3 hr at 36°C was beneficial for increasing

the solubility of the α-crystallin by about 50%, even

after sonication.

Figure 2(A) and (B) shows the change in NBD-PE

probe fluorescence and anisotropy as the ratio of α-

crystallin to lens lipid was increased from 0 to 1±0(w}w), respectively. Note the plateau that occurs

above the α-crystallin to lipid weight ratio of 0±3 to 0±4which indicates that the maximum binding occurs at

this weight ratio. The increase in fluorescence with α-

crystallin binding [Fig. 2(A)] indicates that in the

process of binding, water is excluded from the head

group region of the lipid bilayer. The increase in

anisotropy with α-crystallin binding [Fig. 2(B)]

indicates that the probe becomes less mobile.

4. Discussion

We found that bovine lens lipids have a high

capacity to bind α-crystallin, 0±33 mg α-crystallin (mg

lens lipid)−". Considering that the human lens contains

at most, only 1±2 mg of lipid, from our binding

capacity data from bovine lens material, if we may

speculate about the binding in the human lens, we

calculate that only a small proportion, 0±4 mg, of the

total 32 mg of human lens α-crystallin would be

directly bound to the membrane lipid. It has been

suggested that HMW aggregates of crystallins

associated with human lens membranes could perhaps

assemble on the membrane after binding

(Chandrasekher and Cenedella, 1995). Thus a large

amount of α-crystallin could be indirectly bound to the

membrane via the small amount of α-crystallin bound

directly to the membrane lipids.

This study supports the finding using non-lens

lipids, that intrinsic proteins may not be necessary for

α-crystallin binding (Ifeanyi and Takemoto, 1991a).

The α-crystallin–lipid binding capacity reported in this

paper is about five times higher than previously

reported (Ifeanyi and Takemoto, 1991a) possibly

because we used a much higher concentration of α-

crystallin, 0±5–3 mg α-crystallin ml−" compared to

0±36 mg α-crystallin ml−" used in previous studies and

the amount of lipid used in this study was also often

higher, 2±5 mg ml−", compared to 0±784 mg phospho-

lipid ml−" used in previous studies and}or purified

bovine lipid membranes devoid of protein were used in

this study.

Perhaps more important than the actual amount of

α-crystallin binding, is the possibility that α-crystallin

could stabilize and protect the lipid bilayer. Our

fluorescent probe data [Fig. 2(A)] indicate that upon

bind of α-crystallin, water is excluded from the lipid

headgroup region of the bilayer which could protect

the lipid from hydrophilic oxidants such as H#O#

and

oxidized ascorbate. Exclusion of water and

immobilization of the lipid head groups with α-

crystallin binding would also be expected to decrease

the permeability of the bilayer to hydrophilic cations

and water. This hypothesis is currently being tested

using large unilamellar vesicles. Head group inter-

actions appear to be more important to α-crystallin

binding since binding does not affect the hydrocarbon

structure of the membrane at 36°C (Sato et al., 1996).

At 36°C, lens lipids are near the center of their order

to disorder transition, the most sensitive point for

changes in lipid structure, (Borchman et al., 1993).

Perhaps the role of α-crystallin-lipid binding is to

stabilize the membrane lipid structure as it does to lens

proteins. Further studies are needed to determine

factors influencing α-crystallin–lipid binding, the affect

of α-crystallin on lipid structural stability and the

influence of intrinsic proteins on the binding capacity

and binding constant.

Acknowledgements

Supported by Public Health Service research grantEYO7975 (Bethesda, MD, U.S.A.) and the Kentucky LionsEye Foundation (Louisville, KY, U.S.A.), and an unrestricted

410 D. BORCHMAN AND D. TANG

grant from Research to Prevent Blindness, Inc. Chris Johnshould be acknowledged for his technical help.

References

Borchman, D., Lamba, O. P. and Yappert, M. C. (1993).Structural characterization of human lens membraneclear and cataractous lipid. Exp. Eye Res. 57, 199–208.

Bloemendal, H., Zweers, A., Vermorken, F., Dunia, I. andBenedetti, E. L. (1972). The plasma membranes of eyelens fibers. Biochemical and structural characterization.Cell Diff. 35, 61–7.

Fleschner, C. R. and Cenedella, R. J. (1992). Examination of‘native ’ plasma membrane fractions and its associatedcrystallins. Curr. Eye Res. 11, 739–52.

Cenedella, R. J. and Chandrasekher, G. (1993). Highcapacity binding of alpha crystallins to various bovinelens membrane preparations. Curr. Eye Res. 12,1025–38.

Chandrasekher, G. and Cenedella, R. J. (1995). Proteinassociated with human lens ‘native ’ membrane duringaging and cataract formation. Exp. Eye Res. 60,707–17.

Horwitz, J. (1992). α-Crystallin can function as a molecularchaperone. Proc. Natl. Acad. Sci. U.S.A. 89, 10449–53.

Ifeanyi, F. and Takemoto, L. (1989). Differential binding ofα-crystallins to bovine lens membrane. Exp. Eye Res.49, 143–7.

Ifeanyi, F. and Takemoto, L. (1990a). Specificity of α-crystallin binding to the lens membranes. Curr. Eye Res.9, 259–65.

Ifeanyi, F. and Takemoto, L. (1990b). α-Crystallin fromhuman cataractous versus normal lenses : change inbinding to lens membrane. Exp. Eye Res. 50, 113–6.

Ifeanyi, F. and Takemoto, L. (1991a). Interaction of lens α-crystallin with lipid vesicles. Exp. Eye Res. 53, 535–8.

Ifeanyi, F. and Takemoto, L. (1991b). Involvement of the N-terminal region in α-crystallin–lens membrane rec-ognition. Exp. Eye Res. 52, 305–8.

Liang, J. J. N. and Li, X. (1992). Spectroscopic studies on theinteraction of calf lens membranes with α-crystallins.Exp. Eye Res. 54, 719–24.

Mulders, J. W. M., Stokkermans, J., Leunissen, J. A. M.,Benedetti, E. L., Bloemendal, H. and De Jong, W. W.(1985). Interaction of α-crystallin with plasma mem-branes. Eur. J. Biochem. 152, 721–8.

Mulders, J. W. M., Wojcik, E., Bloemendal, H. and De Jong,W. W. (1989). Loss of high-affinity binding of bovinenuclear α-crystallin. Exp. Eye Res. 49, 149–52.

Peterson, G. L. (1977). A simplification of the protein assaymethod of Lowry et al. which is more generallyapplicable. Anal. Biochem. 83, 346–57.

Ramaekers, F. C. S., Selten-Versteegen, A. E. andBloemendal, H. (1980). Interaction of newlysynthesized α-crystallin with isolated lens plasmamembranes. Biochim. Biophys. Acta 596, 57–63.

Sato, H., Borchman, D., Ozaki, Y., Lamba, O. P., Byrdwell,W. C., Yappert, M. C. and Paterson, C. A. (1996).Lipid–protein interactions in human and bovine lensmembranes by Fourier transform Raman and infraredspectroscopes. Exp. Eye Res. 62, 47–53.

Zhang, W. Z. and Augusteyn, R. C. (1994). On the in-teraction of α-crystallin with membranes. Curr. Eye Res.13, 225–30.