Crosslinking and blue-fluorescence of photo-oxidized calf-lens α-crystallin

&rp,. Eye Res. (1982) 34. 381-388

Crosslinking

and Blue-fluorescence of Photo-oxidized Calf-lens a-Crystallin EIJI

Department

FUJIMORI

of Fine Structure Research, Boston Biomedical Research Institute. 20 Staniford Street, Boston, MA 02114, U.S.A.

(Received 7 February

1981 and accepted 15 June 1981, New York)

In the u.v. (300 nm)-induced photo-oxidation of calf-lens a-crystallin. the polypeptides are crosslinked and blue fluorescent products areformed. The formation of 440/460 nm blue-fluorescent species and the crosslinking of polypeptides are both inhibited by the presence of glutathione. When U.V. (300 nm)-irradiated a-crystallin is further irradiated with U.V. (365 nm) light, both the 440/460 nm fluorescent products and the crosslinking are increased. [The primary photoproduct R’-formylkynurenine. which is produced by the first U.V. (300 nm) irradiation, can absorb U.V. (365 nm) light and is converted to its secondary photoproduct.] Thus the secondary photoproduct of N-formylkynurenine could be involved in both the 440/460 nm fluorescent chromophores and the crosslinking. Thermal stability studies suggest that these two 440/460 nm fluorescent rhromophores may be different forms of the same secondary photoproduct. Key words: photo-oxidation; fluorescence; crosslinking; photoproduct : lens protein ; a-crystallin: IV-formylkynurenine: tryptophan: u.v. light.

1. Introduction During aging and cataractogenesis, lens proteins undergo a number of modifications. Visible fluorescence and near U.V. absorption increase, particularly in senile nuclear cataract (Satoh, Bando and Nakajima, 1973; Augusteyn, 1975). The amount of protein aggregates also increases (Harding and Dilley, 1976). The insoluble aggregates are stabilized through disulfide bonds (Dische and Zil, 1951; Anderson and Spector, 1978) as well as other non-disulfide crosslinks (Buckingham, 1972; Kramps, Hoenders and Wollensak, 1978). The identity of the covalent, non-disulfide crosslinks has not yet been established. Some photochemically-induced chromophores could be associated with these crosslinks and new fluorescence (Lerman, 1980). Almost all fluorescent compounds isolated from human cataractous lens proteins are tryptophan derivatives. Anthranilic acid (Truscott, Faull and Augusteyn, 1977 : Garcia-Castineiras, Dillon and Spector, 1978a; Haard, Hoenders and Ketelaars, 1980) and kynurenine (Dilley and Pirie, 1974; Zigler, Sidbury. Yamanashi and Wolbarsht, 1976) have been found in the nuclear cataractous lens proteins. A small amount of bityrosine was also isolated (Garcia-Castineiras et al., 1978a, b), but its presence was questioned (McNamara and Augusteyn, 1980). Previous studies have also indicated that there is no appreciable decrease in the tryptophan content of cataractous lens proteins (Dilley and Pirie, 1974; Zigler et al., 1976; Truscott and Augusteyn, 1977). However, a decrease of up to 5 o/o (corresponding to the amount of isolated tryptophan derivatives) in the tryptophan content would not he det)ected by conventional methods (Truscott et al., 1977; Haard et al.. 1980). Blue-fluorescence is produced in the photo-oxidation of tryptophan and its residues inlensproteins(Zigman, 1971 ;Pirie, 1972; Borkman, 1977;Fujimori, 1979,1980,1981: Dillon and Spector, 1980). The loss of tryptophan is accompanied by the formation of the primary blue-fluorescent product K-formylkynurenine (FK) (Pirie, 1971: 0011.1835/82/030381+08

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Asquith and Kivett, 1971) as well as other photoproduc*t.s (Halt. ,Llilligan. Ihan from U.C. (300 nm)-irradiated S-carhoxymethyl-lysozyme was also accompanied by caovalentl? crosslinked polymers (Dilley, 1973). Similar crosslinking of lens crgstallins, associat,ed with blue-fluorescence, has been found in a dye-sensitized photo-oxidation system (Goosey, Zigler and Kinoshita, 1980). FK I$ ‘, f ormed in the dye-photosensitized oxidation of tryptophan residues in proteins (Tori and Galiazzo. 1971). Dillon and Spector (1980) have recently shown that, hot,h blue-fluorescence and photopolymerization of lens proteins also occur under anaerobic conditions apparently in a mechanism different from photo-oxidation (cf. Dillon, 1981). In the present study, we have confirmed the u.v. (300 nm)-induced crosslinking of calf-lens a-crystallin under aerobic rondit,ions and then discovered that when subsequently irradiated with U.V. (365 nm) light, U.V. (300 nm)-irradiated a-crystallin (partially crosslinked) undergoes further crosslinking. During these changes in the presence of oxygen, new blue-fluorescent, species are produced. The unique characteristics of these fluorescent products are also reported in this paper.

2. Materials

and Methods

Low-molecular-weight a-crystallin was isolated by gel-filtration with lJltroge1 AcA 34 (LKB) from water-soluble lens proteins of the calf-lens cortex. as previously described (Fujimori, 1978, 1980). Glutathione (GSH) and ascorbic acid were obtained from Sigma. The protein solutions (1 cc of @25 y0 cr-crystallin in 025 M-borate buffer, pH 8.2) in rectangular quartz cells (1 x 1 cm) were irradiated at room temperature under aerobic or anaerobic conditions. The de-aerated solutions were made on a vacuum line by freeze-pump-thaw cycles. Two different wavelengths for U.V. irradiation were chosen: U.V. (300 nm) light absorbed primarily by tryptophan but not by tyrosine or phenylalanine residues and U.V. (365 nm) light absorbed by the photo-oxidized product of tryptophan: FK, but not by tryptophan. For U.V. (300 nm) irradiation, a 150 W Xe arc lamp, which was connected to a Bausch and Lomb high intensity monochromator (band width: 300 + 10 nm). was used at 10 cm from the exit slit of the monochromator. For U.V. (365 nm) irradiat’ion, a high pressure mercury lamp with a near U.V. transmitting filter (Ultraviolet Products, Inc.) was employed at 10 cm from the filter (8-9 mW/cm2). Fluorescence, excitation and phosphorescence spectra were measured using Perkin-Elmer fluorescence spectrophotometers Model 65&lOS and Model MPF-44A (the latter Model for first- and second-derivative fluorescence spectra). For temperature changes, waterwascirculated through the cell holder with a Lauda K4R temperature-regulated circulator. Carefully measured aliquots of non-irradiated-. U.V. (300 nm)-irradiated- and subsequently U.V. (365 nm)-irradiated-ol-crystallin solutions were mixed with 0.06 iv-Tris buffer (pH 6?3), containing 2 y0 sodium dodecyl sulfate (SDS). 5 O0mercaptoethanol and 10 9; glycerol, heated for a few minutes in boiling water and applied to SDS (0.1 9,0)-acrylamide (13 9;) slab gels for electrophoresis. The gels were fixed in 5 :I trichloroacetic acid. stained with Coomassie blue and destained prior to scanning with a Joyce Loebl densitometer.

3. Results and Discussion Crosslinking Subunit profiles of three protein samples, non-irradiated, U.V. (300 nm)-irradiated and subsequently U.V. (365 nm)-irradiated a-crystallin, were studied by SDSpolyacrylamide gel-electrophoresis (Fig. 1). As shown in Fig. 1, dotted curve 1. the

YHOTO-OXIDIZED

E

0

0

C

2-5

3X3

a-CRYSTALLIK

5 Mlgrallon

08

aA

75 (ml)

Frc:. 1. (kl elrctrophoretic patterns of non-irradkrd (dotted curve i), u.v. (3~0 nm)-irradiated (2 hr) (solid ~urvc 2) and subsequently U.V. (365 nm)-irradiated (2 hr) a-crystallin (curve 3). nA and aB arr a-crvstallin subunits, C subunit dimers. D tetramers and E higher polymers. Standard samples are bovine swum a!bumin (67000 dalton) and sldolase (40000 d&on).

non-irradiated control had two bands consisting of subunits, aA (19832 dalton) and aB (20070 dalton) (van der Ouderaa, de Jong and Bloemendal, 1973; van der Ouderaa, de ,Jong, Hilderink and Bloemendal, 1974). U.V. (300 nm) irradiation (2 hr in air) of a-cryst.allin (Fig. 1, solid curve 2) resulted in dimers (band C)> trimers, tetramers (band D) and higher polymers (band E) of the a-crystallin subunits. Higher aggregates failed t’o enter the gel. Since the irradiated sample was treated with mercaptoethanol prior to gel-electrophoresis, the results are consistent with formation of non-disulfide, covalent crosslinks between subunits. The crosslinking progressed with time. With a short period of irradiation (l/2 hr), the major crosslinked species was the dimer. The amount of dimers was highest after irradiation for 1 hr and then gradually decreased with increased amounts of trimers and tetramers. With longer periods of irradiation (510 hr), higher aggregates increased with the complete disappearance of monomeric subunits. The addition of 0.01 M-GSH prior to U.V. (300 nm) irradiation inhibited t.he formation of these polymers. ‘These changes were similar to crosslinking of lens crystallins observed in a photodynamic system (Goosey et al., 1980). When the U.V. (300 nm)-irradiated (2 hr) cr-crystallin was subsequently irradiated for 2 hr with U.V. (365 nm) light, the amount of higher multimers (band D) and aggregates (band E) increased (about 2-fold) compared to that of dimers (about 1.2-fold increase in band C), indicating further crosslinking (Fig. 1, curve 3). With a short’ u.v. (365 nm) irradiation (5 min), dimers immediately increased. When the irradiation was continued beyond 5 min, the amount of monomers and dimers gradually decreased with the increase of higher polymers. It should be noted that little polymers

These u.v.-induced structural changes were accompanied by Huoreacence thangr. It was previously reported that when a-crystallin is irradiat’ed in air with U.V. (300 tm) light, at least four different (two type A and two type H) blue-fluorescent spe&s. Ii&d in Table I. are produced (Fujimori. 1980). Two t,ype A blue-fluorescence bands at, 400 and 435 nm (excited at 350 nm) are produced mainly by t,he primary phot,o-oxidizrcl product FK and differ from each other in the effect of pH (Yirie. 1972 : l!‘ujimori, 1980). GSH (001 M) inhibited the formation of the 400 nm fluorescence but not apparrnt)ly that of the 435 nm fluorescence. In agreement with Dillon and Spector (1980). we have also observed that in the absence of oxygen, another fluorescence at 133 nm (excited at 350 nm), similar to the 435 nm fluorescence but distinct, from the FK fluorescence. is produced. This fluorescence was greatly enhanced in the presence of GSH (Table I). Some photo-adduct with GSH may be responsible for this enhancement. suggesting the possible interaction between tryptophan residues and protein SH-groups in anaerobic protein solutions. TABLE

Blue-$uorescence

I

of U.V. (300 nm)-irradiated a-crystallin ruith and without presence and absence of oxygen

__-~ Fluorescence Type h (excited at 350 nm) 400 nm 435 nm Type B (excited at 400 nm) 440 nm 460 nm

oxygen

No

GASH in the

oxygen

No GAH

GSH

Ko GSH

+* +

++

+1 ++s

+t +t

-

-

GSH

++++ -

* This fluorescence becomes more intense in alkaline solution. t Type B fluorescence is also produced by u.v. (365 nm) irradiation from type A fluorescence. While this conversion is also inhibited by GSH, ascorbic acid selectively inhibits the formation of 440 nm fluorescence but not that of 460 nm fluorescence. $ This fluorescence at 395 nm (preferably excited at 330 nm) does not respond to pH change. 8 This fluorescence has a peak at 433 nm rather than at 435 nm and does not respond to pH change. It is not converted by U.V. (365 nm) irradiation to type B fluorescence.

As also shown in Table 1, two other type B blue-fluorescence bands at 440 and 460 nm (excited at 400 nm) were produced in U.V. (300 nm)-irradiated a-crystallin only in the presence of oxygen, but not in the presence of GSH or in the absence of oxygen. These 440/460 nm fluorescent chromophores are stabilized within the protein framework and dependent upon the protein conformation. Upon addition of protein denaturants (5 M-guanidine-HCl or 1 y0 SDS) after irradiation, these fluorescent bands immediately disappeared. If added prior to irradiation, they were not produced. These type B fluorescent species were also transient photoproducts and converted to other forms upon prolonged irradiation. Two type B fluorescence bands were so closely overlapping that U.V. (300 nm)-

PHOTO-OXIDIZED

450 Wavelength

z-C’RYSTALLIS

500 bwd

3X5

5150

FIG. 2. Normal (---), first-derivative (----) and second-derivative (excited at 400 nm) of U.V. (300 nm)-irradiated (1 hr) a-crystallin.

(-

- --) fluorescence spectra

irradiated (1 hr) a-crystallin, when excited at 400 nm, exhibited an apparently single-peaked fluorescence spectrum around 450 nm (Fig. 2, solid curve). The presence of t,wo distinct 440 and 460 nm fluorescence bands was discovered by the following observations and studies. (a) It was first detected by a red-shift of the fluorescence peak from 440 nm to about 450 nm during the U.V. (300 nm) irradiation of a-crystallin. (b) The second indication of their presence came from the quenching experiments with GSH and ascorbic acid. As the concentration of these anti-oxidants was increased from 601 to 605 M, the progressive quenching of fluorescence was accompanied by a peak shift from 450 to 460 nm. This red-shift indicated a selective quenching, in which the 440 nm fluorescence was more efficiently quenched than the 460 nm fluorescence. (c) Our study of second-derivative fluorescence spectrum gave further support. Firstand second-derivative fluorescence spectra (excited at 400 nm) of U.V. (300 nm)irradiated (1 hr) or-crystallin are shown in Fig. 2. The apparently single peak in the normal fluorescence spectrum (Fig. 2, solid curve) was resolved into two negative peaks having minima at 438 and 463 nm in the second-derivat,ive spectrum (Fig. 2, dash-dot curve). These two correspond to the 440 and 460 nm fluorescent bands. (d) When the excitation wavelength was changed from 400 nm to 420 nm, the 440 nm fluorescence band became a peak, while the 460 nm fluorescence band appeared as a shoulder. This change indicates that these two fluorescent species have different excitation spectra, as shown below in their thermal stability. (e) Two fluorescence bands were also temperature-dependent. The fluorescence and excitation spectra of U.V. (300 nm)-irradiated (1 hr) a-crystallin at liquid nitrogen temperature (1: 1 pH &2 buffer-ethylene glycol) are shown in Fig. 3, curves C and D, respectively. At this low temperature, two excitation peaks for the composite

80 -

Wavelength (nm) FIG. 3. Effect of temperature upon the fluorescence (excited at 400 nm) and excitation spectra (for 450 nm fluorescence) of xv. (300 nm)-irradiated (1 hr) a-crystallin. (A) fluorescence and (B) excitation spectral changes (2 -+ 35 --t 55 + 70 + 3O’C’); (C’) fluorescence and (D) excitation spectra at 77OK (1 : I pH 8.2 buffer-ethylene glyrol).

fluorescence at 450 nm were at 400 and 415 nm with a small shoulder around 420 nm (due to the secondary photoproduct) in addition to a peak at 360 nm (due to the primary photoproduct). At PC, the excitation peak at 422 nm became evident (Fig. 3B, 2%). This excitation spectrum is the same as that at room temperature, previously reported (Fujimori, 1979). Upon heating from 2 to 70°C, the 440 nm fluorescence decreased and was transformed to the 460 nm fluorescence at 35-55 OC (Fig. 3A, 2 -+ 70%). During this transition, another new excitation peak appeared and increased at 432 nm (Fig. 3B, 35 + %Y’C). After cooling from 70 to 3O”C, the new excitation peak at 432 nm (Fig. 3B, 3O”C), as well as the 460 nm fluorescence (Fig. 3A, 3O”C), increased. The above changes indicate that the 460 nm fluorescence, stabilized at higher temperature, has the 432 nm excitation peak different from the 422 nm peak for the 440 nm fluorescence. The thermal conversion from the latter fluorescence to the former suggests that two type B fluorescent chromophores are two different forms of the same photoproduct.

PHOTO-OXIDIZED Relation

between

photocrosslinking

Z-C’RYSTALLIK

3X7

and type R &ue;fEuorescence

The U.V. (300 nm)-induced formation of type B blue-fluorescence only in t,hr presence of oxygen (Table I) is consistent with their source related to the photo-oxidized product FK. FK, produced by U.V. (300 nm) irradiation, could be further excited by subsequent U.V. (300 nm) absorption or energy transfer from excited tryptophan residues to form the secondary photoproduct of FK. Our previous st’udy also demonstrated that type A fluorescence is converted by U.V. (365 nm) irradiation. absorbed by FK, to type B fluorescence (Fujimori. 1979). In the present study, we haveshownthatu.~. (300 nm)-irradiatedir-crystalliniscrosslinkedandthatsubsequent u.v. (365 nm) irradiation of partially crosslinked U.V. (300 nm)-irradiated ol-crystallin increases crosslinking. These results support a relationship between photocrosslinking and type B fluorescence. Furthermore, GSH inhibited both the formation of type H fluorescence and the crosslinking, suggesting that type B blue fluoresence is associated with the crosslinked secondary photoproduct of FK. This participation of FK in photo-oxidized cz-crystallin has been verified by OUI recent’ studies on the phot.oreaction, fluorescence and phosphorescence of FK produced chemically in the ozonization of tryptophan residues in ol-crystallin (Fujimori, 1982). FK and its photoproduct in photo-oxidized cr-crystallinexhibited the same fluorescence and phosphorescence spectra as in ozone-oxidized cl-crystallin. FK in ozone-oxidized a-crystallin also underwent the same photoreactions as found in photo-oxidized a-crystallin, in producing both type B blue fluorescence and increased crosslinking. From this and other findings, we have suggested t’hat FK could form a crosslinkage with an adjacent lysyl amino group (Fujimori, 1981). The exart mechanism offorming (*rosslinks awaits further investigation. ACKNOWLEDGMENT8 I wish to thank Ms Isahella Wang for her valuable technical assistance. This study was supported by research grant EY 01810 from the National Institutes of Health. REFERENCES Anderson. E. 1. and Spector, A. (1978). The state of sulfhydryl groups in normal and cataractous human lens proteins. I. Nuclear region. Exp. Eye Res. 26, 407-17. Asquith, R. S. and Rivett, D. E. (1971). Studies on the photo-oxidation of tryptophan. Biochim. Biophys. Acta 252, 11 l-16. Augusteyn. R. C. (1975). Distribution of fluorescence in the human cataractous lens. Ophthalmic Res. 7. 217-24. Borkman, R. F. (1977). Ultraviolet action spectrum for tryptophan destruction in aqueous solution. Photochem. Photobiol. 26, 163-66. Bucakingham, R. H. (1972). The behaviour of reduced proteins from normal and cataractous lenses in highly dissociating media: crosslinked protein in cataractous lenses. Exp,. Eye Res. 14. 123-9. Bmskingham. R. H. and Pirie, A. (1972). The effect of light on lens proteins in vitro. E.ry. Eye Res. 14. 297-9. Dilley. K. J. (1973). Loss of tryptophan associated with photo-polymerization and yellowing of proteins exposed to light over 300 nm. Biochem. J. 133, 821-6. Dillry. K. lJ. and Pirie. A. (1974). Changes to the proteins of the human lens nucleus in cataract. Exp. Eye Res. 19, 59-72. Dillon, cJ. (1981). The anaerobic photolysis oft ryptophan containing peptides-II. Photochrrrc. Photobid. 33, 137-42.

38X

c:. FI~.IIMOKI

Dillon. J. and Spect,or. A. (1980). A wmparisotr of arl~ol)i~~and anerol~i~ jblloto!,vhi:, IIt I~~II:, protein. &I). E!/:!/,KPS. 31. S-9. Dische, Z. and Zil, H. (1951). Studies on the osiclation of cysteine t,o c.ydintb in Ivns f:rottsltlh during cataract formation. Am. ./. Ophthalmol. 34. i(W 13. Fujimori. E. (1978). Blue-fluorescence of bovine lens proteins. O~hthnlmir f2r.s. 10. L’S!! 65. Fujimori. E. (1979). New photo-c,o;lvrrtit~le rractions of t)lrlr~tlllc,rrsc.rnt a-c,r\-stallin. f’ho/r,them. Photobid. 29. 62.5-8. Fujimori. E. (1980). pH-Deprndent phot,orrac+ions of blur-flrlort~s~rnt ~~itlt’~-c~r.~s~allit~.ti.r/~. Eye lies. 30. 649-57. Fujimori. E. (1981). Blue-fluorescence and crosslinking of fJhl)to~c)xitiizetl pr~~trins. FEHS Left. 135. %7-60. Fujimori. E. (In press). (‘rosslinkingand photorc,action ofozone-oxitlizrd calf-lensa-lr?,st~~ilin. Inwst. Ophthalmol. C’is. Sci. Garcia-Castineiraa. S.. Dillon. J. and Sprct’or, A. (197%). Nor]-trypt,ophan Huorescrtlc~e associated with human lens protein: apparent complexit,y and isolation ofbit,yrosine ant1 anthmnilic acid. Exp. Eyr Res. 26. 161-76. in Garcia-(‘astineiras, S.. Dillon. .J. and Spector, A. (19781)). 1)rtection of bityrosinr cataractous human lens protein. Scirnce 199, 897-9. Goosey, J. D.. Zigler. .I. S.. *Jr. Kinoshita. J. H. (1986). (Iross-linking of lens crystallins in a photodynamic system: a process mediated by singlet oxygen. Sciracp 208. lT?XHO. Haard, P. M. M. van. Hoendrrs. H. J. and Ketrlaars, H. (‘. ,J. (1980). Human lens nuc:lear cataract: hydrogen peroxide action and anthranilic acaid association with lens f)rot,tJins. Ophthalmic Res. 12, S%S. Harding, J. J. and Dilley. K. J. (1976). Structural prot,eins of the mammalian lens: a revi,, with emphasis on changes in development, aging and cataract. &p, Eyye12es.22. l-75. Holt, L. A.. Milligan. B., Rivett. D. E. and Stewart. F. H. C’. (1977). The phototlecorllpositic,n of tryptophan peptides. Biochim. Biophys. dcta 499. 131-8. Jori. G. and Galiazzo. G. (1971). Proflavine-sensitized selective photooxidation of t,r,vptophgl residues in papain. Photorhem. Photohid. 14. 607-19. Kramps. J. A., Hoendrrs. H. J. and Wollrnsa,k, J. (1978). Increase of non-disulphicle crosslinks during progress of nuclear cataract. .V.q). lCye )Ips. 27, 731--S. Lerman, S. (1980). Human ultraviolet radiation cataracts. Ophthulmic Res. 12. 30:%--11. McNamara. M. K. and Augusteyn, R. (1. (1980). 3,3’-Dityrosine in the proteins of smile nuclear cataracts. Exp. Eye Res. 30, 319-21. Pirie. A. (1971). Formation of N-formylkynurenine in proteins from lenses and other sources by exposure to sunlight. Hiochem. .J. 125. ILOB--8. Pirie. A. (1971). Fluorescence of S-formylkvnurenirIr and of proteins exposed 10 sunlight. Biochem. J. 128, 1365-7. Satoh. K., Bando. &MM. and Nakajima. ,A. (1973). Fluorescence in human lens. trl.rl~. &,yp Res. 16, 167-72. Tassin. J. D. and Borkman. R. F. (1980). The photolysis rates of ~omr d- and triprptidrs of tryptophan. Photochem. Photobiol. 32. 577~-85. Truscott, R. J. I$‘, and Augusteyn. It. C’. (1977). Oxidative changes in human lens proteins during senile nuclear cataract formation. Biochim. Biophys. .3ctcf 492. 13%52. Truscott. R. J. W.. Faull, K. and Augusteyn, R. c’. (1977). The identification of anthranilic acid in proteolytic digests of cataractous lens proteins. Ophthalmic Rrs. 9. %69-H. van der Ouderaa. F. ,J.. deJong, W. W. and Bloemendal. H. (1973). The amino acid sequrnc.r of the sA, chain of bovine a-crystallin. Eur. J. Biochem. 39, X17-Z. van der Ouderaa. F. J., de ,Jong, W. W.. Hilderink? A. and Bloemendal, H. (1974). The amino-acid sequence of the aB, chain of bovine a-crystallin. Eur. J. Biochem. 49, 157-68. Zigler. J. S.. Sidbury, .J. B.. Yamanashi. B. S. and Wolbarsht, M. (1976). Studies on brunescent cataracts. I. Antilysis of free and protein-bound amino acids. Ophthalmic Rr’s. 8, 37%87. Zigman, S. (1971). Eye lens color: fbrmation and function. Science 171, 807~~9.