Ophthalmie Res 1990;22:183-186
© 1990 S. Karger AG, Basel 0030-3747/90/0223-0183S2.75/0
Effects of Ultraviolet B Irradiation on Lenticular Riboflavin Metabolism and High-Molecular-Weight-Protein Aggregation Hiroko Hiranoa , Yoshitaka Obarab, Kumiko Katakuraa , Shigeru Onoa ■'Department o f Biochemistry, School o f Medicine, Iwate Medical University, Morioka; bDepartment o f Ophthalmology, Koshigaya Hospital, School o f Medicine, Dokkyo University, Koshigaya, Japan
Key Words. UVB irradiation • Riboflavin • Ester forms of B2 • Riboflavin-binding capacity • HMW protein aggregation • Lens Abstract. We investigated the effects of ultraviolet B (UVB) irradiation on the formation of ester forms of riboflavin and of high-molecular-weight (HMW) protein aggregates and on lenticular riboflavin-binding capacity (LRBC). Esterification of riboflavin decreased as the duration of UV irradiation increased, suggesting the irradiation-induced dénaturation of the apoenzyme of synthetases of ester forms of riboflavin. UVB irradiation of lens homogenate supplemented with riboflavin increased LRBC and the formation of HMW protein aggre gates, while y-crystallin was decreased. These results are consistent with those of our earlier studies in which we obtained data suggesting that, upon exposure of rat lens homogenate to fluorescent light, photosensitized riboflavin may bring about cross-linking of lens protein. Our data demonstrate that the photosensitivity of lenticular riboflavin is increased by longer periods of UV irradiation.
Work in our laboratory has documented that the lens is one of the organs in which riboflavin is metabolized [1-3] and that len ticular riboflavin contributes to the forma tion of coenzyme and acts as a photosensitiz er. In the present study, we investigated the effect of ultraviolet B (UVB) irradiation on the formation of ester forms of riboflavin and on lenticular riboflavin-binding capacity (LRBC). We also examined high-molecular-
weight (HMW) protein aggregates to eluci date the effect of photosensitized lenticular riboflavin [4].
Materials and Methods Experiment I: Effect o f UVB Irradiation on the Formation o f Ester Forms o f Riboflavin and on LRBC Male Wistar strain rats weighing about 250 g were used. Their eyes were excised, and the lenses were carefully removed by a posterior approach and ho
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Introduction
184
Experiment II: Fractionation o f H M W Protein and a-, P- and y-Crystallin in Soluble Protein Lenses were homogenized (100 mg wet weight/ml) in 20 m M sodium phosphate buffer (pH 7.4) contain ing 100 m M NaCl and 25 m M EDTA, and the lens homogenate was divided into 2 groups. Riboflavin (Nakarai Chemical, Japan) was added to 1 group to give a final concentration o f lOpg/ml and the 2 groups were UVB-irradiated (310 nm, 240 erg/ mm2/s) at 4 °C for 0, 20, 40 or 60 min. The lens homogenate was then centrifuged at 10,000# for 60 min to remove insoluble protein and particulate matter. The supernatant was subjected to gel filtra tion at 4 "Con a Sepharose 6B column, 100 X 2.5 cm (Pharmacia Fine Chemicals, Sweden). The elution buffer was 20 m M sodium phosphate buffer (pH 7.4) containing 100 m M NaCl and 25 m M EDTA. Frac tions (3.5 ml) were collected at a flow rate o f 20 ml/h with an LKB 2123 Micro Perpex pump. The position o f HMW protein and a-, (3- and y-crystallin was deter mined by absorbance at 280 nm [5]. HMW protein aggregates were located at the void volume around fraction No. 40. The protein concentration o f each collected fraction was measured from its absorbance at 280 nm and the proportion o f each fraction was expressed as percent o f soluble protein.
Results UVB irradiation of lens homogenate re sulted in increased LRBC and in decreased formation of ester forms of riboflavin (ta ble 1). As shown in table 2, when riboflavin was added to the lens homogenate before
Table 1. Effect o f UVB irradiation on LRBC and on the formation o f ester forms of riboflavin Irradiation time
LRBC nmol/g lens
Ester forms o f riboflavin nmol/g lens/h
0 20 40 60
0.0279± 0.0017 0.3669 + 0.1648* 0.8149±0.2161* 1.0684 ±0.1135*
1.2631 ±0.0078 1.1847 ±0.0747** 1.2119 ± 0.1071 ** 1.1940 ±0.0955**
min min min min
The incubation mixture was irradiated at 4 °C for 20, 40 or 60 min, using an XX-100 type irradiation apparatus (UVP). The UVB wavelength delivered was 310 nm (YM-15 type 240 erg/mm2/s). Each value represents the mean ± SD obtained from 5 experi ments. *p < 0.01, ** p < 0.05, compared to 0 min.
UVB irradiation, the formation of HMW protein aggregates was increased. The Y-crys tallin fraction, on the other hand, 'was de creased.
Discussion Our studies have established that the lens is one of the organs in which riboflavin is metabolized [1, 2] and that lenticular ribo flavin contributes to the formation of coenzyme and acts as a photosensitizer [4], We recently reported [4] that the addition of riboflavin to rat lens homogenate results in a conspicuous increase in HMW protein aggregates. Light exposure, using a fluores cent lamp, revealed that riboflavin acts as a photosensitizer to form HMW protein aggre gates in the lens. Since the lens is exposed to light, it is subjected to oxidative damage by endogenous photosensitizers such as ribo flavin or kynurenine derivatives [6, 7], The effects of UVB irradiation on the esterifica-
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mogenized (60 mg wet weight/ml) in 0.1 M potassium phosphate buffer (pH 7.4) containing 1 m M ATP, 1 m M MgCL and 0.1 mM ZnSCL. To 1 ml o f this homogenate was added 0.01 ml o f 1.5 pCi/ml D-[2IJC]-riboflavin solution (specific activity, 31.0 mCi/mmol; Radio Chemical Centre, UK) and the mixture was irradiated at 4 °C for 20, 40 or 60 min, using an XX-100 type irradiation apparatus (UVP, USA). The UVB wavelength delivered was 310 nm (YM -15 type 240 erg/mm2/s). Thereafter, the mixture was incubated for 60 min at 37 °C. The determina tion o f the ester forms o f riboflavin and o f LRBC was performed on a Sephadex G-15 column [1],
Hirano/Obara/Katakura/Ono
Ultraviolet B Irradiation Lenticular Riboflavin HMW-Protein
185
Table 2. Fractionation o f UVB-irradiated supernatant of lens homogenate supplemented with riboflavin on a Sepharose 6B column Irradiation time
Percent o f soluble protein
0 min
HMW protein
a-crystallin
ß-crystallin
y-crystallin
0.16
13.28
42.25
44.31
20 min
-B : +b 2
0.24 0.48
15.02 15.62
41.51 40.68
43.23 43.22
40 min
- b2 +b 2
0.31 0.76
15.88 17.15
41.10 40.47
42.71 41.46
60 min
- b2 +b 2
0.64 1.57
16.25 18.36
41.91 40.51
41.20 38.56
tion of riboflavin, on LRBC and on the for mation of HMW protein aggregates re mained to be elucidated. Radiation levels of 300-400 nm (UVB), which pass through the cornea but are largely absorbed by the lens, may be cataractogenic. UV radiation expo sure increases directly with hours of sun light and indirectly with geographic lati tude. Although there is a biochemical evi dence that senile cataracts may be attribut able to cumulative UV-induced photooxida tion and cross-linkage of lens proteins, we were unable to find published studies that examined the association between UV irra diation and the formation of HMW protein aggregates in the presence of lenticular ribo flavin. The present study showed that, as the duration of UVB irradiation increased, the synthesis of ester forms of riboflavin de creased. This effect may be attributable to the dénaturation of the apoenzyme of syn
thetases of ester forms of riboflavin such as flavokinase and FAD pyrophosphorylase. HMW proteins are thought to be an inter mediate in the conversion of water-soluble to water-insoluble lenticular proteins [8]. The ratio of HMW protein to other soluble proteins reportedly increases with age in hu mans and cows [9-11], and increased amounts of HMW proteins are found in cataractous lenses, including human senile ca taracts [9]. In the present study, we found that UVB irradiation of lens homogenate supplemented with riboflavin resulted in a significant increase in the formation of HMW protein aggregates and LRBC. The characteristic change occurs in the y-crystallin component. The decrease in y-crystallin corresponded to an increase in the HMW protein aggregates when lens homogenate supplemented with riboflavin was exposed to 60 min of UVB irradiation. This suggests that y-crystallin is closely related to the for
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Lens homogenate was divided into two groups. Riboflavin was added to 1 group to give a final concentra tion of 10 pg/ml and the 2 groups were UVB-irradiated at 4 °C for 0, 20, 40 or 60 min. The supernatant was subjected to gel filtration at 4 °C on a Sepharose 6B column. The relative protein concentrations o f the indi vidual crystallins were measured from the absorbance at 280 nm and expressed as percentage.
mation of HW protein aggregates. The present findings are consistent with our ear lier results when riboflavin-supplemented rat lens homogenate was exposed to fluores cent light [4]. Human lens protein turns brown in direct sunlight as a result of pho tooxidation due to UVB irradiation. Bovine lens crystallins are mainly cross-linked by singlet oxygen photosensitized by riboflavin, methylene blue and rose bengal [12]. The photodynamic system of riboflavin produces a considerable amount of H2O 2 and photo sensitized riboflavin may lead to cross-link ing of lens protein by singlet oxygen and hydrogen radicals [7, 12, 13]. This crosslinkage formation between lens proteins may lead to the formation of HMW protein ag gregates and to an increase in LRBC.
Acknowledgements This research was partly supported by a grant to H.H. from Keiryokai Research Foundation (No. 30). The authors wish to thank Mrs. Kumiko Araya for her expert technical help.
References 1 Ono, S.; Hirano, H.; Sato, Y.: Formation o f flavin adenine dinucleotide and flavin mononucleotide by lens homogenate. Expl Eye Res. 34: 297-301 (1982) . 2 Ono, S.; Hirano, H.: Riboflavin metabolism in the single lens o f rat. Ophthalmic Res. 15: 140-145 (1983) . 3 Hirano, H.; Hamajima, S.; Niitsu, Y.; Oikawa, K.; Ono, S.: Studies on the riboflavin-binding capac ity o f the rat lens. Int. J. Vitam. Nutr. Res. 53: 243-250(1983). 4 Ono, S.; Hirano, H.: Photosensitized acceleration o f riboflavin on the formation o f lenticular HMW-protein aggregation. Int. J. Vitam. Nutr. Res. 57: 401-403 (1987).
Hirano/Obara/Katakura/Ono
5 Bhat, K.S.: Alterations in the lenticular proteins of rats on riboflavin deficient diet. Curr. Eye Res. 2: 829-834 (1982/83). 6 Bando, M.; Mikuni, I.; Obazawa. H.: Acceleration of calcium-induced aggregation o f rat lens soluble protein by photosensitization with 8-methoxypsoralen and 3-hydroxy-L-kynurenine-0-|)-glucosidc. Expl Eye Res. 34: 953-960 (1982). 7 Jemigan, H.M., Jr.; Fukui, H.N.; Goosey, J.D.; Kinoshita, J.H.: Photodynamic effects o f rose bengal or riboflavin on carrier-mediated transport systems in rat lens. Expl Eye Res. 32: 461-466 (1981). 8 Harding, J.J.; Crabbe. M.J.C.: The lens: develop ment, proteins, metabolism and cataracts; in Davson, The eye, vol. IB, p. 207 (Academic Press, London 1984). 9 Jedziniak, J.A.; Kinoshita, J.H.; Yates, E.M.; Benedek, G.B.: The concentration and localization of heavy molecular weight aggregates in aging nor mal and cataractous human lenses. Expl Eye Res. 20: 367-369 (1975). 10 Jedziniak, J.A.; Kinoshita, J.H.; Yates, E.M.; Hocker, L.O.; Bencdek, G.B.: On the presence and mechanism o f formation o f heavy molecular weight aggregates in human normal and catarac tous lenses. Expl Eye Res. 15: 185-192 (1973). 11 Spector, A.; Freund, T.; Li, L.K.; Augusteyn. R.C.: Age dependent changes in the structure of alpha crystallin. Investve Ophthal. 10: 677-686 (1971). 12 Goosey, J.D.; Zigler, J.S., Jr.; Kinoshita, J.H.: Cross-linking o f lens crystallins in a photody namic system: a process mediated by singlet oxy gen. Science 208: *278-1280 (1980). 13 Varma, S.D.; Kumar, S.; Richards, R.D.: Lightinduced damage to ocular lens cation pump: pre vention by vitamin C. Proc. natn. Acad. Sci. USA 76: 3504-3506 (1979).
Received: December 2, 1988 Accepted: October 3, 1989 Dr. Hiroko Hirano Department o f Biochemistry School o f Medicine Iwate Medical University Morioka 020 (Japan)
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186
© 1990 S. Karger AG, Basel 0030-3747/90/0223-0183S2.75/0
Effects of Ultraviolet B Irradiation on Lenticular Riboflavin Metabolism and High-Molecular-Weight-Protein Aggregation Hiroko Hiranoa , Yoshitaka Obarab, Kumiko Katakuraa , Shigeru Onoa ■'Department o f Biochemistry, School o f Medicine, Iwate Medical University, Morioka; bDepartment o f Ophthalmology, Koshigaya Hospital, School o f Medicine, Dokkyo University, Koshigaya, Japan
Key Words. UVB irradiation • Riboflavin • Ester forms of B2 • Riboflavin-binding capacity • HMW protein aggregation • Lens Abstract. We investigated the effects of ultraviolet B (UVB) irradiation on the formation of ester forms of riboflavin and of high-molecular-weight (HMW) protein aggregates and on lenticular riboflavin-binding capacity (LRBC). Esterification of riboflavin decreased as the duration of UV irradiation increased, suggesting the irradiation-induced dénaturation of the apoenzyme of synthetases of ester forms of riboflavin. UVB irradiation of lens homogenate supplemented with riboflavin increased LRBC and the formation of HMW protein aggre gates, while y-crystallin was decreased. These results are consistent with those of our earlier studies in which we obtained data suggesting that, upon exposure of rat lens homogenate to fluorescent light, photosensitized riboflavin may bring about cross-linking of lens protein. Our data demonstrate that the photosensitivity of lenticular riboflavin is increased by longer periods of UV irradiation.
Work in our laboratory has documented that the lens is one of the organs in which riboflavin is metabolized [1-3] and that len ticular riboflavin contributes to the forma tion of coenzyme and acts as a photosensitiz er. In the present study, we investigated the effect of ultraviolet B (UVB) irradiation on the formation of ester forms of riboflavin and on lenticular riboflavin-binding capacity (LRBC). We also examined high-molecular-
weight (HMW) protein aggregates to eluci date the effect of photosensitized lenticular riboflavin [4].
Materials and Methods Experiment I: Effect o f UVB Irradiation on the Formation o f Ester Forms o f Riboflavin and on LRBC Male Wistar strain rats weighing about 250 g were used. Their eyes were excised, and the lenses were carefully removed by a posterior approach and ho
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 8:54:47 AM
Introduction
184
Experiment II: Fractionation o f H M W Protein and a-, P- and y-Crystallin in Soluble Protein Lenses were homogenized (100 mg wet weight/ml) in 20 m M sodium phosphate buffer (pH 7.4) contain ing 100 m M NaCl and 25 m M EDTA, and the lens homogenate was divided into 2 groups. Riboflavin (Nakarai Chemical, Japan) was added to 1 group to give a final concentration o f lOpg/ml and the 2 groups were UVB-irradiated (310 nm, 240 erg/ mm2/s) at 4 °C for 0, 20, 40 or 60 min. The lens homogenate was then centrifuged at 10,000# for 60 min to remove insoluble protein and particulate matter. The supernatant was subjected to gel filtra tion at 4 "Con a Sepharose 6B column, 100 X 2.5 cm (Pharmacia Fine Chemicals, Sweden). The elution buffer was 20 m M sodium phosphate buffer (pH 7.4) containing 100 m M NaCl and 25 m M EDTA. Frac tions (3.5 ml) were collected at a flow rate o f 20 ml/h with an LKB 2123 Micro Perpex pump. The position o f HMW protein and a-, (3- and y-crystallin was deter mined by absorbance at 280 nm [5]. HMW protein aggregates were located at the void volume around fraction No. 40. The protein concentration o f each collected fraction was measured from its absorbance at 280 nm and the proportion o f each fraction was expressed as percent o f soluble protein.
Results UVB irradiation of lens homogenate re sulted in increased LRBC and in decreased formation of ester forms of riboflavin (ta ble 1). As shown in table 2, when riboflavin was added to the lens homogenate before
Table 1. Effect o f UVB irradiation on LRBC and on the formation o f ester forms of riboflavin Irradiation time
LRBC nmol/g lens
Ester forms o f riboflavin nmol/g lens/h
0 20 40 60
0.0279± 0.0017 0.3669 + 0.1648* 0.8149±0.2161* 1.0684 ±0.1135*
1.2631 ±0.0078 1.1847 ±0.0747** 1.2119 ± 0.1071 ** 1.1940 ±0.0955**
min min min min
The incubation mixture was irradiated at 4 °C for 20, 40 or 60 min, using an XX-100 type irradiation apparatus (UVP). The UVB wavelength delivered was 310 nm (YM-15 type 240 erg/mm2/s). Each value represents the mean ± SD obtained from 5 experi ments. *p < 0.01, ** p < 0.05, compared to 0 min.
UVB irradiation, the formation of HMW protein aggregates was increased. The Y-crys tallin fraction, on the other hand, 'was de creased.
Discussion Our studies have established that the lens is one of the organs in which riboflavin is metabolized [1, 2] and that lenticular ribo flavin contributes to the formation of coenzyme and acts as a photosensitizer [4], We recently reported [4] that the addition of riboflavin to rat lens homogenate results in a conspicuous increase in HMW protein aggregates. Light exposure, using a fluores cent lamp, revealed that riboflavin acts as a photosensitizer to form HMW protein aggre gates in the lens. Since the lens is exposed to light, it is subjected to oxidative damage by endogenous photosensitizers such as ribo flavin or kynurenine derivatives [6, 7], The effects of UVB irradiation on the esterifica-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 8:54:47 AM
mogenized (60 mg wet weight/ml) in 0.1 M potassium phosphate buffer (pH 7.4) containing 1 m M ATP, 1 m M MgCL and 0.1 mM ZnSCL. To 1 ml o f this homogenate was added 0.01 ml o f 1.5 pCi/ml D-[2IJC]-riboflavin solution (specific activity, 31.0 mCi/mmol; Radio Chemical Centre, UK) and the mixture was irradiated at 4 °C for 20, 40 or 60 min, using an XX-100 type irradiation apparatus (UVP, USA). The UVB wavelength delivered was 310 nm (YM -15 type 240 erg/mm2/s). Thereafter, the mixture was incubated for 60 min at 37 °C. The determina tion o f the ester forms o f riboflavin and o f LRBC was performed on a Sephadex G-15 column [1],
Hirano/Obara/Katakura/Ono
Ultraviolet B Irradiation Lenticular Riboflavin HMW-Protein
185
Table 2. Fractionation o f UVB-irradiated supernatant of lens homogenate supplemented with riboflavin on a Sepharose 6B column Irradiation time
Percent o f soluble protein
0 min
HMW protein
a-crystallin
ß-crystallin
y-crystallin
0.16
13.28
42.25
44.31
20 min
-B : +b 2
0.24 0.48
15.02 15.62
41.51 40.68
43.23 43.22
40 min
- b2 +b 2
0.31 0.76
15.88 17.15
41.10 40.47
42.71 41.46
60 min
- b2 +b 2
0.64 1.57
16.25 18.36
41.91 40.51
41.20 38.56
tion of riboflavin, on LRBC and on the for mation of HMW protein aggregates re mained to be elucidated. Radiation levels of 300-400 nm (UVB), which pass through the cornea but are largely absorbed by the lens, may be cataractogenic. UV radiation expo sure increases directly with hours of sun light and indirectly with geographic lati tude. Although there is a biochemical evi dence that senile cataracts may be attribut able to cumulative UV-induced photooxida tion and cross-linkage of lens proteins, we were unable to find published studies that examined the association between UV irra diation and the formation of HMW protein aggregates in the presence of lenticular ribo flavin. The present study showed that, as the duration of UVB irradiation increased, the synthesis of ester forms of riboflavin de creased. This effect may be attributable to the dénaturation of the apoenzyme of syn
thetases of ester forms of riboflavin such as flavokinase and FAD pyrophosphorylase. HMW proteins are thought to be an inter mediate in the conversion of water-soluble to water-insoluble lenticular proteins [8]. The ratio of HMW protein to other soluble proteins reportedly increases with age in hu mans and cows [9-11], and increased amounts of HMW proteins are found in cataractous lenses, including human senile ca taracts [9]. In the present study, we found that UVB irradiation of lens homogenate supplemented with riboflavin resulted in a significant increase in the formation of HMW protein aggregates and LRBC. The characteristic change occurs in the y-crystallin component. The decrease in y-crystallin corresponded to an increase in the HMW protein aggregates when lens homogenate supplemented with riboflavin was exposed to 60 min of UVB irradiation. This suggests that y-crystallin is closely related to the for
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 8:54:47 AM
Lens homogenate was divided into two groups. Riboflavin was added to 1 group to give a final concentra tion of 10 pg/ml and the 2 groups were UVB-irradiated at 4 °C for 0, 20, 40 or 60 min. The supernatant was subjected to gel filtration at 4 °C on a Sepharose 6B column. The relative protein concentrations o f the indi vidual crystallins were measured from the absorbance at 280 nm and expressed as percentage.
mation of HW protein aggregates. The present findings are consistent with our ear lier results when riboflavin-supplemented rat lens homogenate was exposed to fluores cent light [4]. Human lens protein turns brown in direct sunlight as a result of pho tooxidation due to UVB irradiation. Bovine lens crystallins are mainly cross-linked by singlet oxygen photosensitized by riboflavin, methylene blue and rose bengal [12]. The photodynamic system of riboflavin produces a considerable amount of H2O 2 and photo sensitized riboflavin may lead to cross-link ing of lens protein by singlet oxygen and hydrogen radicals [7, 12, 13]. This crosslinkage formation between lens proteins may lead to the formation of HMW protein ag gregates and to an increase in LRBC.
Acknowledgements This research was partly supported by a grant to H.H. from Keiryokai Research Foundation (No. 30). The authors wish to thank Mrs. Kumiko Araya for her expert technical help.
References 1 Ono, S.; Hirano, H.; Sato, Y.: Formation o f flavin adenine dinucleotide and flavin mononucleotide by lens homogenate. Expl Eye Res. 34: 297-301 (1982) . 2 Ono, S.; Hirano, H.: Riboflavin metabolism in the single lens o f rat. Ophthalmic Res. 15: 140-145 (1983) . 3 Hirano, H.; Hamajima, S.; Niitsu, Y.; Oikawa, K.; Ono, S.: Studies on the riboflavin-binding capac ity o f the rat lens. Int. J. Vitam. Nutr. Res. 53: 243-250(1983). 4 Ono, S.; Hirano, H.: Photosensitized acceleration o f riboflavin on the formation o f lenticular HMW-protein aggregation. Int. J. Vitam. Nutr. Res. 57: 401-403 (1987).
Hirano/Obara/Katakura/Ono
5 Bhat, K.S.: Alterations in the lenticular proteins of rats on riboflavin deficient diet. Curr. Eye Res. 2: 829-834 (1982/83). 6 Bando, M.; Mikuni, I.; Obazawa. H.: Acceleration of calcium-induced aggregation o f rat lens soluble protein by photosensitization with 8-methoxypsoralen and 3-hydroxy-L-kynurenine-0-|)-glucosidc. Expl Eye Res. 34: 953-960 (1982). 7 Jemigan, H.M., Jr.; Fukui, H.N.; Goosey, J.D.; Kinoshita, J.H.: Photodynamic effects o f rose bengal or riboflavin on carrier-mediated transport systems in rat lens. Expl Eye Res. 32: 461-466 (1981). 8 Harding, J.J.; Crabbe. M.J.C.: The lens: develop ment, proteins, metabolism and cataracts; in Davson, The eye, vol. IB, p. 207 (Academic Press, London 1984). 9 Jedziniak, J.A.; Kinoshita, J.H.; Yates, E.M.; Benedek, G.B.: The concentration and localization of heavy molecular weight aggregates in aging nor mal and cataractous human lenses. Expl Eye Res. 20: 367-369 (1975). 10 Jedziniak, J.A.; Kinoshita, J.H.; Yates, E.M.; Hocker, L.O.; Bencdek, G.B.: On the presence and mechanism o f formation o f heavy molecular weight aggregates in human normal and catarac tous lenses. Expl Eye Res. 15: 185-192 (1973). 11 Spector, A.; Freund, T.; Li, L.K.; Augusteyn. R.C.: Age dependent changes in the structure of alpha crystallin. Investve Ophthal. 10: 677-686 (1971). 12 Goosey, J.D.; Zigler, J.S., Jr.; Kinoshita, J.H.: Cross-linking o f lens crystallins in a photody namic system: a process mediated by singlet oxy gen. Science 208: *278-1280 (1980). 13 Varma, S.D.; Kumar, S.; Richards, R.D.: Lightinduced damage to ocular lens cation pump: pre vention by vitamin C. Proc. natn. Acad. Sci. USA 76: 3504-3506 (1979).
Received: December 2, 1988 Accepted: October 3, 1989 Dr. Hiroko Hirano Department o f Biochemistry School o f Medicine Iwate Medical University Morioka 020 (Japan)
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186
