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Joan E. Roberts, Marilyn Ehrenshaft, Baozhong Zhao, Usha P. Andley, Ronald P. Mason; Immunological Detection Of NFK In Photooxided Lens α-crystallin. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4740.
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Detection of N-formyl Kynurenine (NFK) in photooxided lens α-crystallin using immunological techniques with polyclonal anti-NFK antiserum.
Bovine α-crystallin was incubated without addition of porphyrin or with four porphyrins of biological interest: namely, Uroporphyrin ( endogenous porphyrin) or TPPS, Ce6 and THPP porphyrins used for photodynamic therapy. These samples were either kept in the dark or exposed to UVA irradiation and then examined by both SDS-PAGE/Coomassie staining and anti-NFK western blotting. Human lens epithelial (HLE) cell cultures treated with the above porphyrins were either kept in the dark or exposed to UVA, were stained for α-crystallin, NFK and DNA and were examined by confocal microscopy. Absorption and fluorescent spectroscopic measurements were made of all four porphyrins both with and without α-crystallin.
The Coomassie stained gel of the in vitro photooxidized bovine α-crystallin showed that all four porphyrins caused UVA light-dependent aggregation of α-crystallin monomers into higher molecular weight polypeptides. Western analysis of these same samples also showed UVA light-dependent accumulation of NFK in α-crystallin monomers and polymers. Confocal microscopy of HLE cell cultures indicated that while all four porphyrins photosensitized cellular proteins, they showed variable ability to oxidize α-crystallin tryptophan residues to NFK. While both the TPPS and Uro treated cells do accumulate NFK, only the Ce6 and THPP treated cells show clear and extensive colocalization of NFK with α-crystallin. Absorption and fluorescence spectra showed the four porphyrins varied in their interactions with α-crystallin.
Four porphyrins of biological interest photooxidize α-crystallin in vitro and in HLE cell cultures. Using a newly developed immunological method (Ehrenshaft et al. 2009, 2010), NFK residues could be detected in oxidized lens proteins both in vitro and in HLE cells cultures. Accumulation of the oxidized tryptophan metabolite NFK in α-crystallin protein causes irreversible loss of transparency in the lens, leading to the development of cataracts.
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