Immunological detection of N-formylkynurenine in porphyrin-mediated photooxided lens α-crystallin

Crystallin proteins are responsible for maintaining lens transparency and allowing the lens to focus light undistorted onto the retina. The α-crystallins are the major lens crystallins, and function as both structural proteins and chaperones to protect all lens proteins from damage leading to lens deterioration. Because lens crystallin proteins do not turn over, the damage they accumulate can lead to cataracts, the world's leading cause of blindness. Photosensitizing porphyrins can accumulate in the eye through either endogenous metabolism or through therapeutic or diagnostic procedures. Porphyrin buildup exacerbates lens aging through increased levels of singlet oxygen, resulting in protein polymerization and amino acid residue alteration. Tryptophans oxidize to kynurenine and N-formylkynurenine (NFK) causing irreversible changes in the refractive index of the normally transparent lens, leading to development of cataracts. Additionally, NFK is itself a photosensitizer, and its presence exacerbates lens deterioration. This work uses anti-NFK antiserum to study porphyrin-facilitated photooxidation of α-crystallin tryptophan residues. In vitro experiments show that four biologically interesting porphyrins mediate α-crystallin polymerization and accumulation of both protein radicals and NFK. Confocal microscopy of cultured human lens epithelial cells indicates that while all four porphyrins photosensitize cellular proteins, not all oxidize the tryptophans of cellular α-crystallin to NFK. In this study we used immunological techniques to detect N-formylkynurenine (NFK), a tryptophan oxidation product, in α-crystallin using an anti-NFK antibody. Here we show UVA-irradiated human lens epithelial cells containing the porphyrin THPP (mesotetra (m-hydroxyphenyl) porphine). Fluorescent confocal analysis was used to visualize (clockwise, starting at bottom left) nuclei (blue), NFK (green), and α-crystallin (red), the major lens protein. The final panel (bottom, right) shows the overlay image of the preceding three, and illustrates the potential for irreversible oxidative photodamage that porphyrin accumulation can inflict on the critical eye lens protein α-crystallin. Unirradiated porphyrin-containing cells do not accumulate NFK.