Abstract
Purpose: :
To determine the structure of the UVA–mediated photolysis products of K2P, a Maillard crosslink, and to analyze whether these products can be shown to exist in cataract lens proteins. These data would show that UVA light has caused a specific photochemical reaction to occur in these lenses.
Methods: :
Solutions of purified K2P were irradiated with UVA light (1 KJ/cm2) over 1h, and the photoproducts separated by HPLC on a Prodigy column using a linear gradient from 5 to 45% acetonitrile in 0.1% heptafluorobutyric acid. Two major photoproducts were collected and analyzed by UV and mass spectrometry. Total proteins were also isolated from individual Type I Indian cataract lenses and digested with a battery of proteolytic enzymes over 9 days. The initial peak was collected from a Bio–Gel P–2 column and separated by HPLC. UVA photoproducts of K2P were identified by both LC/MS and subsequent LC/MS/MS.
Results: :
The irradiation of K2P [1–(5–amino–5–carboxymethylamino)–3–hydroxy–2,3–dihydropyridinium] with UVA light caused greater than 80% bleaching as measured by its absorption at 343 nm. After irradiation, HPLC identified one major bleaching product with a max at 295 nm and a mass of 369 Th, and a minor peak with a max at 282 nm and a mass of 352 Th, compared to K2P which had a max at 343 nm and a mass ot 371 Th. Mass spectra of these compounds suggested that they arose by the formation of a third double bond in the pyridinium ring by the removal of either 2 atoms of hydrogen or a molecule of H2O. Total lens proteins from a cataractous human lens were digested and subjected to Bio–Gel P–2 chromatography. HPLC of the initial peak from the Bio–Gel P–2 column using specific ion monitoring showed a peak with a mass of 369 Th and a shoulder at 352 Th. These peaks co–eluted with the K2P photoproducts and had MS spectra that were the same as the two identified K2P photoproducts.
Conclusions: :
UVA light causes the photobleaching of K2P, a significant Maillard crosslink in aged human lens. This occurs by either oxidation or dehydration to form an aromatic pyridinium ring, which absorb at either 282 or 295 nm instead of the 343 nm absorbance maximum of K2P, which explains the observed photobleaching. The identification of these two compounds in cataract lenses argues that UVA photobleaching does occur in these cataract lenses.
Keywords: cataract • protein modifications-post translational • laser