Purchase this article with an account.
H. Guo, B. C. Dutmer, T. M. Gilbert, J. P. Dillon, E. R. Gaillard; The Photoreaction of 3-Hydroxykynurenine With Lysine: A Model for Aging in the Lens. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2426.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The purpose of this study is to investigate a potential aging mechanism in the human lens involving the photochemical attachment of 3-hydroxykynurenine (3-HK) to lens protein using LC/PDA/ESI-MS.
HPLC grade methanol (MeOH) (99.99%), 3-HK, lysine, and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich (St. Louis, MO). 5mM 3-HK and 5mM lysine in 5mL phosphate buffer at pH 7.4 were bubbled with 4% oxygen in argon for 30 mins and then transferred to a 1 cm pathlength quartz cuvette. The sample was positioned 10 cm from a 200 W Xe arc lamp (Oriel Corporation, Stratford, CT) . Aliquots of 200 to 400 µL were removed from the solution after 1 hour, 3 hours, 6 hours, 9 hours and 11 hours photolysis. After taking the UV-visible absorption and fluorescence spectra, the aliquots were dried with Ar and stored at -200C for further LC/ESI-MS analyses (ThermoFinnegan Advantage LC/Surveyor ion trap MS). The reaction mixtures were separated using a Phenomenex Synergi Max-RP C12 4 mm analytical column (150 x 4.6 mm) with a gradient of 5% - 95% CH3OH (0.1% TFA in water) over a period of 20 min and a flow rate of 0.2 mL/min.
Three products which have absorptions between 300nm and 450nm were separated and detected by PDA. These fractions were introduced into the mass spectrometer and have the following properties: m/z = 339 amu with absorption maxima at 295nm and 405nm; m/z = 399 amu with absorption maxima at 270nm and 370nm; m/z = 371 with absorption maxima at 250 nm and 325nm. Collision induced dissociation of these parent ions shows that the fragmentation patterns are consistent with lys reacting with 3-HK in the aromatic ring portion of 3-HK and at least one of the products results from an additional internal Michael addition cyclization. Ab initio calculations of the possible products using molecular mechanics optimizations and TD-DFT calculations (B3PW91/MIDIY++) indicate that these structures are stable photoproducts and show excellent agreement between calculated and experimentally observed energies for electronic transitions.
With aging, modifications in the human lens proteins result in compounds that absorb at 325 nm and out to 550 nm. The present study suggests that these form from the attachment of 3-HK to protein residues such as Lys and Cys via a Michael addition reaction. This would result in a partial melanization of lens protein and eventually lead to opacification of the tissue.
This PDF is available to Subscribers Only