The specific aims for this project are: 1) To isolate and identify some of the molecular species responsible for the photochemically induced age-related fluorescence (particularly in the 360 nm excitation/440 nm emission, and 435 nm excitation/520 nm emission) in the human lens. This will be performed by the isolation and purification of specific fluorescent peptides (enzymatic hydrolysis and chromatography); amino acid composition and sequencing, and further hydrolysis of the peptides to isolate the purified chromophores. Spectroscopic analyses (UV, fluorescence, phosphorescence, GC mass spectrometry and NMR spectroscopy will be performed at certain stages of this procedure to monitor and identify the compounds isolated. 2) To elucidate the molecular mechanisms of photochemical lens damage by studying the photochemistry of purified lens proteins and appropriate model compounds. 3) To probe the distribution of lens protein fractions (alpha, beta, gamma-crystallin and insoluble) using laser excited tryptophan fluorescence decay measurements on intact human lenses (""""""""optical dissection""""""""). Knowing the photochemical reactivities of the various lens protein fractions and their distribution should permit us to predict which region(s) of the intact lens are most susceptible to photochemical damage. 4) In addition to our conventional irradiation source which enables us to bathe whole lenses with photons, we also propose to utilize our laser photolysis apparatus to study localized photodamage in whole lenses and to detect such damage by laser excited fluorescence and ESR spectroscopy. 5) To correlate our in vitro fluorescence studies (on human lenses) with in vivo UV-slit lamp densitography. We have recently demonstrated the feasibility of this approach and we have instituted a study on normal individuals as well as patients on phototherapy. This may enable us to detect changes in lens fluorescence several years prior to their becoming manifest by usual slit lamp examinations. 6) To isolate and identify the photochemical product(s) produced when animals are exposed to UV radiation following a dose of the sensitizer 8-methoxypsoralen. Spectroscopic analyses (as outlined in 1) will be employed. 7) To relate the molecular changes caused by direct and photosensitized UV radiation to morphological events.
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