CATARACT RELATED MODIFICATIONS OF HUMAN LENS CRYSTALLINS

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DESCRIPTION (Investigator's Abstract): Cataract, a common disorder of aging, occurs with increased incidence among persons afflicted with uremia, diabetes, or chronic diarrhea. The rational development of preventive recommendations and therapeutic drugs has been hampered by a lack of knowledge at the molecular level about covalent modifications to lens proteins that either cause or accompany cataract. The proposed investigation will address this need by using new analytical methods based on mass spectrometry to analyze proteins isolated from normal, aged, and cataractous human lenses. In the first level of analysis, a new form of mass spectrometry, electrospray ionization mass spectrometry (ESIMS), will be used to determine the molecular weights of lens proteins with an accuracy of 0.02 percent (5 mass units). Lens proteins will then be proteolytically fragmented into peptides whose molecular weights will be determined with an accuracy of 0.3 mass units by directly-coupled microbore HPLC fast atom bombardment mass spectrometry. The molecular weights of the lens proteins and proteolytic peptides, as well as their chromatographic and electrophoretic properties, will be used to identify, at the molecular level, normal and covalently modified lens proteins. Results for a pool of normal human lenses will be used to make a data bank with which results from similar analyses of aged and cataractous lenses will be compared. Proteins and peptides with molecular weights that are not in the reference data bank will be considered to have undergone covalent modification. Cataractous lenses associated with uremia, diabetes, and chronic diarrhea will be studied by the same procedure so that covalently modified proteins and peptides unique to each type of cataract can be discerned. Lens proteins that have undergone covalent modifications unique to aging and specific forms of cataract will be investigated by a variety of micro analytical methods to identify at the molecular level the type of modification as well as the specific site on the protein which has undergone modification.