There is much evidence indicating that cataract, as well as normal aging of the lens, may occur as disulfide crosslinkages are formed in the lens proteins. These crosslinkages lead to formation of large aggregates, which scatter light and cause the lens to become opaque. The two most important goals of the proposed investigation are to (1) identify specific cysteinyl residues in the crystallins which undergo disulfide bonding to form intermolecular crosslinkages, and (2) identify modifications to crystallins which precede the formation of disulfide crosslinkages. Because lens extracts are mixtures of many different crystallins, initial experiments will use two highly purified models, bovine alpha A2- and gamma II-crystallin, to indicate which disulfide crosslinkages are formed in vitro. These model crystallins will be incubated with potassium cyanate, which is known to cause the formation of cataract in vitro and in vivo. Products of this reaction will be identified and analyzed to improve our understanding of how exposure to cyanate, as well as other substances which change the structure of crystallins, leads to the formation of intermolecular crosslinkages in lens proteins. Since there is some evidence that aspirin inhibits cataractogenisis, the products of the reaction of aspirin and the model crystallins will be identified. The physiological significance of our understanding of the mechanism by which disulfide crosslinkages are formed in vitro will be determined by performing similar analyses on lens proteins isolated from cataractous bovine lens where cataract is induced by the administration of potassium cyanate to a calf. The proposed investigation will then be directed at obtaining similar information from cataractous human lenses. Disulfide-bonded high molecular weight aggregates will be analyzed to determine what modifications have caused a predisposition of these proteins to form disulfide crosslinkages. This detailed investigation of disulfide bonding at the molecular level is possible only because new analytical methods based on HPLC and fast atom bombardment mass spectrometry will be used to identify modified peptides.
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