A major aim of this proposal is to characterize beta-crystallins of the Lens and to study their possible role in normal and cataractous lenses. Initial approach is to establish workable and efficient methods for purification of individual lens beta-crystallins primarily by molecular sieving and ion-exchange chromatography. Each purified protein will be characterized in terms of native and oligomeric structure (PAGE, SDS-PAGE), amino acid composition, N- and C-terminal analyses, peptide mapping, detection of carbohydrate moieties (PAS staining, GLC), immunochemistry, and interaction with glutathione (radio labeling). These techniques are being applied in the case of a 26,000 molecular weight polypeptide which is a subunit of rabbit beta-crystallin and is also the major beta-crystallin polypeptide in a variety of species including bovine and human lenses. This abundant polypeptide is heterogeneous in charge and is ubiquitously present in water-soluble, urea-soluble, and urea-insoluble fractions by lens homogenates. A poly-peptide with the same apparent molecular weight called the main intrinsic protein (MIP) has been found in the lens fiber membrane and gap junction preparations. We would like to establish the identity or non-identity of these two polypeptides by immunological and peptide mapping procedures and in general, to determine if lens crystallins become integrated into lens membranes. Immunochemical techniques will be applied to learn about age and species variations in these proteins. Cross-linking reagents will also be used to study the topology of beta-crystallins in terms of subunit arrangement within each protein (intramolecular interactions) and proximal arrangement of each beta-crystallin with other lens proteins (intermolecular interactions). Utilizing such techniques, the changes in these proteins which occur with aging and cataract in human lenses and in experimental cataract will be studied.