The objective of this research is to identify reagents which will suppress the formation of phase separation cataracts in vitro and ultimately in vivo. These cataracts are produced by the phase separation of proteins in the cytoplasm of human and other mammalian lenses. To achieve this objective we propose three parallel lines of investigation: 1. To inhibit phase separation. The diffusion of the cytoplasmic protein will be restricted either by homogeneously crosslinking the proteins into a stabilized protein network, or by crosslinking within the lens exogeneous organic monomer molecules into a polymer gel network. II. To determine phase diagrams of the constituent lens proteins. The coexistence curves of the individual, purified crystallins will be determined. An explanation will be sought for the striking change in phase separation temperature and opacification properties as a function of position in the lens, using the fact that the relative proportions of individual crystallins vary radically in the lens. III. To determine the interaction constants between lens proteins. The self-association and mixed-interaction constants of crystallins, which govern the short-range ordering of proteins in the normal transparent lens, will be determined in concentrated solutions. The principal methods of analysis in our experiments will include quasielastic light scattering spectroscopy, chemical crosslinking and polymerization techniques, frontal exclusion chromatography, ultra-centrifugation and high pressure liquid chromatography (HPLC).
| Thomson, J A; Schurtenberger, P; Thurston, G M et al. (1987) Binary liquid phase separation and critical phenomena in a protein/water solution. Proc Natl Acad Sci U S A 84:7079-83 |
| Mandal, K; Bose, S K; Chakrabarti, B et al. (1987) Structure and stability of gamma-crystallins. II. Differences in microenvironments and spatial arrangements of cysteine residues. Biochim Biophys Acta 911:277-84 |
| Mandal, K; Bose, S K; Chakrabarti, B et al. (1985) Structure and stability of gamma-crystallins. I. Spectroscopic evaluation of secondary and tertiary structure in solution. Biochim Biophys Acta 832:156-64 |
