STRUCTURE AND STABILITY OF γ-CRYSTALLINS-V. COVALENT AND NONCOVALENT PROTEIN-PROTEIN INTERACTIONS IN PHOTOSENSITIZED REACTIONS

Abstract— Irradiation of γ-crystallins with 300 nm light or with the photosensitizers riboflavin or methylene blue (MB) leads to intermolecular cross-linking and insolubilization. Sodium dodecyl sulfate polyacrylamide gel electrophoresis studies reveal that these cross-links are composed of nondisulfide covalent bonds. The water-insoluble phase is stabilized by noncovalent forces, as denaturants readily dissolve it. High-performance liquid chromatography and electrophoresis results further indicate that the higher multimers are part of this water-insoluble fraction only, with the exception of MB-sensitized reactions, which are also able to produce a water-soluble, high-molecular-weight protein of at least 1 million. Labeling the external sulfhydryl groups with iodoacetamide does not prevent the photoreac-tions; however, a reducing agent such as dithiothreitol does. A mechanism involving initial oxidation and interaction of sulfhydryl groups (forming an intramolecular disulfide) buried within the protein as a necessary precursor to polymerization and precipitation has been proposed in the preceding paper. The present study provides support for this mechanism.