STRUCTURE AND STABILITY OF γ-CRYSTALLINS-V. COVALENT AND NONCOVALENT PROTEIN-PROTEIN INTERACTIONS IN PHOTOSENSITIZED REACTIONS |
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Authors: | Masahiro Kono Krishnagopal Mandal Bireswar Chakrabarti |
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Institution: | Eye Research Institute of Retina Foundation, Boston, MA 02114, USA;Department of Ophthalmology, Harvard Medical School, Boston, MA 02115, USA |
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Abstract: | 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. |
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