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Duckd2-crystallin is a soluble tetrameric lens protein. In the presence of guanidinium hydrochloride (GdnHCl),it undergoes stepwise dissociation and unfolding. Gel-filtra-tion chromatography and sedimentation velocity analysis has demonstrated the dissociation of the tetramer protein to a monomeric intermediate with a dissociation constant of 0.34lM 3 . Dimers were also detected during the dissociation and refoldingprocesses.The sharpenhancement of 1-anilino naphthalene-8-sulfonic acid (ANS) fluorescence at 1M GdnHCl strongly suggested that the dissociated monomers were in a molten globule state under these conditions | Eur. J. Biochem. 270 3988-3995 2003 FEBS 2003 doi 10.1046 j.1432-1033.2003.03787.x Monomeric molten globule intermediate involved in the equilibrium unfolding of tetrameric duck d2-crystallin Hwei-Jen Lee1 Shang-Way Lu1 and Gu-Gang Chang2 1 Department of Biochemistry National Defense Medical Center Taipei Taiwan 2Faculty of Life Sciences National Yang-Ming University Taipei Taiwan Duck ỗ2-crystallin is a soluble tetrameric lens protein. In the presence of guanidinium hydrochloride GdnHCl it undergoes stepwise dissociation and unfolding. Gel-filtration chromatography and sedimentation velocity analysis has demonstrated the dissociation of the tetramer protein to a monomeric intermediate with a dissociation constant of 0.34 IM3. Dimers were also detected during the dissociation and refolding processes. The sharp enhancement of 1 -anilino naphthalene-8-sulfonic acid ANS fluorescence at 1 M GdnHCl strongly suggested that the dissociated monomers were in a molten globule state under these conditions. The similar binding affinity w 60 M of ANS to protein in the presence or absence of GdnHCl suggested the potential assembly of crystallins via hydrophobic interactions which might also produce off-pathway aggregates in higher protein concentrations. The dynamic quenching constant corresponding to GdnHCl concentration followed a multistate unfolding model implying that the solvent accessibility of tryptophans was a sensitive probe for analyzing ỗ2-crystallin unfolding. Keywords ỗ-crystallin lens protein unfolding dissociation argininosuccinate lyase. ỗ2-Crystallin a highly concentrated yet soluble protein in avian and reptile eye lens cess as on important tr md LI ml protein for light refraction 1-3 . Thermodynamic stability for crystallins is essential in maintaining lens transparency 4 . Determining the mechanism of folding and assembly of these proteins is important for understanding how they can form stable transparent structures at high concentrations. The ỗ2-crystallin
