Oxidation-induced Structural Alterations and Its Effect on Chaperone Function of Rat Lens α-Crystallin

An ascorbate-FeCl₃-EDTA-H₂O₂ system was used to oxidize rat lens α-crystallins. Under this oxidative insult, the chaperone activity of α-crystallm toward γ-crystallin was shown to decrease significantly which is quite different from the result reported by Wang and Spector. (Invest. Ophthalmol. Vis. Sci. 1995, 36, 311-321.) Fluorescence spectroscopy and circular dichroism were employed to characterize the structural changes of oxidized α-crystallin. It was found that fluorescence intensity of 1-anilinonaphthalene-8-sulphonate (ANS) bound to oxidized α-crystallin increased comparing to that bound to normal α-crystallin, suggesting oxidation causes the exposure of more hydrophobic regions. Further, α-crystallin's fluorescence intensity in response to tryptophan residues showed a pseudo first order decline. Amino acid analysis of normal versus oxidized α-crystallin confirmed actual decline in tryptophan levels, showing about 80% of tryptophan being modified after 10-hour oxidation. Circular dichroism showed both changes in the secondary and tertiary structures of oxidized α-crystallin, characterized by a large loss of aromatic-type ammo acid interactions and a large loss of β-sheet structure. In conclusion, modified tryptophan, secondary and tertiary structural changes of α-crystallin correlate best with the reduction of chaperone function, the curves all showing a linear slope for 10 hours, then plateaumg. These results indicate that the decrease of α-crystallin chaperone activity is attributed to the structural changes.