- 作者: Yuh Ho, Chien-Lin Lai and Fu-Yung Huang
- 中文摘要: An ascorbate-FeCl3-EDTA-H2O2 system was used to oxidize rat lens α-crystallins. Under this oxidative insult, the chaperone activity of α-crystallin 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 bond 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 amino acid interactions and 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 plateauing. These results indicate that the decrease of α-crystallin chaperone activity is attributed to the structural changes.
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