Fluorescence spectral analysis of the chaperone-like effect of α-crystallin in photomodification of human lens crystallins

Purpose. To gain insight into the mechanism by which human α-crystallin prevents the aggregation of β and γ crystallins induced by photogenerated singlet oxygen or ultraviolet B radiation. Methods. Human α, β and γ-crystallins from young human lenses or recombinant γD and αA were exposed to singlet oxygen (¹O₂) generated using rose bengal and 546.8 nm radiation or to 302 nm radiation. Light scattering was measured at 400 nm. γ-Crystallins were fluorescence labeled with 2-(4′-maleimidylanilino) naphthalene-6-sulphonate (MIANS). Results. Recombinant αA prevented the ¹O₂-induced aggregation of recombinant γD-crystallin, but not the change in tertiary structure of γD. Addition of α to γ-MIANS increased the fluorescence intensity and anisotropy of the latter. Hill plot analysis indicated a cooperative interaction between γ-MIANS and α. UV radiation increased the aggregation of γ-MIANS as shown by an increase in fluorescence anisotropy of the bound label. α-Crystallin completely prevented this radiation-induced anisotropy increase of γ-MIANS. Conclusions. Fluorescence studies on the interaction between α-crystallin and MIANS- labeled β- and γ-crystallin should help define the chaperone-like role of α in preventing aggregation of other crystallins.