Interaction of 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid with alpha-crystallin.
The hydrophobic sites in alpha-crystallin were evaluated using a fluorescent probe 1,1'-bi(4-anilino)naphthalenesulfonic acid (bis-ANS). Approximately one binding site/subunit of alpha-crystallin at 25 degrees C was estimated by equilibrium binding and Scatchard analysis (Kd = 1.1 microM). Based on fluorescence titration, the dissociation constant was 0.95 microM. The number of bis-ANS binding sites nearly doubled upon heat treatment of the protein at 60 degrees C. Likewise, the exposure of alpha-crystallin to 2-3 M urea resulted in increased binding of bis-ANS. Above 3 M urea there was a rapid loss in the fluorescence indicating the loss of interaction between bis-ANS and protein. The alpha-crystallin refolded from 6 M urea showed tryptophan fluorescence emission similar to the native alpha-crystallin. However, the refolded alpha-crystallin showed a 60% increase in bis-ANS binding, suggesting distinct changes on the protein surface resulting from exposure to urea similar to the changes occurring due to heat treatment. The fluorescence of tryptophan in native alpha-crystallin was quenched by the addition of bis-ANS. The quenching was inversely related to the amount of bis-ANS bound to alpha-crystallin. Additionally, the binding of bis-ANS reduced the chaperone-like activity of the protein. Photolysis of bis-ANS-alpha-crystallin complex resulted in incorporation of the probe to both A- and B-subunits, indicating that both subunits in native alpha-crystallin contribute to the surface hydrophobicity of the protein.
Sharma
K K
KK
Mason Eye Institute, Department of Ophthalmology, University of Missouri, Columbia, Missouri 65212, USA. opthks@showme.missouri.edu
Kaur
H
H
Kumar
G S
GS
Kester
K
K
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003459:Crystallins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004735:Energy Transfer; D005456:Fluorescent Dyes; D007700:Kinetics; D007908:Lens, Crystalline / Q000378:metabolism; D046911:Macromolecular Substances; D011489:Protein Denaturation; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D000817:Anilino Naphthalenesulfonates; D003459:Crystallins; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D014364:Tryptophan