Abstract
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.
Highlights
␣, , and ␥-crystallins constitute the major portion of the eye lens fiber cells [1]
We have recently reported that amino acid residues 57– 69 and 93–107 of ␣B-crystallins interact with heat-denaturing alcohol dehydrogenase [37]
The bis-ANS binding to ␣-crystallin was determined by equilibrium dialysis method. ␣-Crystallin was dialyzed against bis-ANS in buffer A at 4 °C for h following a initial incubation for 1 h at °C
Summary
␣, -, and ␥-crystallins constitute the major portion of the eye lens fiber cells [1]. It has been proposed that surface hydrophobic sites in the native ␣-crystallin aggregate are involved in binding of target proteins to ␣-crystallin during chaperone-like activity display [17].
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