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Interaction of 1,1′-Bi(4-anilino)naphthalene-5,5′-Disulfonic Acid with α-Crystallin

1998; Elsevier BV; Volume: 273; Issue: 15 Linguagem: Inglês

10.1074/jbc.273.15.8965

1083-351X

K. Krishna Sharma, Harjeet Kaur, Gopinatha Suresh Kumar, Kathryn Kester,

Enzyme Structure and Function

The hydrophobic sites in α-crystallin were evaluated using a fluorescent probe 1,1′-bi(4-anilino)naphthalenesulfonic acid (bis-ANS). Approximately one binding site/subunit of α-crystallin at 25 °C was estimated by equilibrium binding and Scatchard analysis ( K d = 1.1 μm). Based on fluorescence titration, the dissociation constant was 0.95 μm. The number of bis-ANS binding sites nearly doubled upon heat treatment of the protein at 60 °C. Likewise, the exposure of α-crystallin to 2–3m urea resulted in increased binding of bis-ANS. Above 3m urea there was a rapid loss in the fluorescence indicating the loss of interaction between bis-ANS and protein. The α-crystallin refolded from 6 m urea showed tryptophan fluorescence emission similar to the native α-crystallin. However, the refolded α-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 α-crystallin was quenched by the addition of bis-ANS. The quenching was inversely related to the amount of bis-ANS bound to α-crystallin. Additionally, the binding of bis-ANS reduced the chaperone-like activity of the protein. Photolysis of bis-ANS-α-crystallin complex resulted in incorporation of the probe to both A- and B-subunits, indicating that both subunits in native α-crystallin contribute to the surface hydrophobicity of the protein.