Binding of 1-anilinonaphthalene-8-sulfonic acid to alpha-crystallin

alpha-Crystallin was found to exhibit a time-dependent uptake of the hydrophobic probe, 1-anilinonaphthalene-8-sulfonic acid (ANS), similar to that typically observed with lipid membranes. Analysis of the interaction of ANS with alpha-crystallin revealed two types of interactive processes, partitioning and binding. The predominant process involved partitioning, with a coefficient of 300 M-1. The binding component had the following characteristics: 1 binding site/24 subunits and a Kd of about 9 microM. The binding was unaffected by the number of subunits used in the assembly of the alpha-aggregate, since both the alpha m- and alpha c-forms had similar binding characteristics. No discernible differences were observed in the binding of ANS to homopolymers of alpha A and alpha B subunits, suggesting that the hydrophobic sites to which ANS bound were similar in both the A and B subunits. The majority of the fluorescence was lost when the protein was incubated in 3 M urea, a concentration of denaturant where the protein is still intact, suggesting that the ANS binding sites are located near the surface of the protein. The decrease was attributed to a decrease in the quantum yield of the bound dye.