Interaction of ATP and lens alpha crystallin characterized by equilibrium binding studies and intrinsic tryptophan fluorescence spectroscopy

Abstract

α-Crystallin, the most prevalent protein in vertebrate lenses, is a high molecular weight aggregate composed of αA and αB subunits. Evidence is presented that ATP, a major phosphorus metabolite of the lens binds to α-crystallin extracted from calf lenses. The following parameters were obtained from equilibrium binding studies conducted at 37°C: binding sites per 400 kDa aggregate = 10 and K a = 8.1 · 10 3 M −1; and an essentially identical K a of 7.84 · 103 M −1 and 22 binding sites were determined for a 850 kDa aggregate. The cooperativity parameter, all, approximates unity which denotes that the binding of ligand is at independent sites. Binding was not significant at 22°C and was absent at 4°C. The specificity of the binding site for ATP was established by intrinsic tryptophan fluorescence spectroscopy. In the presence of increasing concentrations of ATP (0.05–0.3 mM), tryptophan fluorescence decreases in a concentration dependent manner to a minimum at 0.2 mM above which there is a non-linear response. Quenching of fluorescence was not evident with P i, AMP or ADP. GTP elicited a minimal quenching of fluorescence only at the highest concentration (0.30 mM). Modulation of both supramolecular organization and lens metabolism is predicted as a consequence of ATP/α-crystallin binding.