DETECTION OF AN UNFOLDING INTERMEDIATE IN ALPHA-UREASE WITH ENHANCED AFFINITY FOR ANSA

Protein aggregation is believed to be due to conformers that expose hy drophobic clusters that promote protein association. Such conformers c an be detected using a fluorescent probe like 8-anilino l-naphthalenes ulfonic acid (ANSA). Here we show that urease exposed to 1.0 M guanidi ne - hydrogen chloride has a higher affinity for ANSA than native or d enatured urease. The binding occurs over a narrow range of denaturant concentration, well below the concentration required to induce denatur ation. The impact of ANSA on urease aggregation was further studied by fluorescence, light-scattering, and activity measurements. We found t hat ANSA modifies urease aggregates and can provide partial protection against inactivation arising from thermally induced aggregation. It s eems that the well-known susceptibility of urease to aggregation is du e to an intermediate that can be populated in the absence of denaturat ion. Such a rationale would explain why folding stability of urease is a poor indicator of long-term stabilization by various media.