Multiple unfolding intermediates of human placental alkaline phosphatase in equilibrium urea denaturation

Alkaline phosphatase is an enzyme with a typical alp hydrolase fold. The co nformational stability of the human placental alkaline phosphatase was exam ined with the chemical denaturant urea. The red shifts of fluorescence spec tra show a complex unfolding process involving multiple equilibrium interme diates indicating differential stability of the subdomains of the enzyme. N one of these unfolding intermediates were observed in the presence of 83 mM NaCl, indicating the importance of ionic interactions in the stabilization of the unfolding intermediates. Guanidinium chloride, on the other hand, c ould stabilize one of the unfolding intermediates, which is not a salt effe ct. Some of the unfolding intermediates were also observed in circular dich roism spectroscopy, which clearly indicates steady loss of helical structur e during unfolding, but very little change was observed for the beta strand content until the late stage of the unfolding process. The enzyme does not lose its phosphate-binding ability after substantial tertiary structure ch anges, suggesting that the substrate-binding region is more resistant to ch emical denaturant than the other structural domains. Global analysis of the fluorescence spectral change demonstrated the following folding-unfolding process of the enzyme: N <----> I-1 <----> I-2 <----> I-3 <----> I-4 <----> I-5 <----> D. These discrete intermediates are stable at urea concentratio ns of 2.6, 4.1, 4.7, 5.5, 6.6, and 7.7 M, respectively. These intermediates are further characterized by acrylamide and/or potassium iodide quenching of the intrinsic fluorescence of the enzyme and by the hydrophobic probes, 1-anilinonaphthalene-8-sulfonic acid and 4,4'-dianilino-1,1'-binaphthyl-5,5 '-disulfonic acid. The stepwise unfolding process was interpreted by the fo lding energy landscape in terms of the unique structure of the enzyme. The rigid central P-strand domain is surrounded by the peripheral a-helical and coil structures, which are marginally stable toward a chemical denaturant.