Renaturation of Yeast Inorganic Pyrophosphatase Denatured in

The renaturation of yeast inorganic pyrophosphatase [EC 3.6.1.1] (PPiase) denatured in guanidine-HCl and urea was studied. The molecular weight of PPiase was esti-mated to be ca. 63,000—70,000 by means of Sephadex G-75 column chromatography in 8 M urea and 6 M guanidine-HCl and by electrophoresis on polyacrylamide gel containing 8 M urea. The activities of PPiase denatured in various concentrations of denaturants were measured in the presence and absence of the denaturants. In the presence of the denaturants, enzymatic activity decreased as the denaturant concentration increased up to 1.5 M guanidine-HCl and 4 M urea. The activities of PPiase denatured in these denaturants were not restored by dilution with buffer. However, the enzymatic activities of PPiase denatured at concentrations higher than 1.5 M guanidine-HCl and 4 M urea were restored by dilution with Tris-HCl buffer (pH 7.5). The recovery of the enzymatic activities of PPiase denatured in 3 to 6 M guanidine-HCl and 6 to 8 M urea was to a level of about 90 % of the native enzyme. Irreversible denaturation of PPiase in lower denaturant concentrations was prevented in the presence of sulfhydryl reagents, dithiothreitol, glutathione, and 2-mercapto