Renaturation of Yeast Inorganic Pyrophosphatase Denatured in Urea and Guanidine Hydrochloride

抄録

The renaturation of yeast inorganic pyrophosphatase [EC 3. 6. 1. 1] (PP_iase) denatured in guanidine-HCl and urea was studied. The molecular weight of PP_iase was estimated 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 PP_iase 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 PP_iase denatured in these denaturants were not restored by dilution with buffer. However, the enzymatic activities of PP_iase 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 PP_iase 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 PP_iase in lower denaturant concentrations was prevented in the presence of sulfhydryl reagents, dithiothreitol, glutathione, and 2-mercaptoethanol. In irreversibly denatured PP_iase, the amount of free SH groups decreased markedly. These results indicated that in lower denaturant concentrations, SH groups in PP_iase are very oxidizable and their oxidation may cause irreversible denaturation. In higher denaturant concentrations, where PP_iase was denatured
completely, the SH groups became less reactive. The conformations of renatured PP_iases were investigated by means of N-bromosuccinimide oxidation, fluorescence emission spectra and circular dichroism spectra. The PP_iase denatured in 6_M guanidine-HCl showed fully restored native conformation, as checked by these methods, although renatured PP_iase gave a trough in the 280nm region of slightly less magnitude than that of PP_iase. On the other hand, PP_iase denatured in 8_M urea showed restored enzymatic activity, but restoration of its conformation was incomplete as compared to PP_iase denatured in 6_M guanidine-HCl. PP_iase renatured from material denatured in lower denaturant concentrations, such as 4_M urea and 1.5_M guanidine-HCl, had quite a different conformation from the native enzyme as judged from CD spectra, N-bromosuccinimide oxidation and fluorescence spectra. Differences in PP_iases denatured in urea and guanidine-HCl were discussed in connection with the possible modification of amino groups in PP_iase by cyanate ions.