Renaturation of Yeast Inorganic Pyrophosphatase Denatured in Urea and Guanidine Hydrochloride

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Abstract
The renaturation of yeast inorganic pyrophosphatase [EC 3.6.1.1] (PP1ase) denatured in guanidine-HCl and urea was studied. The molecular weight of PP1ase 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 PP1ase 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 PP1ase denatured in these denaturants were not restored by dilution with buffer. However, the enzymatic activities of PP1ase 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 PP1ase 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 PP1ase in lower denaturant concentrations was prevented in the presence of sulfhydryl reagents, dithiothreitol, glutathione, and 2-mercapto-ethanol. In irreversibly denatured PP1ase, the amount of free SH groups decreased markedly. These results indicated that in lower denaturant concentrations, SH groups in PP1ase are very oxidizable and their oxidation may cause irreversible denaturation. In higher denaturant concentrations, where PP1ase was denatured completely, the SH groups became less reactive. The conformations of renatured PP1ases were investigated by means of N-bromosuccinimide oxidation, fluorescence emission spectra and circular dichroism spectra. The PP1ase denatured in 6 M guanidine-HCl showed fully restored native conformation, as checked by these methods, although renatured PP1ase gave a trough in the 280 nm region of slightly less magnitude than that of PP1ase. On the other hand, PP1ase denatured in 8 M urea showed restored enzymatic activity, but restoration of its conformation was incomplete as compared to PP1ase denatured in 6 M guanidine-HCl. PP1ase 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 PP1ases denatured in urea and guanidine-HCl were discussed in connection with the possible modification of amino groups in PP1ase by cyanate ions.

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