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Australian Journal of Chemistry Australian Journal of Chemistry Society
An international journal for chemical science
RESEARCH ARTICLE

Denaturation of proteins. IV. N.M.R. studies of ribonuclease-A

JH Bradbury and NLR King

Australian Journal of Chemistry 25(1) 209 - 220
Published: 1972

Abstract

The denaturation of ribonuclease-A by the addition of urea, guanidine hydrochloride, formic acid, and potassium thiocyanate to solutions in water or D2O at 33º has been followed by nuclear magnetic resonance (N.M.R.) spectroscopy.   The complex N.M.R. spectra at low field can be simplified greatly by a difference spectrum obtained by subtracting the spectrum obtained in deuterium oxide from the corresponding spectrum in water, whence the resonances of protons attached to nitrogen are isolated. Binding of urea and guanidine hydrochloride at concentrations well below that needed for unfolding is shown by modification of the C2 histidine resonances due to the histidines located at positions 12 and 119. This confirms that these denaturants inactivate the enzyme by binding at its active site as proposed by Barnard.The unfolding of ribonuclease by urea and guanidine hydrochloride at acid pH is shown to be a two-state process in which the fraction of unfolded molecules (cross-linked random coils) is calculated directly from the relative increase in heights of the various n.m.r, resonances. The unfolding in [D2]formic acid is characterized by the first (major) transition with a midpoint at 8% [D2] formic acid (v/v) and a second (minor) transition centred at 58% [D2]formic acid. In pure formic acid there is evidence of aggregate formation. An intermediate form characterized by a double methionine SCH3 resonance occurs during the first transition. There are therefore a minimum of five different states present during this unfolding. The major unfolding process produced by potassium thiocyanate is followed by a refolding to a non-native ordered form. This unfolding process is incomplete and three different

https://doi.org/10.1071/CH9720209

© CSIRO 1972

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