Effects of denaturants at low concentrations on the reversible denaturation of staphylococcal nuclease

David Shortle, Alan K. Meeker, Sandra L. Gerring

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59 Scopus citations


Three very unstable mutant forms of staphylococcal nuclease were used to quantitate the change in the apparent equilibrium constant for reversible denaturation (Kapp) as a function of denaturant concentration for a variety of different denaturing solutes. The value of this equilibrium constant in the absence of denaturant (Kapp,0) was determined by renaturation of the mutant proteins with a combination of glycerol and calcium ion, the latter of which binds at the active site in the native conformation. Because Kapp,0 fell in the easily measurable range between 0.1 and 1, the change in Kapp, and thus the change in free energy (ΔGapp), at very low concentrations of denaturants could be accurately measured. With guanidine hydrochloride (GuHCl), the rate of change of the apparent free energy of denaturation with respect to denaturant concentration ( d(ΔGapp) dCGuHCl or mGuHCl) was found to be remarkably constant down to zero denaturant concentration, even though this value was different for each of the three proteins. Unlike GuHCl, urea exhibited a slightly reduced value of dΔGapp dCurea at low concentrations. Results with a number of thiocyanate, perchlorate, and iodide salts confirmed that the Hofmeister series holds for concentrations below 0.1 m; that is, with regard to efficacy as a denaturant SCN- . ClO4- > I- and Li+, NH4+ > Na+, K+. However, all of the chaotropic salts analyzed exhibited markedly increased values of d(ΔGapp) dCsalt at concentrations below 0.2 m. One possible explanation for these large deviations from a linear relationship between ΔGapp and salt concentration is that weak binding or adsorption of chaotropic anions is occurring at a saturable number of sites in hydrophobic regions of the denatured state.

Original languageEnglish (US)
Pages (from-to)103-113
Number of pages11
JournalArchives of Biochemistry and Biophysics
Issue number1
StatePublished - Jul 1989

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology


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