TY - JOUR
T1 - Effects of denaturants at low concentrations on the reversible denaturation of staphylococcal nuclease
AU - Shortle, David
AU - Meeker, Alan K.
AU - Gerring, Sandra L.
N1 - Funding Information:
The authors thank Kim Collins for his encouragement and for his careful reading of the manuscript. This work was supported by Grant GM34171 from the National Institutes of Health.
PY - 1989/7
Y1 - 1989/7
N2 - 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.
AB - 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.
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U2 - 10.1016/0003-9861(89)90200-2
DO - 10.1016/0003-9861(89)90200-2
M3 - Article
C2 - 2544138
AN - SCOPUS:0024375552
SN - 0003-9861
VL - 272
SP - 103
EP - 113
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 1
ER -