Effects of denaturants at low concentrations on the reversible denaturation of staphylococcal nuclease.
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 (delta Gapp), 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(delta Gapp)/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 d delta Gapp/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- greater than ClO4- greater than I- and Li+,NH4+ greater than Na+,K+. However, all of the chaotropic salts analyzed exhibited markedly increased values of d(delta Gapp)/dCsalt at concentrations below 0.2 M. One possible explanation for these large deviations from a linear relationship between delta Gapp 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.
Shortle
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Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Meeker
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Gerring
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D000838:Anions; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D005990:Glycerol / Q000494:pharmacology; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007454:Iodides / Q000494:pharmacology; D008836:Micrococcal Nuclease; D009154:Mutation; D010472:Perchlorates / Q000494:pharmacology; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D017670:Sodium Compounds; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D013861:Thiocyanates / Q000494:pharmacology; D014508:Urea / Q000494:pharmacology
D000838:Anions; D006146:Guanidines; D007454:Iodides; D010472:Perchlorates; D017670:Sodium Compounds; D013861:Thiocyanates; D014508:Urea; C031068:sodium perchlorate; D008836:Micrococcal Nuclease; D019791:Guanidine; D005990:Glycerol; D002118:Calcium