The peculiar nature of the guanidine hydrochloride-induced two-state denaturation of staphylococcal nuclease: a calorimetric study.
This work determines the ratio of DeltaH(vH) /DeltaH(cal) for staphylococcal nuclease (SN) denaturation in guanidine hydrochloride (GdnHCl) to test whether GdnHCl-induced denaturation is two-state. Heats of mixing of SN as a function of [GdnHCl] were determined at pH 7.0 and 25 degrees C. The resulting plot of DeltaH(mix) vs [GdnHCl] exhibits a sigmoid shaped curve with linear pre- and post-denaturational base lines. Extending the pre- and post-denaturational lines to zero [GdnHCl] gives a calorimetric DeltaH (DeltaH(cal)) of 24.1 +/- 1.0 kcal/mol, for SN denaturation in the limit of zero GdnHCl concentration. Guanidine hydrochloride-induced denaturation Gibbs energy changes in the limit of zero denaturant concentration (DeltaG degrees (N)(-)(D)) at pH 7. 0 were determined for SN from fluorescence measurements at fixed temperatures over the range from 15 to 35 degrees C. Analysis of the resulting temperature-dependent DeltaG degrees (N)(-)(D) data defines a van't Hoff denaturation enthalpy change (DeltaH(vH)) of 26. 4 +/- 2.8 kcal/mol. The model-dependent van't Hoff DeltaH(vH) divided by the model-independent DeltaH(cal) gives a ratio of 1.1 +/- 0.1 for DeltaH(vH)/DeltaH(cal), a result that rules out the presence of thermodynamically important intermediate states in the GdnHCl-induced denaturation of SN. The likelihood that GdnHCl-induced SN denaturation involves a special type of two-state denaturation, known as a variable two-state process, is discussed in terms of the thermodynamic implications of the process.
Yang
M
M
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77555-1052, USA.
Liu
D
D
Bolen
D W
DW
D002151:Calorimetry; D019791:Guanidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000378:metabolism*; D008956:Models, Chemical; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics
D008836:Micrococcal Nuclease; D019791:Guanidine