Thermodynamic nonideality as a probe of reversible protein unfolding effected by variations in pH and temperature: studies of ribonuclease.
Thermodynamic nonideality arising from the space-filling effect of added sucrose is employed to confirm that the reversible unfolding of ribonuclease A effected by acid may be described as an equilibrium between native and unfolded states of the enzyme. However, the extent of the volume change is far too small for the larger isomer to be the fully expanded state, a result signifying that the acid-mediated unfolding of ribonuclease does not conform with the two-state equilibrium model of protein denaturation. Although the thermal denaturation of ribonuclease A is characterized by a larger increase in volume, quantitative reappraisal of published results on the effects of glycerol on this transition at pH 2.8 (Gekko, K., and Timasheff, S. N., 1981 Biochemistry 20, 4677-4686) leads to an estimated volume increase that is much smaller than that inferred from hydrodynamic studies--a disparity attributed to the dual actions of glycerol as a space-filling solute and as a ligand that binds preferentially to the thermally unfolded form of the enzyme. Even in this unfavorable circumstance the fact that glycerol exerts a net excluded volume effect at least confirms that the thermal unfolding of ribonuclease A is an equilibrium transition between two discrete states. The strengths and limitations of using thermodynamic nonideality as a probe of the two-state equilibrium model of protein denaturation are discussed in the light of these findings.
Shearwin
K E
KE
Department of Biochemistry, University of Queensland, Brisbane, Australia.
Winzor
D J
DJ
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D005990:Glycerol / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D011489:Protein Denaturation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D013395:Sucrose / Q000737:chemistry; D013696:Temperature; D013816:Thermodynamics
D013395:Sucrose; D012259:Ribonuclease, Pancreatic; D005990:Glycerol