pH dependence of the urea and guanidine hydrochloride denaturation of ribonuclease A and ribonuclease T1.
To investigate the pH dependence of the conformational stability of ribonucleases A and T1, urea and guanidine hydrochloride denaturation curves have been determined over the pH range 2-10. The maximum conformational stability of both proteins is about 9 kcal/mol and occurs near pH 4.5 for ribonuclease T1 and between pH 7 and 9 for ribonuclease A. The pH dependence suggests that electrostatic interactions among the charged groups make a relatively small contribution to the conformational stability of these proteins. The dependence of delta G on urea concentration increases from about 1200 cal mol-1 M-1 at high pH to about 2400 cal mol-1 M-1 at low pH for ribonuclease A. This suggests that the unfolded conformations of RNase A become more accessible to urea as the net charge on the molecule increases. For RNase T1, the dependence of delta G on urea concentration is minimal near pH 6 and increases at both higher and lower pH. An analysis of information of this type for several proteins in terms of a model developed by Tanford [Tanford, C. (1964) J. Am. Chem. Soc. 86, 2050-2059] suggests that the unfolded states of proteins in urea and GdnHCl solutions may differ significantly in the extent of their interaction with denaturants. Thus, the conformations assumed by unfolded proteins may depend to at least some extent on the amino acid sequence of the protein.
Pace
C N
CN
Biochemistry Department, Texas A&M University, College Station 77843.
Laurents
D V
DV
Thomson
J A
JA
D001236:Aspergillus oryzae / Q000187:drug effects / Q000201:enzymology; D004722:Endoribonucleases; D006146:Guanidines / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D006163:Ribonuclease T1; D012259:Ribonuclease, Pancreatic; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology*
D006146:Guanidines; D014508:Urea; D004722:Endoribonucleases; D006163:Ribonuclease T1; D012259:Ribonuclease, Pancreatic