Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl alpha-chymotrypsin using different denaturants.
Characteristics and properties of the unfolding free energy change, delta G degrees N-U, as determined by the linear extrapolation method are assessed for the unfolding of phenylmethanesulfonyl chymotrypsin (PMS-Ct). Difference spectral measurements at 293 nm were used to define PMS-Ct unfolding brought about with guanidinium chloride, urea, and 1,3-dimethylurea. All three denaturants were shown to give identical extinction coefficient differences (delta epsilon N-U) between native and unfolded forms of the protein in the limit of zero concentration of denaturant. The independence of delta epsilon N-U on denaturant supports the linear extension of pre- and postdenaturational base lines into the transition zone, allowing evaluation of unfolding equilibrium constants based on the two-state assumption. An expression, based on the linear extrapolation method, was used to provide estimates of delta G degrees N-U for the three denaturants using nonlinear least-squares fitting of the primary data, delta epsilon versus [denaturant]. The three delta G degrees N-U values were identical, within error, suggesting that the free energy change is a property of the protein system and independent of denaturant. It is suggested that the error in delta G degrees N-U determined from use of the linear extrapolation method is significantly larger than commonly reported in the literature.
Santoro
M M
MM
Department of Chemistry, Southern Illinois University, Carbondale 62901.
Bolen
D W
DW
D001665:Binding Sites; D002918:Chymotrypsin / Q000378:metabolism*; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D008433:Mathematics; D008782:Methylurea Compounds / Q000494:pharmacology; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011487:Protein Conformation; D011489:Protein Denaturation; D012997:Solvents; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
D006146:Guanidines; D008782:Methylurea Compounds; D012997:Solvents; D010664:Phenylmethylsulfonyl Fluoride; D014508:Urea; D002918:Chymotrypsin; D019791:Guanidine; C018276:1,3-dimethylurea