alpha-chymotrypsin and 1-3-dimethylurea

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.

Topics: Binding Sites; Chymotrypsin; Guanidine; Guanidines; Kinetics; Mathematics; Methylurea Compounds; Phenylmethylsulfonyl Fluoride; Protein Conformation; Protein Denaturation; Solvents; Thermodynamics; Urea