On the Structural Stability and Solvent Denaturation of Proteins

Denaturation of human and Glycera dibranchiata hemoglobins by the urea and amide classes of denaturants

“…It is possible to examine and predict the midpoints of the transitions from native to denatured or unfolded protein forms by means of the equations of Peller (1959) and Flory (1957) that have been employed with varying success to account for the effects of salts (Mandelkern and Steward, 1964; Robinson and Grant, 1966; Schrier and Schrier, 1967;Von Hippel and Schleich, 1969) and water miscible neutral solutes of the alcohol (Schrier et al, 1965;Herskovits et al, 1970a;Herskovits and Harrington, 1972;Parodi et al, 1973), urea (Herskovits et al, 1970c), and amide (Herskovits et a!., 1970b) classes on the stability of proteins and other biopolymers. The lowering of the melting temperature or denaturation midpoint of a biopolymer, ATm, due to preferential interaction with the denaturing solute of activity, od, per average monomer unit, in the unfolded (i.e., denatured) form is given by the equation first derived by Peller (1959) ™ = T.J -7m = (RTmTm°/mn In (1 + KDav) (3) where Tm and T":°are the midpoints of the denaturation transition temperature in the presence and absence of denaturant, is the enthalpy change of unfolding per monomer unit, is the effective number or fraction of binding sites per monomer unit, and Kb is the association or binding constant of the denaturant with the average monomer unit.…”

“…For Kb calculations with eq 4: = 1.01, 2.88, 7.59, and 20 X 10~2 for each methyl, ethyl, propyl, or butyl group contribution, and Kp = 3.2 and 2.1 X 10~2 for the polar NH(CO)NH2 and CONH2 urea and amide group contributions. The KH* are based on free-energy transfer data (Herskovits et al, 1970a) while the Kp values are calculated using the average denaturation midpoints obtained with urea and formamide of this table and Table I. The Ks values used with eq 5 were based on solubility data of V-acetyl-L-tryptophan ethyl ester data in the various ureas and amides listed (Herskovits et al, 1970b,c).…”

“…This is clearly demonstrated by the trend of the calculated 5m values based on the nonpolar, alkyl chain contributions to the binding constants, " alone, given in the central column (7) of Table IV. In contrast to the behavior of the alcohol series of denaturants (Schrier et al, 1965;Herskovits et al, 1970a) it is not possible to ignore the polar amide group contributions to the binding constants in the case of the lower members of the urea and amide series. This is not to say that hydrophobic effects are absent or have little to do with the denaturation in these solvents; the solubilizing effects of urea and formamide based on model and detergent studies (Nozaki and Tanford, 1963;Wetlaufer et al, 1964;Herskovits et al, 1970b) have been interpreted as being at least partly due to hydrophobic interactions and destabilization of micellar structures (Bruning and Holtzer, 1961; Gratzer and Beaven, 1970).…”

“…For a given series of measurements volumetric dilutions were made using common stock solutions of protein and concentrated denaturing agent, in 5or 10-ml stoppered volumetric flasks as previously described (Herskovits et al, 1970a).…”