title: Partial molar volume, surface area, and hydration changes for equilibrium unfolding and formation of aggregation transition state: high-pressure and cosolute studies on recombinant human IFN-gamma.
abstract: The equilibrium dissociation of recombinant human IFN-gamma was monitored as a function of pressure and sucrose concentration. The partial molar volume change for dissociation was -209 +/- 13 ml/mol of dimer. The specific molar surface area change for dissociation was 12.7 +/- 1.6 nm2/molecule of dimer. The first-order aggregation rate of recombinant human IFN-gamma in 0.45 M guanidine hydrochloride was studied as a function of sucrose concentration and pressure. Aggregation proceeded through a transition-state species, N*. Sucrose reduced aggregation rate by shifting the equilibrium between native state (N) and N* toward the more compact N. Pressure increased aggregation rate through increased solvation of the protein, which exposes more surface area, thus shifting the equilibrium away from N toward N*. The changes in partial molar volume and specific molar surface area between the N* and N were -41 +/- 9 ml/mol of dimer and 3.5 +/- 0.2 nm2/molecule, respectively. Thus, the structural change required for the formation of the transition state for aggregation is small relative to the difference between N and the dissociated state. Changes in waters of hydration were estimated from both specific molar surface area and partial molar volume data. From partial molar volume data, estimates were 25 and 128 mol H2O/mol dimer for formation of the aggregation transition state and for dissociation, respectively. From surface area data, estimates were 27 and 98 mol H2O/mol dimer. Osmotic stress theory yielded values approximately 4-fold larger for both transitions.
authors.affiliation: Department of Chemical Engineering, University of Colorado, Boulder, CO 80309, USA.
authors.forename: J N
authors.initials: JN
authors.lastname: Webb
authors.forename: S D
authors.initials: SD
authors.lastname: Webb
authors.forename: J L
authors.initials: JL
authors.lastname: Cleland
authors.forename: J F
authors.initials: JF
authors.lastname: Carpenter
authors.forename: T W
authors.initials: TW
authors.lastname: Randolph
mesh_terms: D019281:Dimerization; D019791:Guanidine; D006801:Humans; D007371:Interferon-gamma / Q000737:chemistry*; D007700:Kinetics; D008962:Models, Theoretical; D011312:Pressure; D011489:Protein Denaturation; D011994:Recombinant Proteins; D013053:Spectrophotometry; D013499:Surface Properties; D013816:Thermodynamics; D014867:Water
chemical_list: D011994:Recombinant Proteins; D014867:Water; D007371:Interferon-gamma; D019791:Guanidine