Thermal access to amplified chemical potential and the determination of equilibrium constants in protein solutions at subfreezing temperatures.
During rapid cooling of ferric heme protein solutions containing fluoride, locally concentrated ligand cannot fully equilibrate with heme before the temperature drops below 200 K and into the range where energy is insufficient for exchange with iron-bound water. When temperature is then jumped above 200 K, exchange of fluoride for bound water is activated. Between 200 and 240 K, further fluoride complex formation takes place over several minutes; its extent is measured along the kinetic curve by reimmersing the sample into liquid nitrogen and taking EPR spectra. Kinetic curves for replacement of iron-bound water by fluoride in horse aquo-ferrimyoglobin and human aquo-ferrihemoglobin, and corresponding equilibrium constants have been obtained at temperatures between 200 and 240 K. The reaction rates are affected by sucrose. Results indicate that the kinetics of exchange of fluoride for heme-bound water at subfreezing temperatures is protein specific and not diffusion-controlled, and is not affected by the phase transition of ice which takes place at subfreezing temperature. Free energy changes accompanying these reactions are largely continuous as the systems pass from above to below freezing.
Yang
A S
AS
Department of Physics and Biophysics Program, University of Virginia, Charlottesville, 22901, USA.
Brill
A S
AS
D000818:Animals; D005615:Freezing; D006420:Hemeproteins / Q000737:chemistry*; D006736:Horses; D007700:Kinetics; D011487:Protein Conformation; D013696:Temperature; D013816:Thermodynamics; D014783:Viscosity
D006420:Hemeproteins