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 fluor ide, locally concentrated ligand cannot fully equilibrate with heme be fore 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 ac tivated. Between 200 and 240 K, further fluoride complex formation tak es place over several minutes; its extent is measured along the kineti c curve by reimmersing the sample into liquid nitrogen and taking EPR spectra. Kinetic curves for replacement of iron-bound water by fluorid e in horse aquo-ferrimyoglobin and human aquo-ferrihemoglobin, and cor responding equilibrium constants have been obtained at temperatures be tween 200 and 240 K. The reaction rates are affected by sucrose. Resul ts indicate that the kinetics of exchange of fluoride for heme-bound w ater 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 accompanyi ng these reactions are largely continuous as the systems pass from abo ve to below freezing.