Influence of the freezing process upon fluoride binding to hemeproteins

Abstract

Fluoride association with ferric myoglobins and hemoglobins in aqueous buffers above freezing has been well studied. We chose this reaction to investigate the feasibility of observing titration intermediates and estimating dissociation constants at the freezing temperature by electron paramagnetic resonance spectroscopy at cryogenic temperatures. Dependence of apparent dissociation constant upon protein concentration was observed, a factor of four decrease in protein accompanied by about a fourfold increase in the apparent tightness of binding in the range of protein concentration studied. Binding was also found to depend upon cooling rate and concentration of additives (serum albumin, sucrose, glycerol). These effects appear to be associated with freezing-induced concentration of ligand, a process described in the literature. Bands of high concentration of electrolyte accompany solute rejection during ice growth, sweeping by slowing moving macromolecules. Thus, just before being trapped in the solid, the protein can experience a greater concentration of salt than in the original liquid. A mathematical model of this process, based upon simplifying assumptions about nucleation and ice-crystal growth rates in super-cooled solution, shows how the average concentration of mobile solute species can depend upon the concentration of all species present. Semiquantitative computer simulations of the actual, more complex, freezing are also presented and lead to estimates of ice particle size which are then compared with estimates from the former model.