Kinetics of recombinant human brain-derived neurotropic factor unfolding under reversed-phase liquid chromatography conditions.

A model describing the pathway of unfolding of recombinant human brain-derived neurotrophic factor (rhBDNF) under reversed-phase high-performance liquid chromatographic conditions is introduced. The unfolding process is divided into two major steps. The first entails a series of events such as the dissociation of the dimer and the effect of the initial contact of the protein with the stationary phase. The second step is the unfolding of the monomer molecule on the hydrophobic surface exhibiting a rate constant comparable with the time scale of chromatography. The kinetics of rhBDNF unfolding is studied with respect to the type and composition of the organic solvents and as function of the temperature of chromatography. The experimental results validate the suggested multistep unfolding model. The activation energy of monomer unfolding in both solvent systems studied is similar, but the unfolding processes are different in 1-propanol and in acetonitrile.