Thermodynamic parameters associated with guanidine HCl- and temperature-induced unfolding of bFGF

Abstract

Purpose: The thermodynamics and kinetics associated with either thermal- or chaotrope-induced unfolding of recombinant, human basic fibroblast growth factor (bFGF) was characterized using several analytical techniques. Method: Changes in the three-dimensional conformation of bFGF were detected using fluorescence spectroscopy and heparin affinity chromatography, and the observed data analyzed using a simple two-state [N↔D] model to estimate the value of several thermodynamic parameters. Results: Treatment of thermal denaturation data yielded mean values for the T m (temperature when Δ G=0) of 322±21 K (49.3±3.2°C), Δ H m (change in enthalpy when Δ G=0) of 214±23.8 kJ/mol, and Δ S (change in entropy) of 664±40.2 J mol −1 K −1. Treatment of the GnHCl-induced denaturation data yielded mean values of the Δ G (H 2O) (change in free energy in the absence of the denaturant) of 13.0±0.67 kJ/mol and the GnHCl 1/2 (concentration of GnHCl when Δ G=0) of 1.3±0.09 M. Sucrose (0.26 M) was found to be an effective stabilizer against thermal-induced denaturation of bFGF. Renaturation of bFGF which had previously been denatured with 3 M GnHCl was demonstrated following dialysis. However, renaturation of bFGF following heat-induced denaturation was significantly inhibited. Conclusions: Using fluorescence spectroscopy, we have estimated the value of several thermodynamic parameters associated with bFGF unfolding induced by either chemical or thermal means.