THERMODYNAMIC PARAMETERS ASSOCIATED WITH GUANIDINE HCL-INDUCED AND TEMPERATURE-INDUCED UNFOLDING OF BFGF

Purpose: The thermodynamics and kinetics associated with either therma l- or chaotrope-induced unfolding of recombinant, human basic fibrobla st growth factor (bFGF) was characterized using several analytical tec hniques. Method: Changes in the three-dimensional conformation of bFGF were detected using fluorescence spectroscopy and heparin affinity ch romatography, and the observed data analyzed using a simple two-state [N <----> D] model to estimate the value of several thermodynamic para meters. Results: Treatment of thermal denaturation data yielded mean v alues for the T-m (temperature when Delta G = 0) of 322 +/- 21 K (49.3 +/- 3.2 degrees C), Delta H-m (change in enthalpy when Delta G = 0) o f 214 +/- 23.8 kJ/mol, and Delta S (change in entropy) of 664 +/- 40.2 J mol(-1) K-1. Treatment of the GnHCl-induced denaturation data yield ed mean values of the Delta G((H2O)) (change in free energy in the abs ence of the denaturant) of 13.0 +/- 0.67 kJ/mol and the GnHCl(1/2) (co ncentration of GnHCl when Delta G = 0) Of 1.3 + 0.09 M. Sucrose (0.26 M) was found to be an effective stabilizer against thermal-induced den aturation of bFGF. Renaturation of bFGF which had previously been dena tured with 3 M GnHCl was demonstrated following dialysis. However, ren aturation of bFGF following heat-induced denaturation was significantl y inhibited. Conclusions: Using fluorescence spectroscopy, we have est imated the value of several thermodynamic parameters associated with b FGF unfolding induced by either chemical or thermal means. (C) 1998 El sevier Science B.V. All rights reserved.