Reversible thermal denaturation of human FGF-1 induced by low concentrations of guanidine hydrochloride.
Human acidic fibroblast growth factor (FGF-1) is a powerful mitogen and angiogenic factor with an apparent melting temperature (Tm) in the physiological range. FGF-1 is an example of a protein that is regulated, in part, by stability-based mechanisms. For example, the low Tm of FGF-1 has been postulated to play an important role in the unusual endoplasmic reticulum-independent secretion of this growth factor. Despite the close relationship between function and stability, accurate thermodynamic parameters of unfolding for FGF-1 have been unavailable, presumably due to effects of irreversible thermal denaturation. Here we report the determination of thermodynamic parameters of unfolding (DeltaH, DeltaG, and DeltaCp) for FGF-1 using differential scanning calorimetry (DSC). The thermal denaturation is demonstrated to be two-state and reversible upon the addition of low concentrations of added guanidine hydrochloride (GuHCl). DeltaG values from the DSC studies are in excellent agreement with values from isothermal GuHCl denaturation monitored by fluorescence and circular dichroism (CD) spectroscopy. Furthermore, the results indicate that irreversible denaturation is closely associated with the formation of an unfolding intermediate. GuHCl appears to promote reversible two-state denaturation by initially preventing aggregation of this unfolding intermediate, and at subsequently higher concentrations, by preventing formation of the intermediate.
Blaber
S I
SI
Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32306-4380 USA. blaber@sb.fsu.edu
Culajay
J F
JF
Khurana
A
A
Blaber
M
M
D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D016220:Fibroblast Growth Factor 1; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry*; D019791:Guanidine / Q000494:pharmacology*; D006801:Humans; D011489:Protein Denaturation / Q000187:drug effects*; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics
D016222:Fibroblast Growth Factor 2; D016220:Fibroblast Growth Factor 1; D019791:Guanidine