Effect of polyanions on the refolding of human acidic fibroblast growth factor.
Acidic fibroblast growth factor (aFGF) is unstable at physiological temperatures in the absence of polyanions such as heparin. Therefore, the effect of temperature on the kinetics of refolding of aFGF has been examined in the presence and absence of several polyanions. The protein folds into its native state at temperatures up to 30 degrees C without polyanions with an activation energy of approximately 14 kcal/mol, but does not acquire native structure above this temperature. When heparin, inositol hexasulfate, or sulfate ion are present, aFGF refolds below 30 degrees C with a slightly reduced activation energy (10-11 kcal/mol). In addition, the protein now also renatures between 30 and 50 degrees C with activation energies of 1-2 (heparin), 16 (inositol hexasulfate), and 7 (sulfate) kcal/mol. Trace heavy metals appear to inhibit the refolding process, but a molecular chaperone (bovine 70-kDa heat shock cognate protein) and a peptidylprolyl isomerase (the FK506-binding protein) have no effect. It is concluded that the rate of refolding of aFGF at physiological temperatures is probably controlled by the interaction of a native-like state of the protein with an unknown polyanionic species.
Dabora
J M
JM
Department of Pharmaceutical Research, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486.
Sanyal
G
G
Middaugh
C R
CR
D000838:Anions; D002151:Calorimetry; D004492:Edetic Acid / Q000494:pharmacology; D016220:Fibroblast Growth Factor 1 / Q000737:chemistry*; D006493:Heparin / Q000494:pharmacology*; D006801:Humans; D007294:Inositol / Q000031:analogs & derivatives / Q000494:pharmacology; D007700:Kinetics; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013431:Sulfates / Q000494:pharmacology
D000838:Anions; D011994:Recombinant Proteins; D013431:Sulfates; C012036:sodium sulfate; D016220:Fibroblast Growth Factor 1; C013063:inositol hexasulfate; D007294:Inositol; D006493:Heparin; D004492:Edetic Acid