Densimetric determination of equilibrium binding of sucrose octasulfate with basic fibroblast growth factor.
Fibroblast growth factors (FGFs) strongly bind to heparin and are thereby stabilized against deactivation and proteolytic cleavage. Sucrose octasulfate (SOS), which has a chemical structure resembling the repeating unit of heparin, has also been shown to enhance stability of basic FGF against thermal denaturation and to induce a small conformational change. We have examined SOS binding to bFGF using equilibrium dialysis. The difference in SOS concentration across the dialysis membrane was measured using a precision density meter, since the density of SOS differs greatly from that of water. With care, this densimetric technique can measure binding with a precision of +/- 0.1 mol/mol using about 2 mg/ml of protein. These results show that the binding saturates at 2 mol of SOS per mole of bFGF as the SOS concentration increases to 3.6 mM or higher. The effect of SOS on the thermal stability of bFGF was examined using denaturation at a constant heating rate, by both turbidity and differential scanning calorimetry. Since the thermal denaturation is irreversible, the temperature where aggregation abruptly increases was taken to indicate the onset of denaturation. This temperature increased by approximately 12 degrees C as the SOS concentration increased from 0.018 to 3.6 mM and remained constant above 3.6 mM, consistent with our binding data if the binding is specific to the native state.
Arakawa
T
T
Amgen, Inc., Amgen Center, Thousand Oaks, California 91320.
Wen
J
J
Philo
J S
JS
D000595:Amino Acid Sequence; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry* / Q000378:metabolism*; D006358:Hot Temperature; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods; D013395:Sucrose / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics
D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D013395:Sucrose; C053081:sucrose octasulfate