The Stability of bFGF Against Thermal Denaturation

Abstract

The influence of sulphated ligand and pH on thermal denaturation of basic fibroblast growth factor (bFGF) was investigated by differential scanning calorimetry (DSC), and verified by fluorescence spectrophotometry. Purity of bFGF before and after heat denaturation was assessed by SDS-PAGE analysis. In DSC studies the samples were heated to 95 degrees C. The midpoint of the temperature change in the thermogram was designated as Tm. Sulphated ligand experiments were undertaken in potassium phosphate (pH 6.5) and sodium acetate buffers. Control thermograms (with no ligand) showed a Tm at 59 degrees C in potassium phosphate buffer. Higher Tm values were noted as sulphated ligand concentration was increased. Similarly when heparin was added, the Tm moved to a higher temperature. A ratio as low as 0.3:1 of heparin to bFGF, increased the Tm to 90 degrees C, which is a 31 degrees C shift in Tm. The effect of pH on thermal denaturation of bFGF was studied in a citrate-phosphate-borate buffer system. A shift in Tm from 46 to 65 degrees C was observed as the pH is changed from 4 to 8. Changes in protein conformation as a function of pH were monitored by fluorescence spectroscopy. It was found that a pH range from 5 to 9 is optimal for the stability of bFGF formulations. In a stability study it was noted that heparin protected bFGF from thermal denaturation only at high temperature.

Heparin Dodecasaccharide Binding to Platelet Factor-4 and Growth-related Protein-α

“…This may explain why only certain antibodies specific for PF4: heparin are associated with adverse symptomatology (54). Furthermore, even though heparin is usually found to stabilize the conformation of a protein against thermal (55,56) or chemical denaturation (57), it can function in various ways. For example, the addition of heparin tetra-and decasaccharides to a solution of aFGF induces formation of higher molecular weight aggregates (58).…”

Identification of a Novel Domain of Fibroblast Growth Factor 2 Controlling Its Angiogenic Properties

“…Fluorescence emission spectra were collected by excitation at 277 nm. Peak fluorescence was at 310 nm as previously reported for similar experimental conditions (43). Data were corrected for the buffer, FREG-(48 -58), and SCR contributions and for the dilution factor, and its value was plotted as a function of the amount of peptide supplemented.…”

Heparin-functionalized chitosan–alginate scaffolds for controlled release of growth factor

“…Such a strong affinity to heparin is helpful to protect bFGF from denaturation and enzymatic degradation (Vemuri et al, 1994). Therefore, heparin in the ECM plays a major role in storing growth factors, slowing their release while retaining their bioactivity (Baskin et al, 1989).…”