THE STABILITY OF BFGF AGAINST THERMAL-DENATURATION

The influence of sulphated ligand and pH on thermal denaturation of ba sic fibroblast growth factor (bFGF) was investigated by differential s canning calorimetry (DSC), and verified by fluorescence spectrophotome try. Purity of bFGF before and after heat denaturation was assessed by SDS-PAGE analysis. In DSC studies the samples were heated to 95-degre es-C. The midpoint of the temperature change in the thermogram was des ignated as T(m). Sulphated ligand experiments were undertaken in potas sium phosphate (pH 6-5) and sodium acetate buffers. Control thermogram s (with no ligand) showed a T(m) at 59-degrees-C in potassium phosphat e buffer. Higher T(m) values were noted as sulphated ligand concentrat ion was increased. Similarly when heparin was added, the T(m) moved to a higher temperature. A ratio as low as 0.3: 1 of heparin to bFGF, in creased the T(m( to 90-degrees-C, which is a 31-degrees-C shift in T(m ). The effect of pH on thermal denaturation of bFGF was studied in a c itrate-phosphate-borate buffer system. A shift in T(m) from 46 to 65-d egrees-C was observed as the pH is changed from 4 to 8. Changes in pro tein conformation as a function of pH were monitored by fluorescence s pectroscopy. It was found that a pH range from 5 to 9 is optimal for t he stability of bFGF formulations. In a stability study it was noted t hat heparin protected bFGF from thermal denaturation only at high temp erature.