DEAMIDATION OF POLYANION-STABILIZED ACIDIC FIBROBLAST GROWTH-FACTOR

The deamidation of polyanion-stabilized acidic fibroblast growth facto r (aFGF; FGF-1) can be induced by prolonged storage under accelerated conditions of elevated pH and temperature. A urea-isoelectric focusing (urea-IEF) method has been developed to monitor aFGF deamidation in t he presence of highly negatively charged polyanions which are required to maintain the conformational stability of the protein. The kinetics of aFGF deamidation have been established by a combination of urea-IE F and an enzymatic ammonia assay. Native, non-deamidated aFGF (complex ed with heparin) has a half-life of 16 weeks at pH 7, 30 degrees C, an d 4 weeks at pH 8, 40 degrees C. The mitogenic activity and biophysica l properties of deamidated aFGF were compared to the non-deamidated pr otein. These initial deamidation events have no significant effect on the protein's overall conformation, thermal stability, interaction wit h heparin, or bioactivity. At longer times, however, limited aggregati on of the protein was observed after prolonged storage under some cond itions. N-terminal protein sequencing of the protein's first 21 amino acid residues have identified one of the deamidation sites in a flexib le, peptide-like region of the protein (Asn(8)-Tyr(9)).