MAJOR DEGRADATION PRODUCTS OF BASIC FIBROBLAST GROWTH-FACTOR - DETECTION OF SUCCINIMIDE AND ISO-ASPARTATE IN-PLACE OF ASPARTATE

The degradation products of basic fibroblast growth factor (bFGF) were isolated by ion exchange HPLC (HP-IEC) and characterized. The predomi nant product at pH 5 was a succinimide in place of aspartate(15) as de termined by LC/MS, N-terminal sequencing, and susceptibility to degrad ation at pH >6.5. The rate of appearance of the succinimidyl-bFGF at 2 2 degrees C was comparable to that reported for small peptides, consis tent with a high flexibility predicted for asp(15)-gly. Tryptic mappin g together with [H-3]-methylation indicated that iso-aspartate was for med at the position of asp(15). Size exclusion HPLC indicated the pres ence of intact and truncated dimers and trimers which associated throu gh disulfide linkages. Two truncated monomer forms were found that co- eluted by HP-IEC; the cleavages were determined to be at asp(28)-pro a nd asp(15)-gly using LC/MS and N-terminal sequencing. These degradatio n products which occurred at sites that are away from receptor or hepa rin binding domains of bFGF remained bioactive in a cell proliferation assay.