Deamidation of polyanion-stabilized acidic fibroblast growth factor.
The deamidation of polyanion-stabilized acidic fibroblast growth factor (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 the 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-IEF and an enzymatic ammonia assay. Native, non-deamidated aFGF (complexed with heparin) has a half-life of 16 weeks at pH 7, 30 degrees C, and 4 weeks at pH 8, 40 degrees C. The mitogenic activity and biophysical properties of deamidated aFGF were compared to the non-deamidated protein. These initial deamidation events have no significant effect on the protein's overall conformation, thermal stability, interaction with heparin, or bioactivity. At longer times, however, limited aggregation of the protein was observed after prolonged storage under some conditions. N-terminal protein sequencing of the protein's first 21 amino acid residues have identified one of the deamidation sites in a flexible, peptide-like region of the protein (Asn8-Tyr9).
Volkin
D B
DB
Department of Pharmaceutical Research, Merck Research Laboratories, West Point, PA 19486.
Verticelli
A M
AM
Bruner
M W
MW
Marfia
K E
KE
Tsai
P K
PK
Sardana
M K
MK
Middaugh
C R
CR
D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D000641:Ammonia / Q000737:chemistry; D000818:Animals; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004355:Drug Stability; D016220:Fibroblast Growth Factor 1 / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D051379:Mice; D008934:Mitogens / Q000737:chemistry / Q000494:pharmacology; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014508:Urea / Q000737:chemistry
D000577:Amides; D008934:Mitogens; D011994:Recombinant Proteins; D016220:Fibroblast Growth Factor 1; D000641:Ammonia; D014508:Urea