title: Strategy for fluorescent labeling of human acidic fibroblast growth factor without impairment of mitogenic activity: a bona fide tracer.
abstract: Here we describe, for the first time, the design and characterization of a bona fide fluorescently labeled mutant of the human acidic fibroblast growth factor (aFGF). The aFGF-Cys2 mutant was recombinantly synthesized by substituting the second amino acid with a reactive cysteine whose sulfhydryl group's side chain reactivity facilitated the covalent binding of a fluorescent probe as a thiolyte monobromobimane. Using a combination of biophysical and functional assays, we found that the fluorescently labeled mutant aFGF is characterized by essentially the same global folding, mitogenic activity, and association behavior with heparin, its physiological activator, as the unlabeled wild-type protein. We used this new tracer protein mutant to determine the association behavior of aFGF with heparin in the presence of high concentrations of albumin that mimicked more closely the plasma medium in which aFGF is naturally located and in which it has evolved to function. By exposing the aFGF-Cys2-heparin complex to increasing concentrations of albumin up to physiological plasma levels, we were able to demonstrate that macromolecular crowding does not affect the stoichiometry of the interaction. In summary, the dimeric aFGF-Cys2-heparin complex might represent a biologically relevant complex in physiological media.
authors.affiliation: Department of Biochemistry and Molecular Biology I, Universidad Complutense, 28040 Madrid, Spain.
authors.forename: M José
authors.initials: MJ
authors.lastname: Feito
authors.forename: Mercedes
authors.initials: M
authors.lastname: Jiménez
authors.forename: Concha
authors.initials: C
authors.lastname: Fernández-Cabrera
authors.forename: Germán
authors.initials: G
authors.lastname: Rivas
authors.forename: Guillermo
authors.initials: G
authors.lastname: Giménez-Gallego
authors.forename: Rosa M
authors.initials: RM
authors.lastname: Lozano
mesh_terms: D001643:Bridged Bicyclo Compounds / Q000378:metabolism*; D003470:Culture Media / Q000494:pharmacology; D003545:Cysteine / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D016220:Fibroblast Growth Factor 1 / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D008934:Mitogens / Q000378:metabolism* / Q000494:pharmacology; D050505:Mutant Proteins / Q000378:metabolism; D017510:Protein Folding / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D001643:Bridged Bicyclo Compounds; D003470:Culture Media; D005456:Fluorescent Dyes; D008934:Mitogens; D050505:Mutant Proteins; D016220:Fibroblast Growth Factor 1; D003545:Cysteine; C028381:monobromobimane