Stoichiometry of heparin binding to basic fibroblast growth factor.
Fibroblast growth factors (FGFs) strongly bind to heparin and are thereby stabilized against deactivation and proteolytic cleavage. We have investigated the interactions of basic fibroblast growth factor (bFGF) with low- and high-molecular-weight heparin using size exclusion chromatography with on-line light scattering, absorbance, and refractive index detection. When heparin-bFGF mixtures with excess heparin are chromatographed using eluant that does not contain heparin, essentially all the protein is seen to elute as a complex with the heparin, indicating strong binding such that the complex does not dissociate significantly during chromatography (approximately 20 min). Combining the data from the light scattering, absorbance, and refractive index chromatograms allows us to determine the molecular weight of the protein component of the complex, and therefore to measure the number of bFGF molecules bound per heparin. A series of samples were prepared with a constant concentration of bFGF and variable amounts of a low-molecular-weight heparin (LMWH, M(r) = approximately 5000). At bFGF: heparin ratios above 1.5, a mix of complexes containing 3, 2, and 1 bFGF molecules is observed, with an average of 2.2 bFGF molecules per complex. Since the amount of bFGF incorporated into complexes implies an average of 2.5 +/- 0.3 bFGF molecules per heparin, there is only one heparin molecule per complex. The coexistence of complexes of different size when bFGF is in excess implies that the LMWH molecules are heterogeneous with respect to their ability to bind bFGF. When a high-molecular-weight heparin (HMWH, M(r) = 15,000) is used, complexes averaging 6.3 bFGF molecules per HMWH molecule are seen, while the overall amount of bFGF appearing in complexes implies six to seven sites per HMWH. These data show that the protein molecules can be packed very closely together. Both types of heparin give a heparin mass of 2300 Da per bFGF binding site, which corresponds approximately to an octasaccharide.
Arakawa
T
T
Amgen Inc., Amgen Center, Thousand Oaks, California 91320.
Wen
J
J
Philo
J S
JS
D002850:Chromatography, Gel; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006493:Heparin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008433:Mathematics; D008970:Molecular Weight; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D006493:Heparin