Mach H, Volkin D B, Burke C J, Middaugh C R, Linhardt R J, Fromm J R, Loganathan D, Mattsson L
Department of Pharmaceutical Research, Merck Research Laboratories, West Point, Pennsylvania 19486.
Biochemistry. 1993 May 25;32(20):5480-9. doi: 10.1021/bi00071a026.
The binding of human acidic fibroblast growth factor (aFGF) to heparin has been analyzed by a variety of different approaches to better elucidate the nature of this protein/sulfated polysaccharide interaction. Static and dynamic light scattering as well as analytical ultracentrifugation analyses indicates that 14-15 molecules of a FGF can bind to a 16-kDa heparin chain, with approximately 10 of these bound relatively uniformly to high-affinity sites. The dissociation constants of these latter sites are estimated to be approximately 50-140 nM on the basis of surface plasmon resonance experiments in which the association and dissociation rates of aFGF interaction with immobilized heparin were measured. The size of the binding site of a FGF on heparin was also determined by heparin lyase digestion of a FGF/heparin complexes followed by isolation and characterization of protected oligosaccharides. The smallest aFGF-protected oligosaccharide comigrated with delta UA2S(1-->4)-alpha-D-GlcNp2S6S(1-->4)-alpha-L-IdoAp-2S( 1-->4)-alpha-D-GlcNp2S6S (where delta UA represents 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid and S is sulfate). Thus, aFGF appears to bind at high density (one molecule every 4-5 polysaccharide units) and with high affinity to heparin. This potentially provides a concentrated, stabilized storage form of the growth factor that can be released for receptor-mediated cellular activation in response to the proper stimuli. It is also possible that close proximity of aFGF molecules on the highly sulfated regions of heparan chains may be involved in the induction of receptor aggregation as suggested by Ornitz et al. [Ornitz, D. M., Yayon, A., Flanagan, J. G., Svahn, C. M., Levi, E., & Leder, P. (1992) Mol. Cell. Biol. 12, 240-247].
人们已通过各种不同方法分析了人酸性成纤维细胞生长因子（aFGF）与肝素的结合情况，以更好地阐明这种蛋白质/硫酸化多糖相互作用的本质。静态和动态光散射以及分析超速离心分析表明，14 - 15个aFGF分子可与一条16 kDa的肝素链结合，其中约10个分子相对均匀地结合到高亲和力位点。基于表面等离子体共振实验（其中测量了aFGF与固定化肝素相互作用的结合和解离速率），这些后一种位点的解离常数估计约为50 - 140 nM。aFGF在肝素上的结合位点大小也通过对aFGF/肝素复合物进行肝素酶消化，随后分离和表征受保护的寡糖来确定。最小的aFGF保护寡糖与δUA2S(1→4)-α-D-GlcNp2S6S(1→4)-α-L-IdoAp-2S(1→4)-α-D-GlcNp2S6S（其中δUA代表4 - 脱氧-α-L-苏式-己-4-烯吡喃糖醛酸，S为硫酸根）一起迁移。因此，aFGF似乎以高密度（每4 - 5个多糖单元一个分子）且高亲和力与肝素结合。这可能提供了一种生长因子的浓缩、稳定储存形式，可在适当刺激下释放以进行受体介导的细胞激活。正如Ornitz等人所指出的[Ornitz, D. M., Yayon, A., Flanagan, J. G., Svahn, C. M., Levi, E., & Leder, P. (1992) Mol. Cell. Biol. 12, 240 - 247]，硫酸乙酰肝素链高度硫酸化区域上aFGF分子的紧密接近也可能参与受体聚集的诱导。
