Surface plasmon resonance analysis to evaluate the importance of heparin sulfate groups' binding with human aFGF and bFGF.
Human acidic and basic fibroblast growth factors (aFGF and bFGF) are classic and well characterized members of the heparin-binding growth factor family. Heparin is generally thought to play an extremely important role in regulating aFGF and bFGF bioactivities through its strong binding with them. In order to unravel the mechanism of the interactions between heparin and FGFs, and evaluate the importance of heparin sulfate groups' binding with FGFs, surface plasmon resonance analyses were performed using IAsys Cuvettes System. Heparin and its regioselectively desulfated derivatives were immobilized on the cuvettes. aFGF and bFGF solutions with different concentrations were pipetted into the cuvettes and the progress of the interaction was monitored in real-time by Windows-based software, yielding kinetic and equilibrium constants for these interactions. In addition, in order to reduce the delicate difference among the cuvettes, inhibition analyses of mixture of FGFs and immobilized native heparin by modified heparins were also done. The data from these two methods were similar, indicating that all sulfate groups at 2-O, 6-O and N- in heparin were required for the binding to aFGF; and that their contribution to the binding was in the order 2-O, N- and 6-O-sulfate group. In contrast, definite contribution of the 6-O-sulfate group to the binding with bFGF was most apparent, while the other two sulfate groups appeared to be necessary in the order 2-O and N-sulfate group. These methods established here can be used for analysing the effect of sulfate groups in heparin on the binding with other human FGF members or other heparin-binding proteins.
Wu
Xiao-feng
XF
College of Animal Sciences, Zhejiang University, Huajiachi Campus, Hangzhou 310029, China. wuxiaofeng@zju.edu.cn
Xu
Ya-xiang
YX
Shen
Guo-xin
GX
Kamei
Kaeko
K
Takano
Ryo
R
Hara
Saburo
S
D000327:Adsorption; D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D005346:Fibroblast Growth Factors / Q000737:chemistry / Q000378:metabolism*; D006493:Heparin / Q000737:chemistry / Q000378:metabolism*; D006497:Heparan Sulfate / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013237:Stereoisomerism; D020349:Surface Plasmon Resonance
D005346:Fibroblast Growth Factors; D006493:Heparin; D006497:Heparan Sulfate; D011994:Recombinant Proteins; D012965:Sodium Chloride