Capillary electrophoresis and enzyme solid phase assay for examining the purity of a synthetic heparin proteoglycan-like conjugate and identifying binding to basic fibroblast growth factor.
Interaction of basic fibroblast growth factor (bFGF) with heparin/heparan sulfate proteoglycans protects the growth factor against proteolytic degradation and is essential for its cellular activity. Although the structural requirements of heparin and heparan sulfate for the high-affinity binding to bFGF have been extensively examined, studies on intact heparin proteoglycans are limited. In this report, the purity and the binding ability of a heparin proteoglycan-like molecule-the heparin-bovine serum albumin (heparin-BSA) conjugate-was examined using capillary zone electrophoresis (CZE). Furthermore, the affinity of bFGF binding to the heparin-BSA conjugate was studied using an enzyme solid-phase assay. Chondroitin sulfate, dermatan sulfate, hyaluronan, heparan sulfate and variously sulfated disaccharides derived from heparin and heparan sulfate were also studied for their ability to compete with the binding of bFGF to heparin. Heparin-BSA conjugate was synthesized by reductive amination and, following precipitation with 1.5 vols of ethanol-sodium acetate, it was obtained free of contaminating heparin. Heparin-BSA-bFGF conjugate was obtained following incubation of heparin-BSA with bFGF for 2 h at 37 degrees C. Intact heparin, heparin-BSA and heparin-BSA-bFGF conjugates were completely resolved by CZE using 50 mM phosphate, pH 3.5, as operating buffer, reversed polarity (30 kV) and detection at 232 nm. Competitive solid phase assay showed that, among the glycosaminoglycans tested, heparin exhibits the highest affinity binding to bFGF (IC(50) = 6.4 nM). Heparan sulfate showed a lower affinity as compared with that of heparin, whereas all other glycosaminoglycans and heparin/heparan sulfate-derived disaccharides tested showed minute effects. The developed CZE method is rapid and accurate and can be easily used to identify bFGF-interacting heparin preparations of biopharmaceutical importance.
Dimitrellos
V
V
Department of Chemistry, Section of Organic Chemistry, Biochemistry and Natural Products, Laboratory of Biochemistry, University of Patras, 261 10 Patras, Greece.
Lamari
F N
FN
Militsopoulou
M
M
Kanakis
I
I
Karamanos
N K
NK
D019075:Electrophoresis, Capillary / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism*; D006493:Heparin / Q000302:isolation & purification* / Q000378:metabolism; D011509:Proteoglycans / Q000302:isolation & purification* / Q000378:metabolism
D011509:Proteoglycans; D016222:Fibroblast Growth Factor 2; D006493:Heparin