Characterization of keratinocyte growth factor binding to heparin and dextran sulfate

Binding of keratinocyte growth factor (KGF) with heparin (molecular weight of 5000) and dextran sulfate (molecular weight of 8000) was studied using an online monitoring of size-exclusion chromatography with light scattering, refractive index, and uv absorbance detectors. This technique allows the determination of the molecular weight of KGF eluting as complexes with the above polymers. When mixtures of KGF with heparin were injected into the column, two peaks of heparin/KGF complexes were observed. The first peak corresponded to, on average, 3.4 KGF per complex and the second peak to an average of about 2 KGF per complex. These results suggest that the heparin/ KGF complex is heterogeneous, consisting of 1,2,3, and 4 KGF molecules per complex. To calculate the number of heparin molecules in these complexes, the rate of disappearance of free KGF was determined as heparin was added. The average number of KGF bound to 1 mol of heparin was calculated to be about 2 mol, suggesting that only one heparin molecule is present in these complexes. The heparin binding of two KGF mutants, i.e., (C1, 15S)KGF (with substitutions of serine for cysteines 1 and 15) and d28KGF (lacking 28 N-terminal amino acid residues), was essentially identical to that of the native sequence KGF. A similar experiment was carried out for KGF binding to dextran sulfate. The molecular weight of the complex corresponded to 2 to 2.6 KGF molecules per complex. The rate of disappearance of free KGF as the dextran sulfate added showed 2-3 mol of KGF bound to 1 mol of dextran sulfate, consistent with the idea that the complex contains only 1 dextran sulfate molecule.