CHARACTERIZATION OF KERATINOCYTE GROWTH-FACTOR BINDING TO HEPARIN ANDDEXTRAN SULFATE

Binding of keratinocyte growth factor (KGF) with heparin (molecular we ight of 5000) and dextran sulfate (molecular weight of 8000) was studi ed using an online monitoring of size-exclusion chromatography with li ght scattering, refractive index, and uv absorbance detectors. This te chnique allows the determination of the molecular weight of KGF elutin g as complexes with the above polymers, When mixtures of KGF with hepa rin 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 t he number of heparin molecules in these complexes, the rate of disappe arance of free KGF was determined as heparin was added. The average nu mber of KGF bound to 1 mol of heparin was calculated to be about 2 mel , suggesting that only one heparin molecule is present in these comple xes. The heparin binding of two KGF mutants, i.e., (C1,15S)KGF (with s ubstitutions 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 corres ponded to 2 to 2.6 KGF molecules per complex. The rate of disappearanc e 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 comple x contains only 1 dextran sulfate molecule. (C) 1996 Academic Press, I nc.