Binding of hIL-10 to heparin agarose.

(A) Amino acid sequence of the C-terminus of human IL-10. Basic residues appear in gray. The sequence −LRRCHR-(105-110), which is similar to one consensus site, XBBXBX (where B represents a cationic lysine, arginine, or histidine residue and X represents a hydropathic residue) and is described by several investigators16,18-20in GAG-binding proteins, is underlined. The sequence is taken from Moore et al13 and Vieira et al.14 (B) High-affinity binding of hIL-10 to heparin. Factor Xa-cleaved products were loaded onto a heparin–agarose column, and the binding of hIL-10 was analyzed on SDS-PAGE (14%) and visualized with Coomassie blue staining. Lanes 1 and 10, molecular weight markers in kd; lane 2, factor Xa-cleaved products before loading onto the heparin column (C); lane 3, run through from the column (RT); lane 4, wash from the column (W); lanes 5 to 8, eluate after 100 mmol/L NaCl; lanes 9 and 11 to 13, eluate after 300 mmol/L NaCl; lanes 14 to 17, elute after 600 mmol/L NaCl; lane 18, elute after 1 mol/L NaCl. MBP–hIL-10 fusion protein, MBP, factor Xa, and hIL-10 are indicated.