BINDING OF THE GROWTH-FACTOR GLYCYL-L-HISTIDYL-L-LYSINE BY HEPARIN

Evidence is presented that the growth factor glycyl-histidyl-lysine (G HK) binds to heparin, and the interaction has been characterized by [H -1]NMR spectroscopy. H-1 chemical shifts indicate that GHK interacts w ith both the carboxylic acid and the carboxylate forms of heparin. The chemical shift data are consistent with a weak delocalized binding of the triprotonated (ImH(+), GlyNH(3)(+), LysNH(3)(+)) form of GHK by t he carboxylic acid form of heparin. As the pD is increased and the car boxylic acid groups are titrated, chemical shift data indicate that am monium groups of GHK are hydrogen bonded to heparin carboxylate groups , while the histidyl imidazolium ring occupies the imidazolium-binding site of heparin. Evidence for site-specific binding includes displace ment of chemical shift titration curves for heparin to lower pD, incre ased shielding of specific heparin protons by the imidazolium ring cur rent and displacement of chemical shift titration curves for GHK to hi gher pD. Specific binding constants were determined for binding of the (ImH(+), GlyNH(3)(+)), LysNH(3)(+)), (ImH(+), GlyNH(2), LysNH(3)(+)) and (Im, GlyNH(3)(+), LysNH(3)(+)) forms of GHK by the carboxylate for m of heparin from chemical shift vs. pD titration data.