Copper complexes of glycyl-histidyl-lysine and two of its synthetic analogues: chemical behaviour and biological activity

Copper complex formation equilibria of glycyl-L-kistidyl-r-lysine (Gly-His- Lys, GHK) and of two synthetic analogues, where the histidine residue was r eplaced with a synthetic amino acid (L-spinacine or L-1,2,3,4-tetrahydro-is oquinoline-3-carboxylic acid), have been carefully investigated using diffe rent experimental techniques: potentiometry, solution calorimetry, W-VIS sp ectrophotometry, circular dichroism and electron paramagnetic resonance spe ctroscopies. All the ligands formed complexes having different stoichiometr ies and stabilities: evidence for the formation of binuclear species is als o shown. The structures of the main complexes are discussed. It is suggeste d that the lateral lysine amino group participates in complex formation, bu t only at alkaline pH values: at physiological pH this group is protonated and available for possible interactions with cellular receptors. The above tripeptides have been tested for their enzymatic stability in human serum: the synthetic compounds showed no significant degradation for at least 3 h. Finally, their activity as growth factor has been studied in vitro. The tw o synthetic analogues showed an activity comparable to or even higher than that of GHK, thus suggesting their possible use as additives in cell cultur e media, even in the presence of serum. Relevant information on the GHK act ion mechanism as cell growth factor has been obtained: the formation of cop per complexes, driven by the first (Gly) residue, appears necessary while t he second residue (His) does not appear to play a specific role; the presen ce of the free side chain of the third residue (Lys) appears to be of funda mental importance. (C) 2001 Elsevier Science B.V. All rights reserved.