Ternary Cu²⁺ Complexes of Human Serum Albumin and Glycyl-l-histidyl-l-lysine

Human serum albumin (HSA) and the growth factor glycyl-l-histidyl-l-lysine (GHK) bind Cu²⁺ as part of their normal functions. GHK is found at its highest concentration in the albumin-rich fraction of plasma, leading to speculation that HSA and GHK form a ternary Cu²⁺ complex. Although preliminary evidence was presented 40 years ago, the structure and stability of such a complex have remained elusive. Here, we show that two ternary Cu(GHK)N_Im^HSA complexes are formed between GHK and the imino nitrogen (N_Im) of His side chains of HSA. We identified His3 as one site of ternary complex formation (conditional binding constant K_Cu(GHK)N^Cu(GHK) = 2900 M^-1 at pH 7.4), with the second site (K_Cu(GHK)N^Cu(GHK) = 1700 M^-1) likely being supplied by either His128 or His510. Together with the established role of HSA as a molecular shuttle in the blood, these complexes may aid the transport of the exchangeable Cu²⁺ pool and the functional form of GHK.