The Cu(II) and Zn(II) binding abilities of Gly-His-Thr-Asp-amide (GHTD-am), a tetrapeptide coreleased from the pancreas along with insulin, were studied using UV-vis and circular dichroism spectroscopies, potentiometry, and calorimetry. GHTD-am is a very strong Cu(II) chelator, forming a three-nitrogen complex with a conditional affinity constant K-c at pH 7.4 of 4.5 X 10(12) M-1. The fourth coordination site can be occupied by a solvent molecule or a ternary ligand, such as imidazole, with K-c on the order of several hundred reciprocal molar. The Zn(II) binding ability of GHTD-am is relatively weak, with K-c values at pH 7.4 of 3.0 X 10(4) and 2.0 x 10(3) for the first and second GHTD-am molecule coordinated, respectively. These results are discussed in light of the modes of interactions of Zn(II) and Cu(II) ions with insulin. A direct effect of GHTD-am on the Zn(II) interactions with insulin is unlikely, but its Cu(II) complex may have a biological relevance because of its high affinity and ability to form ternary complexes.