Binding of copper(II) to thyrotropin-releasing hormone (TRH) and its analogs

Abstract

Spectroscopy (UV-Vis, 1 H NMR, ESR) and electrochemistry revealed details of the structure of the Cu(II)-TRH (pyroglutamyl-histidyl-prolyl amide) complex. The 1 H NMR spectrum of TRH has been assigned. NMR spectra of TRH in the presence of Cu(II) showed that Cu(II) initially binds TRH through the imidazole. TRH analogs, pGlu-His-Pro-OH, pGlu-(1-Me)His-Pro-amide, pGlu-His-(3,4-dehydro)Pro-amide, pGlu-His-OH, pGlu-Glu-Pro-amide, and pGlu-Phe-Pro-amide provided comparison data. The stoichiometry of the major Cu(II)-TRH complex at pH 7.45 and greater is 1:1. The conditional formation constant (in pH 9.84 borate with 12.0 mM tartrate) for the formation of the complex is above 10 5 M −1 . The coordination starts from the 1-N of the histidyl imidazole, and then proceeds along the backbone involving the deprotonated pGlu-His amide and the lactam nitrogen of the pGlu residue. The fourth equatorial donor is an oxygen donor from water. Hydroxide begins to replace the water before the pH reaches 11. Minority species with stoichiometry of Cu-(TRH) x (x = 2-4) probably exist at pH lower than 8.0. In non-buffered aqueous solutions, TRH acts as a monodentate ligand and forms a Cu(II)-(TRH) 4 complex through imidazole nitrogens. All the His-containing analogs behave like TRH in terms of the above properties.

The rotating ring-disk electrochemistry of the copper(II) complex of thyrotropin-releasing hormone

“…This is clearly not the case here with TRH. We determined earlier that equatorially bound water in Cu(II)-TRH(H À2 )(H 2 O) can lose one proton [20]. This deprotonation is likely responsible for the shift E 1/2 with pH.…”

“…Shown in Fig. 2, the absorption band does not shift with varying pH in the range of pH 7.4-11 [20], while the E 1/2 displays a linear decrease with a slope of À70 mV/pH. When either of the two reagents is in excess, the numerical value of slope gets closer to À59 mV.…”

“…The conditional Cu(II)-TRH(H À2 )(H 2 O) binding constant at pH 9.8 has been estimated to be at least 10 5 M À1 [20]. This corresponds to 90% or more bound at concentrations of 1.00 mM each Cu(II) and TRH.…”

“…The unique structure of TRH makes it interesting from a coordination chemistry and electrochemistry perspective. The probable structure of Cu(II)-TRH has been determined by spectroscopic and electrochemical means [20]. Briefly, Cu(II) binds to TRH through the pGlu amide nitrogen, the pGlu-His peptide bond nitrogen, the 1 N of the His imidazole and a water.…”

“…Preliminary electrochemical investigations in conjunction with determining the structure of the Cu(II)-TRH complex [20] showed that the electrochemistry of this complex is not simple. Because of the implications for electrochemical detection, and for the functioning of the hormone in vivo, we have investigated the electrochemistry of the complex.…”