Faculty of Chemistry, University of Wrocław, Joliot-Curie 14, 50-383 Wroclaw, Poland.
Inorg Chem. 2011 Aug 15;50(16):7489-99. doi: 10.1021/ic2002942. Epub 2011 Jul 19.
The stability constants, stoichiometry, and solution structures of copper(II) complexes of neuropeptide gamma (NPG) (D(1)-A-G-H(4)-G-Q-I-S-H(9)-K-R-H(12)-K-T-D-S-F-V-G-L-M(21)-NH(2)) and acethyl-neuropeptide gamma (Ac-D(1)-A-G-H(4)-G-Q-I-S-H(9)-K-R-H(12)-K-T-D-S-F-V-G-L-M(21)-NH(2)) were determined in aqueous solution. For both peptides the additional deprotonations were observed; therefore, the potentiometric data calculations for NPG were only made in 2.5-7.4 pH range. For Ac-NPG one additional deprotonation was observed, likely hydroxy group of Ser residue, and the potentiometric data calculations in the 2.5-10.5 pH range may be performed. The potentiometric and spectroscopic data (UV-vis, CD, EPR) for the neuropeptide gamma show that a D(1) residue stabilizes significantly the copper(II) complexes with 1N {NH(2),β-COO(-)}, 2N {NH(2),β-COO(-),N(Im)}, and 3N {NH(2),β-COO(-),2N(Im)} coordination modes as the result of coordination through the β-carboxylate group. The Ac-NPG forms with the copper(II) ions the 3N {3N(Im)} complex in a wide 4.5-7.5 pH range. At higher pH deprotonation and coordination of the sequential amide nitrogens occur. Metal-catalyzed oxidation of proteins is mainly a site-specific process in which amino acids at metal-binding sites to the protein are preferentially oxidized. To elucidate the products of the copper(II)-catalyzed oxidation of NPG and Ac-NPG the liquid chromatography-mass spectrometry method (LC-MS) and the Cu(II)/H(2)O(2) as a model oxidizing system were employed. For solutions containing a 1:4 peptide-hydrogen peroxide molar ratio oxidation of the methionine residue to methionine sulphone was observed. For the 1:1:4 Cu(II)-NPG-H(2)O(2) system oxidation of two His residues and cleavage of the G(3)-H(4) and R(11)-H(12) peptide bonds were detected, supporting involvement of His(4) and His(12) in binding of the copper(II) ions. Oxidations of three histidine residues to 2-oxohistidines and fragmentations of Ac-NPG near the His (H(4), H(9),H(12)) residues support participation of the histidyl-imidazole nitrogen atoms in coordination of the metal ions.
在水溶液中测定了神经肽 γ（NPG）（D（1）-A-G-H（4）-G-Q-I-S-H（9）-K-R-H（12）-K-T-D-S-F-V-G-L-M（21）-NH（2））和乙酰神经肽 γ（Ac-D（1）-A-G-H（4）-G-Q-I-S-H（9）-K-R-H（12）-K-T-D-S-F-V-G-L-M（21）-NH（2））与铜（II）配合物的稳定常数、化学计量比和溶液结构。对于两种肽，观察到额外的去质子化；因此，仅在 2.5-7.4 pH 范围内对 NPG 进行了电位测定数据计算。对于 Ac-NPG，观察到一个额外的去质子化，可能是丝氨酸残基的羟基，并且可以在 2.5-10.5 pH 范围内进行电位测定数据计算。神经肽 γ 的电位和光谱数据（UV-vis、CD、EPR）表明，D（1）残基通过β-羧酸盐基团的配位，显著稳定了具有 1N{NH（2），β-COO（-）}、2N{NH（2），β-COO（-），N（Im）}和 3N{NH（2），β-COO（-），2N（Im）}配位模式的铜（II）配合物。Ac-NPG 在较宽的 4.5-7.5 pH 范围内与铜（II）离子形成 3N{3N（Im）}配合物。在较高的 pH 值下，会发生连续酰胺氮的去质子化和配位。蛋白质的金属催化氧化主要是一个特异性位点的过程，其中金属结合部位的氨基酸优先被氧化。为了阐明铜（II）催化氧化 NPG 和 Ac-NPG 的产物，采用了液相色谱-质谱法（LC-MS）和 Cu（II）/H（2）O（2）作为模型氧化体系。对于含有 1:4 肽-过氧化氢摩尔比的溶液，观察到甲硫氨酸残基向甲硫氨酸砜的氧化。对于 1:1:4 Cu（II）-NPG-H（2）O（2）体系，检测到两个 His 残基的氧化和 G（3）-H（4）和 R（11）-H（12）肽键的断裂，支持 His（4）和 His（12）参与铜（II）离子的结合。三个组氨酸残基被氧化为 2-氧代组氨酸，以及 Ac-NPG 靠近 His（H（4）、H（9）、H（12））残基的片段化，支持组氨酸咪唑氮原子参与金属离子的配位。
