Determination of a Binding Site of Cu, Ni, Mg, and Ca Metal Ions with Angiotensin II Peptide by Electrospray Tandem Mass Spectrometry

Abstract

Angiotensin II (Ang II) is the main active hormone in the renin-angiotensin blood pressure regulation system. 1 Ang II is an octapeptide hormone (Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8, DRVYIHPF) that has been studied for several decades in the structure-dependent activity of the biological pathway of the Ang II receptor. [2][3][4][5][6][7][8][9][10][11][12][13] Free Ang II molecular structures in solutions have been investigated with a variety of techniques. The results have been reported as β-turn, random coil, hair-pin and other structures.2-8 The structures of Ang II complexed with a receptor have been reported as a hair-pin or an extended structure in NMR and crystallography studies. [9][10][11][12][13] It is evident that several reports are not consistent with each other and that no clear agreement exists regarding the biologically active structure of Ang II during the biological process.For the study of blood pressure control and metal-peptide interaction, the influences of metal cations on the biological activity of Ang II have been also investigated. , and Ca 2+ ) and the Ang II molecule to explain the biological effect in terms of a gas-phase structural difference. We present the results of a tandem mass spectrometry and multiple mass spectrometry study regarding the interaction of metal ions with the Ang II molecule, the C-terminus amidated Ang II molecule (DRVYIHPF-NH 2 ), and two of its segments: the N-terminus tetrapeptide Asp1-Arg2-Val3-Tyr4-NH2 (DRVY-NH2) and the N-terminus hexapeptide Asp1-Arg2-Val3-Tyr4-Ile5-His6-NH2 (DRVYIH-NH2). Two N-terminus segments are used to confirm the [metal II + Ang II] 2+ complex formation efficiency and the metal binding site. Experimental SectionThe gas-phase [metal II + Ang II] 2+ cation complexes were produced by an electrospray ionization source. The experimental MS/MS and MS/MS/MS spectra for fragmentation pattern analysis were obtained using a Thermo Finnigan LTQ mass spectrometer (Thermo Electron Corp., San Jose, CA, USA). This mass spectrometer is a linear ion trap mass spectrometer equipped with an atmospheric pressure-ionization source. LTQ conditions. The samples were introduced into the electrospray interface by a direct infusion method using a microsyringe pump (SGE, Australia) at a flow rate of 5 µL min. The MS/MS spectra were acquired with experimental conditions of an isolation width of 0.5 -1 mass unit, an activation time of 30 msec and q z = 0.25. In MS/MS or MS/MS/MS mode, the parent ion molecules were manually selected one by one, and each was subjected to collision-induced dissociation (CID).Reagents. Ang II human (96%, Fluka), DRVYIHPF-NH2 (> 90%, Peptron, Daejeon, Korea), DRVY-NH2 (> 90%, Peptron, Daejeon, Korea), DRVYIH-NH2 (> 90%, Peptron, Daejeon, Korea), CuCl 2 ․2H 2 O (99%, Sigma-Aldrich Korea), Ni(NO 5 ) 2 ․ 6H2O (97%, Junsei chemical Co., Tokyo, Japan), CaCl2․2H2O (98%, Daejung Chemical Korea), Mg(NO3)2․6H2O (98%, Junsei chemical Co., Tokyo, Japan), and H2O (HPLC grade, Merck) were used in experiments. Ang II was dissolved in water to...

Copper-Catalyzed Tyrosine Nitration

“…In Figure a, the peaks of Ang I monocoordinated copper(II) are observed, demonstrating the presence of Ang I–Cu 2+ during the Ang I nitration. According to the tandem MS results shown in Supporting Information S-9, Cu 2+ should bind to the carbonyl group on the backbone between valine and tyrosine, in accordance with the literature . The structure of Ang I–Cu 2+ is shown in Scheme .…”

“…According to the tandem MS results shown in Supporting Information S-9, Cu 2+ should bind to the carbonyl group on the backbone between valine and tyrosine, in accordance with the literature. 60 The structure of Ang IÀCu 2+ is shown in Scheme 3. Because it has been reported that amino acid monocoordinated Cu 2+ shows enhanced activity for the decomposition of H 2 O 2 , 61 it can be proposed that both peptidecoordinated and noncoordinated Cu 2+ may play a role in the copper-catalyzed tyrosine nitration.…”

Antioxidant promotion of tyrosine nitration in the presence of copper(ii)

“…According to our previous studies, 20 copper(II) can be coordinated by Ang I and the binding happens close to the tyrosine residue of Ang I. The same binding site between Ang I and copper(II) was also reported by Kim et al 35 In Fig. 1(a), the copper coordinated Ang I is observed as a small peak after the nitrated Ang I ((Ang I + NO 2 ) 3+ ).…”