Metal-catalyzed oxidation of protein methionine residues in human parathyroid hormone (1-34): formation of homocysteine and a novel methionine-dependent hydrolysis reaction

Abstract

The oxidation of PTH(1-34) catalyzed by ferrous ethylenediaminetetraacetic acid (EDTA) is site-specific. The oxidation of PTH(1-34) is localized primarily to the residues Met[8] and His[9]. Beyond the transformation of Met[8] and His[9] into methionine sulfoxide and 2-oxo-histidine, respectively, we observed a hydrolytic cleavage between Met[8] and His[9]. This hydrolysis requires the presence of Fe(II) and oxygen and can be prevented by diethylenetriaminepentaacetic acid (DTPA) and phosphate buffer. Conditions leading to this site-specific hydrolysis also promote the transformation of Met[8] into homocysteine, indicating that the hydrolysis and transformation of homocysteine may proceed through a common intermediate.

Figure 1

Representation of the sequences of human parathyroid hormones (PTH(1-34) and PTH(1-34) Met[8]Ala mutant). The dashed lines indicate the digestion sites obtained by means of trypsin in ammonium bicarbonate buffer (50 mM, pH 8). The tryptic fragments are noted as F1–F5.

Figure 2

Schematic representation of the processes used to oxidize and digest PTH(1-34). Pathways a and b describe processes performed under air and Ar, respectively.

Figure 3

Percentage of conversion of the tryptic fragment F5 into products P1 and P2 after oxidation of PTH(1-34) under the experimental conditions Exp. 1–6 as described in Section 2.2 and schematized in Figure 2. The bars indicate the lowest and highest ratios of transformation of F5 into P1 and P2 over five replicates.

Figure 4

CID mass spectrum obtained by means of a SYNAPT mass spectrometer of product P1 (m/z 736.4, doubly charged) generated after oxidation of PTH(1-34).

Figure 5

CID mass spectrum obtained by means of a SYNAPT mass spectrometer of product P2 (m/z 906.4) generated after oxidation of PTH(1-34).

Figure 6

LC-MS analysis of oxidized PTH(1-34) (24.3 μM) in air: (A) control, no oxidation; (B) oxidation in the presence of [Fe^II/EDTA]^2– (100 μM) and H₂O₂ (100 μM); DTPA (500 μM) was added to the solution prior to digestion; (C) oxidation in the presence of Fe^II and DTPA (100 μM); H₂O₂ (100 μM) and DTPA (500 μM) were added to the solution prior to digestion; (D) oxidation in the presence of [Fe^II/EDTA]^2– (100 μM) and H₂O₂ (100 μM); (E) oxidation in the presence of [Fe^II/EDTA]^2– (100 μM); DTPA (500 μM) was added to the solution prior to digestion. In all cases catalase (0.07 μM) was added to the solution prior the digestion.

Figure 7

LC-MS analysis of oxidized PTH(1-34) (24.3 μM) under Ar in the presence of [Fe^II/EDTA]^2– (100 μM) and H₂O₂ (100 μM): (A) no DTPA was added prior to open the tube to air; (B) DTPA (500 μM) was added in the solution prior exposure of the solution to air and LC-MS analysis. In all the case catalase (0.07 μM) was added under Ar.

Figure 8

LC-MS analysis of oxidized PTH(1-34) (24.3 μM): (A) PTH(1-34) preoxidized with H₂O₂ was exposed to [Fe^II/EDTA]^2– (100 μM) and H₂O₂ (100 μM) under Ar. Catalase (0.07 μM) was added to the solution prior to the digestion, (B) control.

Figure 9

LC-MS analysis of oxidized PTH(1-34) (24.3 μM) under Ar in the presence of DTPA (100 μM) and H₂O₂ (100 μM). Catalase (0.07 μM) was added under Ar.

Figure 10

LC-MS analysis of oxidized PTH(1-34) (24.3 μM) by H₂O₂ and following by Fenton oxidation according to the experimental protocol Exp. 1.

Figure 11

LC-MS analysis of oxidized PTH(1-34) (24.3 μM) and PTH(1-34) Met[8]Ala (24.6 μM) under air: (A) oxidation of PTH(1-34) in the presence of [Fe^II/EDTA]^2– (100 μM), H₂O₂ (100 μM); DTPA (500 μM) was added to the solution prior to digestion. The chromatogram highlights product P2. (B) Control of nonoxidized PTH(1-34) Met[8]Ala. The MS/MS spectrum of the tryptic fragment F5A is presented in Figure S10 (Supporting Information). (C) Oxidation of PTH(1-34) Met[8]Ala in the presence of [Fe^II/EDTA]^2– (100 μM), H₂O₂ (100 μM); DTPA (500 μM) was added to the solution prior to digestion. (D) oxidation of PTH(1-34) Met[8]Ala in the presence of [Fe^II/EDTA]^2– (100 μM), H₂O₂ (100 μM), DTPA (500 μM) was added in the solution prior to digestion. Chromatogram D shows the absence of P2A (m/z 846.4). The absolute intensity for the most abundant species is displayed in each chromatogram.

Figure 12

Oxidation of PTH(1-34) in the presence of H₂O¹⁸/H₂O¹⁶ (80:20, v:v). Mass spectrum in the range of the m/z of product P2 (m/z 906.4, when generated in the presence of 100% H₂O¹⁶).

Figure 13

CID mass spectrum obtained by means of a FT-ICR mass spectrometer of product P4 (m/z 721.4, doubly charged) generated after oxidation of PTH(1-34). X stands for the homocysteine residue.

Figure 14

CID mass spectrum obtained by means of a FT-ICR mass spectrometer of product P4-NEM (m/z 721.4, doubly charged) generated after oxidation of PTH(1-34). “u₁” and “u₂” stand for the homocysteine and C-terminal lysine residues derivatized with NEM, respectively.

Scheme 1

Postulated Reaction Mechanism to Rationalize the Formation of P2 during Oxidation of PTH(1-34) by [Fe^II(Ln)]^m– and Oxygen. (Ln = EDTA, H₂O, or Acetate). R₁ and R₂ Represent the Peptide Sequences SVSEIQL and NLGKHLNSMERVEWLRKKLQDVHNF, Respectively

Scheme 2

Postulated Reaction Mechanism to Rationalize the Transformation of Met[8] into Homocysteine during Oxidation of PTH(1-34). R₁ and R₂ Represent the Peptide Sequences SVSEIQL and HNLGKHLNSMERVEWLRKKLQDVHNF, Respectively