Ca2+ Complexation With Relevant Bioligands in Aqueous Solution: A Speciation Study With Implications for Biological Fluids

Abstract

A speciation study on the interaction between Ca²⁺ and ligands of biological interest in aqueous solution is reported. The ligands under study are l-cysteine (Cys), d-penicillamine (PSH), reduced glutathione (GSH), and oxidized glutathione (GSSG). From the elaboration of the potentiometric experimental data the most likely speciation patterns obtained are characterized by only protonated species with a 1:1 metal to ligand ratio. In detail, two species, CaLH₂ and CaLH, for systems containing Cys, PSH, and GSH, and five species, CaLH₅, CaLH₄, CaLH₃, CaLH₂, and CaLH, for system containing GSSG, were observed. The potentiometric titrations were performed at different temperatures (15 ≤ t/°C ≤ 37, at I = 0.15 mol L^-1). The enthalpy and entropy change values were calculated for all systems, and the dependence of the formation constants of the complex species on the temperature was evaluated. ¹H NMR spectroscopy, MALDI mass spectrometry, and tandem mass spectrometry (MS/MS) investigations on Ca²⁺-ligand solutions were also employed, confirming the interactions and underlining characteristic complexing behaviors of Cys, PSH, GSH, and GSSG toward Ca²⁺. The results of the analysis of ¹H NMR experimental data are in full agreement with potentiometric ones in terms of speciation models and stability constants of the species. MALDI mass spectrometry and tandem mass spectrometry (MS/MS) analyses confirm the formation of Ca²⁺-L complex species and elucidate the mechanism of interaction. On the basis of speciation models, simulations of species formation under conditions of some biological fluids were reported. The sequestering ability of Cys, PSH, GSH, and GSSG toward Ca²⁺ was evaluated under different conditions of pH and temperature and under physiological condition.

Keywords: 1H NMR spectroscopy; Ca2+; biological ligands; mass spectrometry; potentiometry; sequestration; speciation in biological fluids; thermodynamic parameters.

FIGURE 1

Ligands under study.

FIGURE 2

Speciation diagrams of Ca²⁺-ligand (L) systems at t = 15 °C (dotted lines) and t = 37 °C (solid lines), C_M = 2 mmol L⁻¹, C_L = 4 mmol L⁻¹, I = 0.15 mol L⁻¹ in NaCl (A) L = Cys (B) L = PSH (C) L = GSH (D) L = GSSG.

FIGURE 3

¹H NMR spectra on solutions containing Ca²⁺ (M) and Cys(L) at C_M = 7.5 mmol L⁻¹, C_L = 10 mmol L⁻¹, t = 25 °C, I = 0.15 mol L⁻¹ in NaCl, 1.94 ≤ pH ≤ 10.46.

FIGURE 4

¹H NMR spectra on solutions containing Ca²⁺ (M) and GSSG(L) at C_M = 8 mmol L⁻¹, C_L = 6 mmol L⁻¹, t = 25  °C, I = 0.15 mol L⁻¹ in NaCl, 2.23 ≤ pH ≤ 10.00.

FIGURE 5

Experimental (□) and calculated (O) chemical shift values (in ppm) vs. pH of (A) CH on Ca²⁺(M)-Cys(L) solutions, (B) CH_3-a on Ca²⁺(M)-PSH(L) solutions, (C) CysCHα on Ca²⁺(M)-GSH(L) solutions, (C) CysCHα on Ca²⁺(M)-GSSG(L) solutions.

FIGURE 6

MS/MS spectrum of (A) Cys and (B) PSH in the presence of Ca²⁺.

FIGURE 7

MS/MS spectrum of (A) GSH and (B) GSSG in the presence of Ca²⁺.

FIGURE 8

Ca-Cys, GSH, GSSG species in biological fluids at t = 37 °C, I = 0.15 mol L⁻¹. (A) Plasma conditions (pH = 7.4); (B) lens water conditions (pH = 7.2); (C) lens cataractous water conditions (pH = 7.2).

FIGURE 9

Bar plot of ΔG, ΔH, and TΔS referring to Ca²⁺-Cys (A), Ca²⁺-PSH (B), Ca²⁺-GSH (C), and Ca²⁺-GSSG (D) species at t = 25 °C, I = 0.15 mol L⁻¹ in NaCl, according to overall formation reaction.

FIGURE 10

Comparison of sequestering ability of Cys, PSH, GSH, and GSSG toward Ca²⁺ under physiological conditions (pH = 7.4, t = 37 °C, I = 0.15 mol L⁻¹).