Evaluation of [Co(gly)3]- as a 35Cl- NMR Shift Reagent for Cellular Studies

Abstract

We studied the efficacy of the tris-glycinatocobaltate(II) complex ([Co(gly)3]-) as a shift reagent (SR) for chloride by 35Cl NMR spectroscopy and compared to that of Co2+(aq). Due to the relatively low thermodynamic stability of [Co(gly)3]-, a 1:3 Co(II)/gly stoichiometric solution at physiological pH is approximately a 2:1 mixture of [Co(gly)2(H2O)2] and [Co(gly)(H2O)4]+. This SR was found to be stable up to higher pH values than Co2+(aq), better preventing Co(OH)2 formation at alkaline pH. No significant differences in the 35Cl- NMR chemical shift induced by Co(II)/gly or Co2+(aq) were observed in the presence of physiological concentrations of either Ca2+ or Mg2+, or of either Na+ or K+. Although Co2+(aq) was almost twice as effective as Co(II)/gly in shifting the 35Cl- NMR resonance at the same high ρ ([SR]/[Cl-]) value and low ionic strength, Co2+(aq) showed a significant decrease (p < 0.05) in the 35Cl- chemical shift at higher ionic strength. Line widths at half-height were significantly (p < 0.05) less for Co(II)/gly than for Co2+(aq) at ρ values in the range 0.066−0.40. Intracellular chloride was clearly detectable by 35Cl NMR spectroscopy in human skin fibroblast cells suspended in medium containing 40 mM Co(II)/gly SR. We determined that, although Co2+(aq) provides a larger shift than Co(II)/gly at the same ρ value, there are significant advantages for using Co(II)/gly, such as pH stability, ionic strength independent chemical shifts, narrow 35Cl- NMR resonances, and reduced cellular toxicity, as a SR in biological systems.