Defining Chemical Species in Complex Environments Using K-Edge X-ray Absorption Spectroscopy:  Vanadium in Intact Blood Cells and Henze Solution from the TunicateAscidia ceratodes

Abstract

A K-edge X-ray absorption spectrum (XAS) fitting approach has been developed to speciate elements of interest in complex materials and used here to model the storage of biological vanadium within whole blood cells from the tunicate Ascidia ceratodes. The response of the K-edge XAS of solution-phase V(III) to increasing c(sulfate) at constant pH 1.8 produced specific and systematic effects in the preedge transition at 5468.8 eV (preedge transitions: 1s-->4A2 at 5464.9 +/- 0.1 eV, 1s-->4T2 at 5466.9 +/- 0.1 eV, and 1s-->4T1 at 5468.8 +/- 0.1 eV for 11 different V(III)/sulfate solutions). In contrast, variations in acidity (as pH) at constant c(sulfate) systematically modified the V(III) preedge XAS at 5466.9 eV. The energy position of the K-edge absorption maximum also serially shifted -0.32 eV/pH unit, from 5483.7 eV (pH 3.0) to 5484.7 eV (pH 0.3). Fits to the V-K XAS of two samples of A. ceratodes whole blood cells representing dozens of animals implied storage of V(III) ions in four predominant solution regimes: approximately 10% high sulfate/pH 0 acid; approximately 40% high sulfate/pH 1.8 acid; approximately 40% moderate sulfate/pH 1.8 acid; approximately 10% moderate sulfate/pH 3 acid. For lysed blood cells, the best fit represented 63% of the V(III) in a pH 1.6 sulfate-free environment and a further 16% in acidic sulfate solution. Nearly 18% of lysed cells vanadium(III) appeared in a tris(catecholate)-like environment. A detailed speciation of biological vanadium complex ions was calculated from these fits by application of the known equilibrium constants governing V(III) and sulfate in acidic aqueous solution. The utility of blood cell V(III) to ascidians is discussed. Fits to K-edge XAS spectra using the XAS spectra of appropriate models are suggested to be generally applicable to elucidating the state of metal ions in a wide variety of complex environments.

Vanadium haloperoxidases: From the discovery 30 years ago to X-ray crystallographic and V K-edge absorption spectroscopic studies

“…The XANES of these organisms, when compared to that of suitable model compounds, showed that the vanadium was predominantly in the V(3+) state in an octahedral environment, while the EXAFS revealed the nearest neighbours to be oxygens. In subsequent studies, the V XANES features were used to quantify the amounts of V(3+) ions associated with the biological catecholate-based chelator tunichrome, and water and sulphate, as well as mixtures of the latter in various species [114][115][116][117][118], and a detailed mechanism for the reduction of vanadate to V(3+) was proposed [119]. In this particular context, the role of sulphur in the vanadium metabolism was also investigated with sulphur K edge XANES [120][121][122].…”

The uptake and fate of vanadyl ion in ascidian blood cells and a detailed hypothesis for the mechanism and location of biological vanadium reduction. A visible and X-ray absorption spectroscopic study

“…Each final spectrum represents the average of 16 to 20 individual scans. The vanadium K-edge XAS spectra of all the solid and solution models used herein have been reported previously [15,22,26,31,38].…”

“…In contrast, incubation with DTT plus VO 2+ produced a significant new feature at 5469 eV in the vanadium K-edge XAS. This result proved consistently true in each of the three experiments.Both aqua vanadyl ion itself and sulfate ligation of V(III) produce intensity near 5469 eV in vanadium pre-K-edge XAS spectra [31]. However, the new 5469 eV feature stemming from the VO 2+ -incubated blood cells is far too intense to reflect changes in intravacuolar c sulfate , and thus indicates newly endocytosed vanadyl ion.…”

“…Each final spectrum represents the average of 16 to 20 individual scans. The vanadium K-edge XAS spectra of all the solid and solution models used herein have been reported previously [15,22,26,31,38].Fits to the vanadium K-edge spectra were carried out using the program DATFIT4, which is part of the EXAFSPAK software package mentioned above. The blood cell vanadium K-edge spectra were first further processed within the program Kaleidagraph (Synergy Software, Reading PA) to remove extraneous curvature before carrying out the fits.…”

“…In addition, the pre-edge energy position of the new intracellular vanadyl feature in all three VO 2+ -incubated samples is shifted −0.3 eV from that of VOSO 4 in pH 1 H 2 SO 4 solution, and −0.2 eV from that of VOCl 2 in 1 M HCl solution ( Figure 3, inset b; the latter data are not shown). Thus, a significant proportion of uptaken vanadyl ion was shunted to an environment quite different from the pentaaqua ion,, that is typically found as a minority species in the acidic vacuolar milieu of A. ceratodes signet ring cells (see Supplemental Figure 3) [10,22,31,44]. This finding was subjected to more detailed analysis using model-based curve-fitting of the blood cell XAS spectra in terms of known complexes, discussed below.…”