Quantitative, chemically specific imaging of selenium transformation in plants

Quantitative, chemically specific images of biological systems would be invaluable in unraveling the bioinorganic chemistry of biological tissues. Here we report the spatial distribution and chemical forms of selenium in Astragalus bisulcatus (two-grooved poison or milk vetch), a plant capable of accumulating up to 0.65% of its shoot dry biomass as Se in its natural habitat. By selectively tuning incident x-ray energies close to the Se K-absorption edge, we have collected quantitative, 100-μm-resolution images of the spatial distribution, concentration, and chemical form of Se in intact root and shoot tissues. To our knowledge, this is the first report of quantitative concentration-imaging of specific chemical forms. Plants exposed to 5 μM selenate for 28 days contained predominantly selenate in the mature leaf tissue at a concentration of 0.3–0.6 mM, whereas the young leaves and the roots contained organoselenium almost exclusively, indicating that the ability to biotransform selenate is either inducible or developmentally specific. While the concentration of organoselenium in the majority of the root tissue was much lower than that of the youngest leaves (0.2–0.3 compared with 3–4 mM), isolated areas on the extremities of the roots contained concentrations of organoselenium an order of magnitude greater than the rest of the root. These imaging results were corroborated by spatially resolved x-ray absorption near-edge spectra collected from selected 100 × 100 μm2 regions of the same tissues.

Sulfur K-edge x-ray absorption spectroscopy: A spectroscopic tool to examine the redox state of S-containing metabolites in vivo

The sulfur K-edge x-ray absorption spectra for the amino acids cysteine and methionine and their corresponding oxidized forms cystine and methionine sulfoxide are presented. Distinct differences in the shape of the edge and the inflection point energy for cysteine and cystine are observed. For methionine sulfoxide the inflection point energy is 2.8 eV higher compared with methionine. Glutathione, the most abundant thiol in animal cells, also has been investigated. The x-ray absorption near-edge structure spectrum of reduced glutathione resembles that of cysteine, whereas the spectrum of oxidized glutathione resembles that of cystine. The characteristic differences between the thiol and disulfide spectra enable one to determine the redox status (thiol to disulfide ratio) in intact biological systems, such as unbroken cells, where glutathione and cyst(e)ine are the two major sulfur-containing components. The sulfur K-edge spectra for whole human blood, plasma, and erythrocytes are shown. The erythrocyte sulfur K-edge spectrum is similar to that of fully reduced glutathione. Simulation of the plasma spectrum indicated 32% thiol and 68% disulfide sulfur. The whole blood spectrum can be simulated by a combination of 46% disulfide and 54% thiol sulfur.

X-ray absorption fine structure as a monitor of zinc coordination sites during oogenesis of Xenopus laevis.

The x-ray absorption fine structure (XAFS) zinc K-edge steps for intact stages I,II and V,VI Xenopus laevis oocytes demonstrate that the zinc concentration is about 3 and 1 mM, respectively. However, the chi(k) function for the early stage oocytes differs markedly from that for the late one. Analysis of the XAFS data for stage I,II oocytes indicates that zinc is bound to 2.0 +/- 0.5 sulfur atoms at an average coordination distance of 2.29 +/- 0.02 angstroms and 2.0 +/- 0.5 nitrogen or oxygen (N/O) atoms at 2.02 +/- 0.02 angstroms. In marked contrast, in stage V,VI oocytes, zinc is bound to 4.1 +/- 0.4 N/O atoms at an average distance of 1.98 +/- 0.01 angstroms. Our previous studies demonstrated that 90% of the zinc in stage VI oocytes is sequestered within yolk platelets, associated with a single molecule, lipovitellin, the proteolytically processed product of vitellogenin. XAFS analysis of yolk platelets, lipovitellin, and vitellogenin demonstrates that zinc is bound to 4.0 +/- 0.5 N/O ligands at an average distance of 1.98 +/- 0.01 angstroms in each case, identical to that of stage V,VI oocytes. The higher shell contributions in the Fourier transforms indicate that two of the N/O zinc ligands are His in both stage V,VI and I,II oocytes. The results show that in stage I,II oocytes, there is a high concentration of a zinc protein whose zinc coordination site likely is composed of (His)2(Cys)2, such as, e.g., TFIIIA. As the oocytes develop, the predominant zinc species becomes one that exhibits the (His)2(N/0)2 zinc site found in lipovitellin. Hence, the ligands to the zinc atoms in intact oocytes and the changes that take place as a function of oogenesis and after their fertilization, during embryogenesis, now can be examined and explored.