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K-edge X-ray absorption and EPR spectroscopies were used to test the variation in blood cell vanadium between and within specimens of the tunicate Ascidia ceratodes from Bodega Bay, California. Intracellular vanadium was speciated by fitting the XAS spectra of whole blood cells with linear combinations of the XAS spectra of models. Blood cell samples representing one specimen each, respectively, revealed 92.5 and 38.7% of endogenous vanadium as [V(H(2)O)(6)](3+), indicating dissimilar distributions. Conversely, vanadium distributions within blood cell samples respectively representing one and six specimens proved very similar. The derived array of V(III) complexes was consistent with multiple intracellular regions that differ both in pH and c(sulfate), both within and between specimens. No systematic effect on vanadium distribution was apparent on mixing blood cells. EPR and XAS results indicated at least three forms of endogenous vanadyl ion, two of which may be dimeric. An inverse linear correlation was found between soluble and complexed forms of vanadyl ion, implying co-regulation. The EPR A value of endogenous vanadyl ion [A(0)=(1.062+/-0.008)x10(-2) cm(-1)] was marginally different from that representing Monterey Bay A. ceratodes [A(0)=(1.092+/-0.006) x10(-2) cm(-1)]. Comparisons indicate that Bodega Bay A. ceratodes maintain V(III) in a more acidic intracellular environment on average than do those from Monterey Bay, showing variation across populations. Blood cell vanadium thus noticeably diverges at all organismal levels among A. ceratodes.

The 1-H nuclear magnetic resonance spectrum of living tunicate blood cells was examined in an attempt to develop a biophysical assay for the native vanadium chromogen. The living cell spectrum was found to exhibit a broad 21 ppm downfield Gaussian signal which, however, disappears immediately upon cell disruption. Examination of the properties of this extremely low field signal revealed that it corresponds to a labile vanadium (III) aquo complex contained in the cell vacuoles, that vanadium(III) concentrations are rigidly regulated within these vacuoles, and that artifact formation does occur in the hemolysate. The living cell spectrum also indicates the number of ligand-bound vanadium(III) coordination sites in the native blood pigment. Results are discussed relation to the possible functions of the vandium chromogen.