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J Biol Chem, Vol. 273, Issue 38, 24498-24503, September 18, 1998


Vanadium K-edge X-ray Absorption Spectroscopy Reveals Species Differences within the Same Ascidian Genera
A COMPARISON OF WHOLE BLOOD FROM ASCIDIA NIGRA AND ASCIDIA CERATODES*

Patrick FrankDagger and Keith O. HodgsonDagger §

From the Dagger  Department of Chemistry, Stanford University, Stanford, California 94305-5080 and the § Stanford Synchrotron Radiation Laboratory, Stanford Linear Accelerator Center, Stanford University, Stanford, California 94309

Kenneth Kustin, and William E. Robinsonparallel

From the  Department of Chemistry, Brandeis University, Waltham, Massachusetts 02254-9110 and the parallel  Environmental, Coastal, and Ocean Sciences Department, University of Massachusetts, Boston, Massachusetts 02125-3393

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Vanadium K-edge x-ray absorption spectroscopy (XAS) was used to examine whole blood preparations from the tunicates Ascidia nigra and Ascidia ceratodes. Each XAS spectrum exhibits a rising edge inflection near 5480 eV characteristic of vanadium(III) and an intensity maximum at 5484.0 eV. In A. ceratodes blood cells, intrinsic aquo-VSO4+ complex ion is indicated by an inflection feature at 5476 eV in the first derivative of the vanadium K-edge XAS spectrum, but this feature is notably absent from the first derivative of the vanadium K-edge spectrum of blood cells from A. nigra. A strong pre-edge feature at 5468.6 eV also uniquely distinguishes the vanadium K-edge XAS spectrum of A. nigra blood cells, implying that vanadyl ion represents ~25% of the endogenous vanadium. However, the energy position of the rising edge inflection of the vanadium K-edge XAS spectrum of A. nigra (5479.5 eV) is 1 eV lower than that of A. ceratodes (5480.5 eV), the reverse of any expected shift arising from the endogenous vanadyl ion. Thus, in contrast to A. ceratodes, a significant fraction of the blood cell vanadium(III) in A. nigra is apparently in a ligation environment substantially different from that provided by water. These novel species-related differences may have taxonomic significance.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The bioinorganic chemistry of vanadium in ascidians has been of increasing interest in the past several years, resulting in a steady clarification of problems regarding the distribution and intracellular status of this metal ion (1-4). In solitary phlebobranch ascidians, vanadium is concentrated within blood cells up to several million times the ambient seawater concentration. In addition, blood cell vanadium is typically reduced from oceanic V(V)-vanadate to V(IV) or V(III) (5-20). There is no evidence for V(V) within blood cells, although vanadium in this oxidation state has been detected in blood plasma (20).

We reported previously (13) on the results of a K-edge x-ray absorption spectroscopic study of vanadium in the blood cells of the solitary ascidian Ascidia ceratodes, which is found along the Pacific coast of North America (21). From vanadium K-edge extended x-ray absorption fine structure and both vanadium and sulfur (22, 23)1 K-edge spectra, the predominant form of intracellular vanadium within A. ceratodes blood cells was found to be the trivalent complex aquo-ion [VSO4(H2O)4-5]+, with six oxygen (or nitrogen) ligands at 1.99 ± 0.01 Å. This finding was anticipated by the results of prior 1H NMR (9, 10) and extended x-ray absorption fine structure (11) studies on whole blood cells. The extended x-ray absorption fine structure results now include measurement of four independent whole blood cell samples ranging across 15 years (11, 13). The immediate ligand atom environment of V(III), i.e. six oxygens (or nitrogens) at 1.99 Å, was found to be preserved when the blood cells were lysed to produce fresh Henze solution.

We now report the first extension of this exploration to include whole blood cell preparations of the closely related tunicate Ascidia nigra. This solitary ascidian ranges along the warm waters of the southern Atlantic coast of North America and the Caribbean (25-27). The blood cells of A. nigra are known to be rich in vanadium, principally as V(III) (14, 27). From SQUID2 magnetometry studies, it was shown that A. nigra blood cell vanadium also includes a moderate percentage (<= 20%) of vanadyl ion (14), in contrast to A. ceratodes (9-13, 28). Here, we report the first results of vanadium K-edge XAS experiments performed on intact whole blood cell preparations from A. nigra, which were designed to explicitly examine the in vivo status of this metal. Comparative analysis of the vanadium K-edge XAS spectra of A. nigra and A. ceratodes whole blood was used to test the hypothesis that a single universal V(III) ligation environment is common to the blood cells of all vanadium-containing ascidian species.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Sample Preparation-- A. ceratodes specimens were collected from Monterey Bay, CA, and blood samples were prepared for vanadium K-edge XAS examination as described in detail elsewhere (13). A. nigra specimens were collected from Key Largo, FL, and shipped the same day to the University of Massachusetts (Boston). Animals were apportioned into four approximately equal-sized groups, which were each maintained in a 20-gallon aquarium containing reconstituted seawater (Instant Ocean; 32per thousand ) at 26 °C for <96 h before sample preparation.

A. nigra blood was taken by cutting away part of the tunic from the basal region of the body. The blood that pooled was collected using a sterile disposable plastic Pasteur pipette and transferred to an ice-cold acid-clean 50-ml centrifuge tube containing 0.1 mg of dithiothreitol to prevent cell agglutination. A slow flow of argon was maintained over the collected blood during the entire workup.

Once ~30 ml of blood was obtained (5-11 animals), the sample was mixed by inverting the capped tube several times. The whole blood was then layered onto an ice-cold solution of 30% ethylene glycol and 0.5 M NaCl contained in a 15-ml acid-clean glass centrifuge tube. The tube contained a 140-µl Lucite XAS sample cell held vertically at the bottom within a specialized nylon holder designed to funnel descending blood cells into the open mouth of the XAS sample cell. The ethylene glycol/NaCl solution overlaid the Lucite cell by ~1 cm. Approximately 8 ml of whole blood could be introduced into the centrifuge tube at one time. The apparatus was then centrifuged at 1000 × g for 8 min. The supernatant was discarded; a fresh charge of 8 ml of whole blood was added; and the centrifugation procedure was repeated until the Lucite XAS cell was observed to be full. The XAS cell was removed from the centrifuge tube, capped, and immediately frozen in liquid nitrogen. Cells were then stored at -80 °C until shipped to Stanford University frozen within a cold (<=  approximately -100 °C) shipping Dewar.

XAS Data Collection-- Vanadium K-edge spectra of the inorganic vanadium model complexes and blood cells from A. ceratodes were measured as has been reported in detail (13). Vanadium K-edge spectra for A. nigra blood cell preparations were measured on Stanford Synchrotron Radiation Laboratory wiggler beam line 7-3, using a wiggler field of 18 kG. Data were collected under dedicated operating conditions of 3 GeV and 60-90 mA of current. The incident x-ray beam was energy-resolved using a Si[220] double crystal monochromator, which was detuned 50% at 6143 eV to minimize harmonic contamination. Vanadium K-edges were measured as x-ray fluorescence excitation spectra using an argon-filled fluorescence ionization chamber detector (Stern-Heald-Lytle detector) with a Ti filter and Soller slits set at 90 ° from the x-ray beam. The final spectrum is the average of 16 scans.

Vanadium foil calibration spectra were obtained after every three to four data scans by transmission using an in-line nitrogen-filled ionization chamber as detector. All XAS samples were held at 15 K using an Oxford Instruments CF1208 continuous flow liquid helium cryostat.

Raw vanadium K-edge data were processed as described in detail elsewhere (13, 29). Reproducibility in K-edge position is about ±0.1-0.2 eV from time to time. Resolution at the vanadium K-edge is ~0.6 eV. The XAS edge spectra were calibrated to the first inflection point on the rising edge of the spectrum of a vanadium foil standard, which was assigned to 5464.0 eV.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

K-edge x-ray absorption spectra involve the x-ray-induced energy-dependent successive promotion of a 1s electron into valence shells, higher energy bound states, and eventually into the continuum (29-33). Vanadium K-edge spectra are observed in the x-ray absorption energy region above ~5450 eV. In centrosymmetric ligation environments, the lowest energy transitions, involving 1s right-arrow 3d excitations, are symmetry-forbidden and of low intensity. In lower symmetries, valence shell d-p mixing allows enhanced intensity in these transitions (31, 32). At energies above the 1s right-arrow 3d excitations, the principal rising edge occurs, which can include transitions to higher energy bound states such as 1s right-arrow 4p, 1s right-arrow np. Multiple scattering features and other resonance transition features may also occur in this energy region (3, 33-35). The energy position of the rising K-edge of vanadium may shift in response to changes in the ligation environment (up to about ±2.5 eV) and oxidation state (up to about ±5 eV/unit oxidation state at constant ligation and symmetry) for ligation with oxygen donor atoms (33, 36).

The K-edge spectra of selected vanadium complexes (Fig. 1) illustrate the points outlined above. For example, the intensity maximum of the vanadium K-edge XAS spectrum of vanadium(III), at 5484.2 eV for the hexaaquo-ion (in 1 M HCl), shifts to 5481.8 eV on triscatecholate ligation. The -2.4-eV shift in energy implies that within the triscatecholate ligation environment, the effective charge on the vanadium(III) absorber is lowered relative to vanadium(III) within the ligation environment provided by water molecules alone. Likewise, on oxidation of vanadium(III)-triscatecholate to the analogous vanadium(IV) complex, the intensity maximum of the vanadium K-edge shifts 5.0 eV, from 5481.8 to 5486.8 eV. The V(IV)-triscatecholate vanadium K-edge XAS maximum is also 2.6 eV higher in energy than is the K-edge intensity maximum of aquo-vanadium(III). In no case thus far examined has a ligation change alone shifted the K-edge XAS energy position of the intensity maximum of a V+ complex more than does reduction to the analogous V(-1)+ complex for vanadium within oxygen donor atom ligands.


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  		Fig. 1.   K-edge XAS spectra of biologically relevant vanadium complexes. (------), V(III) sulfate (pH 1.8 solution); (- - -), K3V(catecholate)3 (solid dispersed in BN); - · - ·, vanadyl sulfate (0.1 M sulfuric acid solution); (-----), (Et3NH)2V(catecholate)2 (solid dispersed in BN). The rising edge energy position varies with change in ligation and oxidation state. Relative to the vanadium K-edge XAS of the hexaaquo-ion, tris-chelation of vanadium(III) by catechol results in a 2-eV shift in the rising K-edge to lower energy. The vanadyl ion XAS K-edge exhibits an intense pre-edge transition at 5468.8 eV since the lack of centrosymmetry in the [V(IV)=O]2+ ion results in valence orbital d-p mixing. The inset shows that the low intensity pre-edge 1s right-arrow 3d features of the three pseudo-octahedral vanadium complexes are also diagnostic for ligation and oxidation state.

Vanadyl ion ([V(IV)=O]2+) is unique among the vanadium species shown in Fig. 1 in having a very short vanadium-oxo bond and in exhibiting an intense and diagnostic pre-edge feature at 5468.8 eV. This vanadyl ion XAS pre-edge feature displays nearly 10 times the intensity of the 1s right-arrow 3d feature of, for example, pseudo-octahedral vanadium(III,IV) (Fig. 1, inset). This intensity arises from valence shell d-p mixing due to the lack of centrosymmetry within vanadyl ion (31-33) and will dominate the pre-edge energy region of the K-edge XAS spectrum of any V(III)/V(IV) mixture containing more than ~5% of vanadyl species.

In Fig. 2, the vanadium K-edge spectra of whole blood cells from the tunicates A. nigra and A. ceratodes exhibit both similarities and differences in the status of endogenous vanadium. Thus, the K-edge XAS spectrum of vanadium in A. ceratodes is almost identical to that of vanadium(III) in sulfuric acid (pH 1.8) (cf. Fig. 1). The ligation of sulfate to A. ceratodes blood cell vanadium(III) can be inferred from the presence of a shoulder found at 5475.5 eV on the rising edge of the vanadium K-edge spectrum (13). This shoulder has been consistently observed in K-edge XAS spectra of solution-state vanadium(III) ion when [sulfate] is significant, but has not been observed when this counterion is excluded. In addition, the 5475.5-eV shoulder disappears from the vanadium K-edge spectra of lysed A. ceratodes blood cells, indicating loss of the endogenous vanadium-sulfate interaction. Thus, the vanadium in A. ceratodes blood cells is represented by >= 90% solution-phase aquo-vanadium(III) sulfate, as corroborated by both prior whole cell 1H NMR (9, 10) and sulfur K-edge XAS (22, 23)2 spectroscopies. However, the rising edge of the vanadium K-edge XAS spectrum of A. nigra blood cells does not exhibit a 5476-eV shoulder. Therefore, in contrast with A. ceratodes, in A. nigra blood cells, V(III) is apparently uncomplexed by sulfate. This point is amplified below.


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  		Fig. 2.   Vanadium K-edge XAS spectra of whole blood cells from A. nigra (------) and A. ceratodes (- - -). The rising edge of each spectrum around 5478 eV is consistent with predominantly vanadium(III), although minor differences in spectral line shape are visible. The relatively strong pre-edge feature at 5468.6 eV in the spectrum of A. nigra indicates a significant percentage of endogenous vanadyl ion, however. As shown in the inset, the contribution from vanadyl ion to the pre-edge portion of the A. nigra XAS spectrum is overwhelming and indicates that it constitutes ~25% of blood cell vanadium.

The K-edge XAS of endogenous vanadium in A. nigra blood cells also shows contrasting evidence of a significant vanadyl ion fraction in the rather strong pre-edge feature at 5469 eV (Fig. 2 and inset). Comparison with the spectra in Fig. 1 indicates that the XAS pre-edge feature of inorganic vanadyl ion in solution exhibits a normalized intensity of ~0.4 unit of absorption. A direct inference can thus be made that vanadyl ion represents ~25% of the vanadium in the A. nigra blood cell sample in Fig. 2. This value is comparable to the 10 or 20% vanadyl ion found in whole or freeze-dried blood cell samples, respectively, from A. nigra using SQUID magnetometry (14).

The endogenous vanadyl ion within A. nigra blood cells can also be further characterized by the presence or absence of ligation. In Fig. 3, the vanadium K-edge spectra of the biscatecholate complex of vanadyl ion is compared with that of pentaaquo-vanadyl ion in 0.1 M sulfuric acid solution. Catechol (1,2-dihydroxybenzene) produces a chelation environment about vanadyl ion consisting of a square plane of four phenolate oxygens (37). The differences in vanadyl ion ligation environment are directly reflected in the intensity and energy positional differences in the respective pre-edge features. In Fig. 3 (inset), the first derivatives of the spectra in the pre-edge region show the relative 1.1-eV difference in the energy of the intensity maxima. The other obvious differences in the respective vanadyl K-edge XAS spectral line shapes are passed over with note. The vanadium K-edge XAS pre-edge feature of vanadyl bisacetylacetonate (not shown) occurs at virtually the identical energy position and intensity of that of the biscatecholate complex, marking the relative insensitivity of this feature to the details of chelation.


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  		Fig. 3.   Vanadium K-edge spectra of vanadyl sulfate (------); solution in 0.1 M sulfuric acid) and K2VO(catecholate)2 (- - -); solid dispersed in BN). Inset, the first derivatives of the pre-edge portions of the spectra. Note the negative 1.1-eV shift in relative energy position of the intense pre-edge feature on vanadyl bis-ligation. The increased intensity of the pre-edge feature on ligation probably follows a positive difference in d-p valence orbital mixing in the vanadium center (32). The vanadium K-edge XAS pre-edge feature of vanadyl bisacetylacetonate (not shown) is virtually identical in shape, position, and intensity to that of the biscatecholate.

A detailed comparison among the first derivatives of the pre-edge portions of the vanadium K-edge XAS spectra of A. nigra, A. ceratodes, and inorganic vanadyl sulfate in 0.1 M sulfuric acid solution is shown in Fig. 4. The A. nigra spectrum conspicuously exhibits a vanadyl feature that is virtually indistinguishable from that of the inorganic pentaaquo-vanadyl ion. That is, the first derivative line shape and the energy position of maximum intensity (the first derivative zero) are essentially identical to those characteristics of the first derivative K-edge XAS spectrum of inorganic vanadyl ion. Therefore, the most likely status of endogenous vanadyl ion within A. nigra blood cells is not very different from that of the inorganic solution-phase pentaaquo-ion. Note also the presence of blood cell-derived first derivative vanadium K-edge XAS features at 5464.5 and 5466.5 eV, which are not likewise reproduced in the vanadyl ion spectrum. These features are indicative of endogenous vanadium(III) (13).


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  		Fig. 4.   Comparative first derivative pre-edge XAS spectra of blood cells from A. nigra (------), A. ceratodes (- - -), and vanadyl sulfate in aqueous 0.1 M sulfuric acid solution (-----). The near positional identity of the vanadyl sulfate feature with the intense pre-edge feature from A. nigra blood cells indicates that the endogenous blood cell vanadyl ion is probably the pentaaquo-ion and is not bis-chelated. The respective blood cell spectra share similar weak features at 5464.5 and 5466.5 eV, which are not likewise present in the spectrum of vanadyl ion and which are indicative of vanadium(III). Note that the plot is double y scale.

The possibility that the observed A. nigra vanadyl ion represents a population of lysed blood cells can be eliminated by reference to the known vanadium K-edge spectrum of fresh Henze solution (11, 13), which does not include features from vanadyl ion. This result indicates that large amounts of vanadyl ion are not immediately formed on blood cell lysis. Rather, vanadyl ion K-edge XAS features appear only when Henze solution has been extensively exposed to oxygen and more alkaline media (11). The A. nigra blood cell samples discussed here showed no evidence of the sort of extensive oxidative lysis necessary to produce significant amounts of vanadyl ion from V(III) and indeed showed no visible evidence of blood cell lysis at all. Therefore, the observed vanadyl ion XAS pre-edge feature very likely reflects the internal contents of intact blood cells.

Further comparative examination of the two blood cell spectra (Fig. 2) reveals that the A. nigra spectrum exhibits a lower intensity rising edge than the A. ceratodes spectrum and subtle differences in line shape that can be observed throughout the vanadium rising K-edge energy region (5474-5482 eV). These differences are rendered more obvious in the first derivatives of the respective blood cell vanadium K-edge spectra (Fig. 5).


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  		Fig. 5.   First derivative of the vanadium K-edge spectra of A. nigra (------) and A. ceratodes (-----). Differences in spectral details include the obvious A. nigra-derived vanadyl ion feature at 5469 eV. The prominent feature in the A. ceratodes first derivative XAS spectrum near 5476 eV, indicating an endogenous vanadium(III)-sulfate interaction, is completely missing from the spectrum of A. nigra. Although the energy positions of the intensity maxima are identical at 5484.0 eV, the energy positions of the rising edge inflections (the first derivative maxima) differ by ~1 eV, at ~5479.5 eV in A. nigra and 5480.5 eV in A. ceratodes.

As discussed above, the presence of an endogenous V(III)-sulfate interaction is clearly indicated in the inflection feature at 5476 eV in the first derivative vanadium K-edge XAS A. ceratodes blood cell spectrum of Fig. 5. However, this feature is clearly absent from the analogous first derivative vanadium K-edge XAS spectrum of A. nigra blood cells. Therefore, endogenous vanadium(III) within intact A. nigra blood cells is not appreciably ligated by sulfate. None of the A. nigra blood cell samples in our hands thus far examined by means of vanadium K-edge XAS has exhibited a sulfate-derived 5476-eV inflection feature.3 This finding almost certainly does not reflect wholesale adventitious blood cell lysis, but instead indicates the status of endogenous vanadium.

In further contrast, the rising edge inflection comes at 5479.5 eV in the vanadium K-edge XAS spectrum of A. nigra blood cells, but at 5480.5 eV in that of A. ceratodes (cf. Fig. 5). Despite this 1.0-eV difference, the energy positions of the respective intensity maxima are identical at 5484.0 eV (i.e. the first derivative zero). The difference in the positions of the rising edge inflections indicates that at least some of the vanadium(III) within A. nigra blood cells is likely to be in a different, possibly more covalent ligation environment than the vanadium(III) within A. ceratodes blood. To our knowledge, these findings establish the first observed species level variation in the in vivo status of blood cell vanadium(III).

The direction of the observed shift in vanadium rising K-edge XAS inflection in the spectrum from A. nigra blood cells is opposite the expected energy shift from any contribution that might be due to the presence of ~25% vanadyl ion. Thus, as V(IV), an appreciable vanadyl ion fraction would be expected to shift a composite vanadium(III,IV) rising edge XAS inflection to higher energy than that of V(III) alone. This is illustrated in Fig. 6a, in which the vanadium K-edge spectrum of A. nigra blood cells is compared with a composite vanadium K-edge XAS addition spectrum, numerically generated by linear combination of the vanadium K-edge XAS spectra of vanadyl sulfate in 0.1 M sulfuric acid solution and vanadium(III) in 40% methanolic perchloric acid (pH 1.5) at a ratio of 24:76, respectively. The vanadyl ion pre-edge absorption intensity of the A. nigra vanadium K-edge XAS spectrum is reproduced reasonably well by the composite spectrum. However, the rising edge of the composite model XAS spectrum is ~0.5 eV to higher energy than that of the blood cell XAS spectrum, as suggested above. These conclusions also follow from a comparison of the respective first derivative spectra (Fig. 6b).


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  		Fig. 6.   Vanadium K-edge XAS spectra (a) and first derivative vanadium K-edge XAS spectra (b) of the whole blood cell sample from A. nigra in Fig. 2 (------) and a numerically generated spectrum consisting of a linear 24:76 combination of the vanadium K-edge XAS spectra of vanadyl sulfate in 0.1 M sulfuric acid solution and vanadium(III) in 40% methanolic perchloric acid (pH 1.5), respectively (-----). The pre-edge portion of the A. nigra absorption XAS spectrum, which is dominated by vanadyl ion, is relatively well reproduced by the addition model spectrum. However, the ~0.5-eV difference in rising edge energy indicates that there is an endogenous vanadium(III) component contributing to the A. nigra blood cell XAS spectrum that is not accounted for by aquo-V(III) ion.

Therefore, the observed net shift to lower energy of the A. nigra blood cell K-edge XAS position, relative to those of both A. ceratodes and the numerical model, indicates an inherent overcompensation of any shift to higher eV that might have been produced by the vanadyl ion endogenous within A. nigra blood cells. This overcompensation must be produced by a novel vanadium(III) complex also within A. nigra blood cells. This endogenous V(III) complex must produce a vanadium K-edge XAS rising edge inflection at a lower energy than that of aquo-vanadium(III).

We surmise two storage possibilities for this novel vanadium(III) complex. 1) The vanadium(III) fraction in A. nigra blood cells is stored in at least two ligation environments, at least one of which is significantly different from acidic aqueous solution. 2) The vanadium(III) fraction in A. nigra blood cells is entirely stored within a biological ligation array that provides a donor environment only somewhat stronger than that provided by water molecules alone. We consider possibility 2 to be less likely because much of the endogenous vanadium(III) is liberated as the free metal ion on blood cell lysis (27).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Kustin et al. (38) were first to question the then predominant view that there was a single "vanadocyte" in ascidian blood. Oltz et al. (39) demonstrated, using fluorescence-activated cell sorting, that at least three blood cell types (signet ring, compartment, and morula) contained vanadium in both A. nigra and A. ceratodes. This finding has been corroborated for A. ceratodes (20), and similar distributions have been reported for Ascidia ahodori (40-42). Of the three cell types, the vast majority of vanadium was found present in the signet ring cells (39, 40). In contrast, uncomplexed tunichrome, a trimer of L-3,4-dihydroxyphenylalanine and/or L-3,4,5-trihydroxyphenylalanine that has undergone a significant amount of secondary metabolic processing, was predominantly present in only the morula cells. Tunichrome contains a wealth of ortho-disposed aromatic phenolic groups that could readily serve as a chelating site for vanadium (39). Low molecular weight 3,4-dihydroxyphenylalanine-containing proteins, which have been isolated and partially described (4, 43), constitute an alternative potential binding locale for endogenous vanadium. Whether tunichrome-complexed vanadium or other analogous complexes are present in one or more tunicate blood cells has yet to be demonstrated.

The blood cells from A. nigra and A. ceratodes fall into similar morphological classes, although individual cell types may differ in detail (20, 25-27, 44-47) and differ widely in distribution (47, 48). In morula and signet ring cells from A. nigra, the absolute vanadium concentrations were much higher than in the comparable cells of A. ceratodes (39). The relative proportion of total vanadium distributed between morula and signet ring cell populations also differed between the two species. The A. ceratodes morula cell population accounted for a relatively higher proportion of the blood cell vanadium compared with A. nigra. Therefore, any differential percentage of complexed blood cell vanadium(III) dividing these species cannot be facilely assigned to morula cells, despite the attraction of this hypothesis, since little or no vanadium(III) in A. ceratodes blood cells is chelated. In A. ahodori, vanadium predominates in signet ring cells, with a lesser although significant presence in morula cells (40); and in Ascidia sydneiensis samea, vanadium is largely concentrated in signet ring cells, and very little or none is found in morula cells (49, 50).

These sorts of differences, i.e. the relative distribution of vanadium in different blood cell types in different species, may explain the variation we have observed between A. nigra and A. ceratodes in the status of blood cell vanadium as reflected in K-edge XAS spectra. However, it is clear that the analytical correlation of the percent of free or complexed vanadium(III) and of the percent of vanadyl ion with blood cell type will require examination of intact whole blood cell samples for which detailed cell counts are available. The susceptibility of ascidian blood cells to disruption makes difficult an analysis based upon physically separated blood cell subpopulations due to the possibility of systematic loss artifacts (39, 40, 49, 50).

Although it is possible that a single blood cell type may contain two or more different vanadium environments, isolated in different cell organelles, this hypothesis seems unlikely in view of the fact that at least three different cell types contain vanadium. It is more reasonable to suppose, at this stage, that the differences we have observed reflect separate vanadium environments in different cell types. These differences could be explained either by relative differences in blood cell distributions or else by absolute differences in the concentration of sequestered vanadium in each of the cell types in each species.

Vanadium content and oxidation state have been used to propose phylogenetic relationships among tunicate suborders (e.g. vanadium is absent in stolidobranchs, but is present in aplousobranchs and phlebobranchs), families, and genera, although some exceptions apparently exist (8, 15, 51, 52, 54). Blood cell vanadium in phlebobranchs is dominated by the trivalent ion, with only a few percent present as V(IV), whereas in aplousobranchs, blood cell vanadium is predominantly V(IV) (15, 16). However, SQUID magnetometry and now vanadium K-edge XAS have shown that a significant vanadyl ion concentration can exist in blood cells of the phlebobranch A. nigra, marking a distinct contrast with the status of vanadium in A. ceratodes blood cells. A further organismal contrast can now also be made at the level of blood cell vanadium(III). That is, it appears that an observable fraction of A. nigra blood cell vanadium(III) is chelated, as compared with little or no chelation of blood cell V(III) in A. ceratodes. Thus, taxonomic distinctions based upon blood cell vanadium ligation status and oxidation state are now possible at the subgenera level.

Relevant in this regard is the possibility of in vivo protein-bound vanadium, raised by the isolation of such materials from lysates of A. sydneiensis samea blood cells (56). However, the alkaline conditions of lysis (pH 8.5) and dialysis (pH 7.4) seem favorable for artifactual complexation of freed vanadium (III,IV) ions by newly associated blood cell proteins, which may occur even at low pH (12, 57). Therefore, some care is warranted in crediting this report (5).4

The question remains as to whether any of the endogenous vanadyl ion within A. nigra blood is in the form of oxo-bridged dimers or polymers. The results from EPR and SQUID experiments were consistent with the presence of monomeric vanadyl ion in these cells (14, 38, 58).3 However, the SQUID measurements also indicated a diamagnetic component, consistent with either vanadate or a vanadyl dimer with an S = 0 ground state (24, 59-62). Vanadium K-edge XAS experiments are planned to elucidate this question.

The clear and pronounced differences observed in blood cell vanadium storage between the two closely related species A. nigra and A. ceratodes were unexpected. The vanadium K-edge XAS characteristics distinguishing the blood cells of A. nigra from those of A. ceratodes suggest new taxonomically relevant variations in blood cell vanadium storage discernible even in closely related tunicates. This discovery also portends an even wider range of vanadium environments in other more distantly related ascidian species. For example, reports of vanadium-containing granules in blood cells from the species Phallusia and Ciona (53, 55) may reflect sites where biological vanadium is stored as an aggregated complex.

From the results reported herein, it now seems likely that all species of phlebobranch ascidians will display different vanadium K-edge XAS spectral patterns and thus different vanadium storage distributions, even if all species have similar biochemical mechanisms responsible for vanadium uptake and storage. Finally, we reiterate the need for restraint in the natural desire to extend the specific conclusions drawn from an examination of the blood cells of any one species of tunicate to all other ascidian species and other vanadium-containing organisms.

    ACKNOWLEDGEMENTS

We thank Linda Linebaugh and Lisa McIntosh for skilled technical assistance in microscopic examination of the A. nigra blood cell samples.

    FOOTNOTES

* This work was supported by National Science Foundation Grant CHE94-23181 and National Institutes of Health Grant RR-01209 (to K. O. H) and by Massachusetts Institute of Technology Sea Grant G92-RB-33 (to W. E. R. and K. K.). XAS spectra were measured at the Stanford Synchrotron Radiation Laboratory, which is supported by the Department of Energy, Office of Basic Energy Sciences, Divisions of Chemical and Materials Sciences. The Biotechnology Program at the Stanford Synchrotron Radiation Laboratory is supported by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program, and by the Department of Energy, Office of Biological and Environmental Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: SQUID, super-conducting quantum interference device; XAS, x-ray absorption spectroscopy.

1 P. Frank, B. Hedman, and K. O. Hodgson, submitted for publication.

3 P. Frank, K. O. Hodgson, K. Kustin, and W. E. Robinson, unpublished results.

4 It is worth repeating here the conclusion evinced by the workers in Ref. 5: "The properties of the vanadium in vanadocyte hemolysates do not give any conclusive information about the properties of vanadium in the much more condensed system inside the vanadocyte." We know of no reason to doubt the continued force of this conclusion.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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