Several species of marine tunicates store oxygen-sensitive VIII in blood cells. A sensitive colorimetric VIII assay was used to survey the leading candidates for the native reducing agent of vanadate in tunicates (i.e., An-type tunichromes, glutathione, NADPH, and H2S) in reactions with VV or VIV ions under anaerobic, aqueous conditions at acidic or neutral pH. Except for the case of An-1 and VV ions in pH 7 buffer, the assay results for the biogenic reducing agents clearly showed that appreciable quantities of VIII products were not generated under the conditions tested. Therefore, the assay results place new limits on hypothetical mechanisms of VIII formation in vivo. For reactions between An-1 and VV ions in pH 7 buffer, low levels of VIII products could not be ruled out because of an interfering peak in the colorimetric assays. For similar reactions between VV ions and An-1, or an An-1,2 mixture, in mildly to moderately basic media, the product mixtures precipitated as greenish black solids. Analyses of the precipitated V/An mixtures using vanadium K-edge X-ray absorption spectroscopy (XAS) showed that the major products were tris(catecholate)-type VIV complexes (65 +/- 6%) and bis(catecholate)-type VIVO complexes (20 +/- 4%). XAS analysis of the V/An-1 product mixture also provided evidence of a minor VIII component (9 +/- 5% of total V), notable for possible relevance to tunicate biochemistry. The combined results of XAS studies, spectrophotometric studies [Ryan, D. E., et al. (1996) Biochemistry 35, 8640-8650], and EPR studies [Grant, K. B., et al. (1996) J. Inorg. Biochem. (manuscript in preparation)] consistently establish that reactions between tunichromes (Mm-1 or An-1) and VV ions generate predominantly VIV-tunichrome complexes in neutral to moderately basic aqueous media.

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Tunichromes are yellow, polyphenolic tripeptides prevalent in blood cells of tunicates (suborders phlebobranchia and stolidobranchia). Spectrophotometric studies of reactions between tunichrome Mm-1 and VV or VIV ions were conducted in vitro in various media to crudely approximate cellular conditions: deionized water, aqueous methanol, and aqueous buffers at pH 2 and 7. Catechol was used in parallel studies for comparison to tunichrome and was found to be a good model for tunichrome reactivity. For VIV in pH 7 buffer, both catechol and Mm-1 formed complexes with VIV ions, and no redox products were found. For VV in pH 2 buffer, both catechol and Mm-1 were oxidized by VV ions. Room temperature EPR qualitatively showed that Mm-1 in pH 2 buffer reduced VV ions to free VIV ions. For VV in pH 7 buffer, Mm-1 was oxidized by VV ions and formed VIV complexes. At higher concentrations, the VIV complexes were observed by low temperature EPR [Grant, K. B. (1994) Dissertation, Columbia University; Grant, K. B., et al. (1996) J. Inorg. Biochem. (manuscript in preparation)]. Using a colorimetric assay for VIII, we found that reactions between Mm-1 and VV or VIV ions in pH 7 buffer clearly did not generate appreciable quantities of VIII products. Thus, the colorimetric VIII assay resolved the issue of VIII product formation raised in EPR studies of Mm-1 [cf. Ryan, D. E., et al. (1992) Biochemistry 35, 8651-8661]. Overall, the results provide insights into tunichrome-vanadium chemistry and identify conditions which promote complexation and/or redox reactions in vitro.