Further characterization of tunicate and tunichrome electrochemiluminescence.

Electrophoresis, size exclusion chromatography, fluorescence, and electrochemiluminescence (ECL) data obtained from the cell-rich perivisceral fluid ('blood') of the tunicate, Molgula occidentalis, suggests that a yellowish protein or protein subunit of < 6.5 kDa is probably responsible for the low level intrinsic ECL reported previously. Variable potential ECL scans and spectrofluorometric analyses directly indicated that chlorophylls from ingested phytoplankton were not significant contaminants of M. occidentalis blood samples. Chlorophylls were also examined indirectly from monocultures of dinoflagellate and diatom species to determine their ECL levels. While ECL was observed in these cultures, high concentrations (10(6) organisms/mL) of dinoflagellates or diatoms were needed to produce ECL levels comparable to those observed for M. occidentalis blood. Additional work with two synthetic tunichrome isomers indicated 10-fold increases in ECL when the '2,3,4-tunichrome' was reacted with Tl+ in 1:1 and 1:2 metal ion to ligand molar ratios. The '3,4,5-tunichrome' isomer exhibited a titration curve with Hg2+ suggestive of the existence of at least two Hg2+ binding sites and a 9- to 10-fold increase in ECL output.

Electrochemiluminescence from tunicate, tunichrome-metal complexes and other biological samples.

Low level intrinsic electrochemiluminescence (ECL) was induced from body fluids and homogenized tissues of oysters and several species of tunicates. No significant ECL was detected in human blood cell lysates, or bovine haematin, but minor ECL was observed in avian blood cell lysates. Both terrestrial grass and seagrass exhibited ECL, which is probably attributable to chlorophyll, since dead (brown) grass did not demonstrate ECL. It was postulated that organic-metal complexes in marine invertebrates were, at least in part, responsible for the intrinsic ECL, since such animals are known to be rich in organically bound metals. However, alternative biochemical mechanisms for the observed ECL, which do not involve metal chelates, are possible. Various metal ions were added to the invertebrate preparations to determine whether exogenous metals could enhance or inhibit the ECL reactions. Strongly oxidizing metal ions such as Ag+, Au+, Cu2+, Hg2+ and Sb2+ at > or = 100 ppm severely inhibited the intrinsic ECL response. No statistically significant ECL enhancement due to addition of metal ions was noted. ECL "profiles' were generated which demonstrated differences in the ECL responses of individual tunicate preparations to the presence of various exogenous metal ions. Differences in ECL profiles may represent differences in types or levels of endogenous metal chelates or other biochemical constituents. In addition, synthetic tunichromes (tunicate pigments) were analysed for ECL in the presence and absence of various added metal ions. One synthetic tunichrome isomer demonstrated a specific ECL interaction with Hg2+, while the other demonstrated broader ECL activity with several metal ions.