Comparison of the benzyl viologen and bimane HPLC assays for the determination of sulfide-oxidizing capability in the tissues of hydrothermal vent and non-vent polychaetes

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Abstract
Two commonly used methods for in vitro measurement of tissue-level sulfide oxidation in animals were compared using homogenates of hydrothermal vent (Paralvinella sulfincola and P. palmiformis) and non-vent (Nereis virens and Nephtys caeca) polychaetes. All examined worms showed heat-labile sulfide-oxidizing ability and rates were slightly higher in the two vent species. A previously observed discrepancy between the results of the spectrophotometric benzyl viologen (BV) and bimane high-performance liquid chromatography (HPLC) assays was confirmed. We explain this discrepancy firstly by the fact that H2S removal measured by the bimane HPLC assay is only the first step in a cascade of several possible sulfur-oxidation steps recorded by BV. Secondly, we show that the low H2S/protein ratio used in the bimane HPLC assay can result in undersaturation of the catalyst responsible for H2S oxidation. The latter can lead to underestimation of potential oxidation rates and may be as important as the nonspecificity of BV in explaining differences between assay results. Tissue-level sulfide oxidation is clearly widespread in marine invertebrates, but the catalyst(s) responsible remain(s) unidentified. The fact that sulfide-oxidation rates in vent polychaete tissues are similar to rates in non-vent species and appear to reflect a common basal level of sulfide-oxidizing activity in many animal tissues suggests that other defense mechanisms may be more important in the adaptation of these worms to the hydrothermal milieu.