HPLC analysis of nonprotein thiols in planktonic diatoms: pool size, redox state and response to copper and cadmium exposure

Abstract

A sensitive method was developed to analyze low molecular weight thiols involved in metal homeostasis and detoxification in phytoplankton. The aims of this study were to (1) separate and measure all relevant thiols in a single HPLC run, (2) measure redox states of the thiols and (3) identify specific responses of thiols (pools, redox) to heavy metals by testing diatoms with different metal tolerances (Ditylum brightwellii, Phaeodactylum tricornutum, Skeletonema costatum andThalassiosira pseudonana). Copper or cadmium were dosed at a maximum tolerable, species-dependent level, to exponential phase cells growing in artificial medium (14‰ salinity). Loss of cell viability was monitored by the decrease of fluorescein fluorescence after a 24-h metal exposure. Thiols in extracts of exposed cells and controls were labeled with monobromobimane. Picomoles of cysteine, glutathione, γ-glutamylcysteine and phytochelatins (PCs) were detected and separated by reversed-phase high performance liquid chromatography. Cysteine increased in all species after metal exposure. The species with the largest glutathione pools in the control (P. tricornutum) synthesized the largest PC pools upon metal exposure, however, at a ∼ 40% loss of glutathione. A considerable increase of glutathione was observed inD. brightwellii upon metal exposure. However, it produced little PC (and only with Cu). In controls (∼ 3 pM Cu2+), PC2 was detectable inS. costatum, P. tricornutum andT. pseudonana. Oxidized thiol fractions were recovered by the reductants DTT and TCEP, both performing identically. Compared to the other thiols, cysteine had low redox ratios. InD. brightwellii glutathione and PC redox ratios were lower than inP. tricornutum, S. costatum andT. pseudonana. It was expected that Cu-induced oxidative stress would decrease the thiol redox ratios, however, this was not observed.