Coumarins give misleading absorbance with Ellman’s reagent suggestive of thiol conjugates

Abstract

In the course of a screening for phytochelatins in cadmium-exposed bryophytes in the terrestrial mosses Polytrichum formosum and Atrichum undulatum we detected compounds with absorption properties and retention times similar to phytochelatins when applying the commonly used standard method RP-HPLC and post-column derivatization with thiol-specific DTNB (Ellman) reagent. Moreover, as with phytochelatins known in other plants, the concentrations of these compounds increased slightly after Cd stress. The concentration of the precursor glutathione (gamma-ECG), however, increased in the presence of Cd. In order to verify the identity of these putative phytochelatins we performed LC-ESI-MS analyses as well as 1H NMR on extracts from P. formosum and A. undulatum. Spectroscopic investigations indicated that the detected compounds were neither phytochelatins nor other thiol compounds. From the results of HPLC-1H NMR and mass spectrometry we concluded that at least one of these substances was a coumarin, probably a 5,8-dihydroxy-7-methoxycoumarin-5-beta-glucopyranoside, which has already been described for A. undulatum and P. formosum. The results of our investigations prove that under the basic pH conditions essential for the Ellman test for thiol compounds, coumarins show comparable UV/VIS absorption properties. Therefore, a positive post-column Ellman reaction cannot unambiguously prove the presence of thiol-containing compounds in plants.

Chromatographic and mass spectrometric analysis of glutathione in biological samples

“…Derivatization of GSH usually increases the method specificity and sensitivity. Recently, several HPLC methods utilizing pre-column derivatization with colorimetric reagents, such as Ellman's [121][122][123][124] or Sanger's [125] reagents, have been described. Ellman's reagent is widely used for the analysis of thiols in biological samples via the determination of the liberated thionitrobenzoate anion [126].…”

Heavy metals and thiol pool in three strains of Tetracladium marchalianum

“…The flow-rate was 0.8 mL · min -1 . In order to exclude the detection of other non-thiolic compounds absorbing at 410 nm under these separation conditions, as shown in BERLICH et al (2002), the post column derivatization was repeated with KH 2 PO 4 without Ellman reagent as a negative control.…”

Sulfur assimilation and glutathione metabolism under cadmium stress in yeast, protists and plants

“…2, reaction 4). PCs have been detected in some yeast [15][16][17]105], higher plants [20,106,107], algae [18], and protists [19,108], but not in bryophytes [109,110]. Heavy metals such as Zn 2+ , Hg 2+ , Cd 2+ , Fe 3+ , Al 3+ , Cu 2+ and Pb 2+ act as enzyme activators, Cd 2+ being the most potent [12,14a,111].…”