Measurement of thiol-containing amino acids and phytochelatin (PC2) via capillary electrophoresis with laser-induced fluorescence detection.

Jonathan J Hart, Ross M Welch, Wendell A Norvell, Leon V Kochian

Index: Electrophoresis 23(1) , 81-7, (2002)

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Abstract

An analytical method for determining thiols and phytochelatins using high-performance capillary electrophoresis coupled with laser-induced fluorescence detection is presented. The technique utilizes the labeling of thiols with the fluorescent reagent 5-bromomethylfluorescein (5-BMF), which is excited by a 488 nm argon ion laser and fluoresces at 515 nm. The paper describes the determination of the optimal conditions for reaction of 5-BMF with thiols as well as the parameters for electrophoresis runs that produce optimal electropherogram peaks. The technique is shown to be very sensitive for cysteine, cysteinyl-glycine, gamma-glutamyl-cysteine, glutathione and (gamma-glutamylcysteinyl)2-glycine (PC2). Concentrations as low as 25 nmol/L and amounts as low as 1 fmol were detected for glutathione. Sensitivity for detection of PC2 was somewhat lower. The method was shown to be simple, rapid and accurate and should facilitate measurement of thiol-containing amino acids, peptides and phytochelatin (PC2) in small volumes of extracts obtained from biological tissue.