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Measurement of thiol‐containing amino acids and phytochelatin (PC 2 ) via capillary electrophoresis with laser‐induced fluorescence detection
Author(s) -
Hart Jonathan J.,
Welch Ross M.,
Norvell Wendell A.,
Kochian Leon V.
Publication year - 2002
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683(200201)23:1<81::aid-elps81>3.0.co;2-v
Subject(s) - capillary electrophoresis , thiol , laser induced fluorescence , fluorescence , chemistry , phytochelatin , chromatography , biochemistry , glutathione , optics , enzyme , physics
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, γ‐glutamyl‐cysteine, glutathione and (γ‐glutamyl‐cysteinyl) 2 ‐glycine (PC 2 ). Concentrations as low as 25 nmol/L and amounts as low as 1 fmol were detected for glutathione. Sensitivity for detection of PC 2 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 (PC 2 ) in small volumes of extracts obtained from biological tissue.