|Hart, J - CORNELL UNIVERSITY|
Submitted to: Electrophoresis
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 8, 2001
Publication Date: January 1, 2002
Citation: HART, J.J., NORVELL, W.A., WELCH, R.M., KOCHIAN, L.V. MEASUREMENT OF THIOL-CONTAINING AMINO ACIDS AND PHYTOCHELATINS VIA CAPILLARY ELECTROPHORESIS WITH LASER-INDUCED FLUORESCENCE DETECTION. ELECTROPHORESIS. 2002. v. 23. p. 81-87. Interpretive Summary: Thiol-containing compounds play important roles in the biochemical functioning of plant cells. Cysteine and glutathione are examples of common thiol-containing compounds that carry out critical biochemical tasks. Phytochelatins are a family of compounds that contain multiple thiol groups and function to control toxic heavy metals and regulate cellular levels of essential micronutrient metals. Because of their fundamental roles, accurate measurement of these and other thiol-containing compounds is desirable. An analytical method for determining thiols, including phytochelatins, using high-performance capillary electrophoresis (HPCE) coupled with laser-induced fluorescence (LIF) detection is presented in this paper. HPCE offers advantages over other chemical detection techniques, including simple instrumentation, the ability to process very small samples and short run times. The technique presented in this paper utilizes the labeling of thiols with a fluorescent reagent (5-BMF) which i detected by LIF. The paper describes the determination of the optimal conditions for reaction of 5-BMF with thiols as well as the parameters for measurement runs that produce optimal detection signals. The technique is shown to be very sensitive for cysteine, cysteinyl-glycine, gamma-glutamyl-cysteine, glutathione and PC2 (a phytochelatin). Extremely low concentrations and amounts of glutathione were detected using this method. 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 compounds, including phytochelatins, particularly when limited amounts of tissue are available for analysis.
Technical 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 epaper describes the determination of the optimal conditions for reaction o 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-glutamyl-cysteinyl)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 PCs in small volumes of extracts obtained from biological tissue.