Measurement of thiol-containing amino acids and phytochelatin (PC2) via capillary electrophoresis with laser-induced fluorescence detection.
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.
Hart
Jonathan J
JJ
United States Plant, Soil, and Nutrition Laboratory, United States Department of Agriculture, Agricultural Research Service, Cornell University, Ithaca, NY 14853, USA. jjh16@cornell.edu
Welch
Ross M
RM
Norvell
Wendell A
WA
Kochian
Leon V
LV
D000596:Amino Acids / Q000032:analysis* / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000295:instrumentation* / Q000379:methods; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D005978:Glutathione; D007834:Lasers; D008667:Metalloproteins / Q000032:analysis* / Q000737:chemistry; D009842:Oligopeptides / Q000032:analysis / Q000737:chemistry; D054811:Phytochelatins; D010940:Plant Proteins / Q000032:analysis / Q000737:chemistry; D012680:Sensitivity and Specificity; D013438:Sulfhydryl Compounds / Q000032:analysis* / Q000737:chemistry
D000596:Amino Acids; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008667:Metalloproteins; D009842:Oligopeptides; D010940:Plant Proteins; D013438:Sulfhydryl Compounds; C081567:5-(bromomethyl)fluorescein; D054811:Phytochelatins; D005978:Glutathione