Fluorimetric determination of monobromobimane and o-phthalaldehyde adducts of gamma-glutamylcysteine and glutathione: application to assay of gamma-glutamylcysteinyl synthetase activity and glutathione concentration in liver.
The reversed-phase HPLC separation of fluorescent o-phthalaldehyde (OPA) derivatives has been applied to the assay of hepatic gamma-glutamylcysteine and glutathione (GSH) levels and the enzymes producing these peptides. The method has been compared to the assay using monobromobimane (MB) as the derivatizing agent. The OPA method has the advantage of faster derivatization, the lack of need to adjust the pH, isocratic separation and selectivity for GSH and gamma-glutamylcysteine. The MB method requires pH adjustment following derivatization and gradient elution chromatography. MB is also non-selective, yielding fluorescent derivatives of all biological thiols and more interfering peaks on the chromatogram. MB-based analyses are also approximately sixty times more expensive per sample. MB yields fluorescent degradation products on exposure to light. OPA adducts are stable for up to ten days when stored at -20 degrees C. OPA detection is sensitive to 12.5 pmol in the sample, at a signal-to-noise ratio of 2.5. The two methods correlate well. Hepatic gamma-glutamylcysteine synthetase in the same liver preparation was found to be 4.85 +/- 0.47 nmol min-1 mg-1 protein by the OPA method and 4.42 +/- 0.52 nmol min-1 mg-1 protein by the MB method. GSH concentrations were found to be 90.4 +/- 6.5 nmol/mg protein for the OPA method and 92.5 +/- 3.4 for the MB method.
Yan
C C
CC
Department of Pharmacology, College of Medicine, University of Arizona, Tucson 85724, USA.
Huxtable
R J
RJ
D000818:Animals; D001643:Bridged Bicyclo Compounds; D002851:Chromatography, High Pressure Liquid / Q000379:methods / Q000706:statistics & numerical data; D003600:Cytosol / Q000737:chemistry / Q000201:enzymology; D004151:Dipeptides / Q000032:analysis*; D004355:Drug Stability; D005456:Fluorescent Dyes; D005721:Glutamate-Cysteine Ligase / Q000378:metabolism*; D005978:Glutathione / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D008099:Liver / Q000737:chemistry* / Q000201:enzymology* / Q000648:ultrastructure; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D009764:o-Phthalaldehyde
D001643:Bridged Bicyclo Compounds; D004151:Dipeptides; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D009764:o-Phthalaldehyde; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; C017341:gamma-glutamylcysteine; C028381:monobromobimane