Suppression of thiol exchange reaction in the determination of reduced-form thiols by high-performance liquid chromatography with fluorescence detection after derivatization with fluorogenic benzofurazan reagent, 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate and 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole.
The derivatization of the reduced-form thiols with SBD-F (7-fluoro-2,1,3-benzoxadiazole-4-sulfonate) and ABD-F (4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole) was studied. The yields of the derivatives of the reduced-form thiols (cysteine, homocysteine, reduced-form glutathione) with SBD-F at 60 degrees C for 45 min in the borate buffer (pH 9.3) were significantly decreased in the presence of the oxidized-form thiols (cystine, homocystine, oxidized-form glutathione) because of the thiol exchange reaction between the reduced-form and the oxidized-form thiols. The use of ABD-F at low temperature enabled the suppression of these thiol exchange reactions, and the recommended conditions were below 5 degrees C for 90 min in borate buffer (pH 9.3). These results suggest that ABD-F is a preferred derivatization reagent for the accurate determination of the reduced-form thiols in samples containing the oxidized-form thiols. In addition, it was also suggested that the derivatization of the reduced-form thiols should also be performed at low temperature when derivatization reagents such as o-phthalaldehyde (OPA) and monobromobimane (BrB) are used.
Santa
Tomofumi
T
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Japan. santa@mol.f.u-tokyo.ac.jp
Aoyama
Chiaki
C
Fukushima
Takeshi
T
Imai
Kazuhiro
K
Funatsu
Takashi
T
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D005464:Fluorobenzenes / Q000737:chemistry*; D010069:Oxadiazoles / Q000737:chemistry*; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D005456:Fluorescent Dyes; D005464:Fluorobenzenes; D010069:Oxadiazoles; D013438:Sulfhydryl Compounds; C037802:7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate; C046364:4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole