Simultaneous determination of thiols and disulfides by high-performance liquid chromatography with fluorescence detection
Simultaneous determination of thiols and disulfides by high-performance liquid chromatography with fluorescence detection
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荧光检测高效液相色谱同时测定硫醇和二硫化物
DOI：
10.1016/s0003-2670(00)82313-2
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发表时间：
1988
影响因子：
6.2
通讯作者：
K. Imai
中科院分区：
文献类型：
--
作者：
T. Toyo′oka;S. Uchiyama;Yukio Saito;K. Imai
建议同时测定硫醇和二硫化物需要4-（氨基磺酰基）-7-氟-2，1，3-苯并恶二唑（ABD-F）作为硫醇的柱前衍生试剂和7-氟-2，1，3-苯并恶二唑-4-磺酸铵（SBD-F）作为二硫化物的柱前衍生试剂，然后进行色谱分析。将溶液中的硫醇和二硫化物用ABD-F在含有InM二钠-EDTA的pH 9.3硼酸盐缓冲液中在60°C下处理5分钟。用乙酸乙酯除去过量的ABD-F后，在三正丁基膦（还原剂）存在下，在60°C下用SBD-F处理水相中剩余的二硫化物20分钟。通过反相色谱法分离由此产生的ABD-硫醇和SBD-硫醇，并通过荧光测定法（380 nm激发，510 nm发射）检测。SBD-半胱氨酸、SBD-同型半胱氨酸、ABD-同型半胱氨酸、ABD-半胱氨酸、SBD-谷氨酸、ABD-同型半胱氨酸、SBD-N-乙酰半胱氨酸、ABD-谷胱甘肽和ABD-N-乙酰半胱氨酸通过从0.15 M H3 PO 4/CH 3CN（96：4）至0.15 M H3 PO 4/CH 3CN（85：15）的线性梯度洗脱30分钟，然后用0.15 M H3 PO 4/CH 3CN（85：15）等度洗脱10分钟，得到很好的分离。衍生物的检测限在0.09-0.9 pmol范围内。用该法测定大鼠组织中巯基和二硫化物，在肾中测得半胱氨酸（0.75 μmol g ~（-1））和胱氨酸（0.62 μmol g ~（-1）），在肝、脾、心和睾丸中测得还原型谷胱甘肽（1.4-3.4 μmol g ~（-1））。
The proposed simultaneous determination of thiols and disulfides requires 4- (aminosulfonyl)- 7-fluor-2,1,3,-benzoxadiazole (ABD-F) as the pre-column derivatization reagent for thiols and ammonium 7-fluoro-2,1,3,-benzoxadiazole-4-sulfonate (SBD-F) for disulfides followed by chromatography. The thiols and disulfides in solution are treated with ABD-F at 60°C for 5 min in pH 9.3 borate buffer containing 1 nM disodium-EDTA. After removal of excess of ABD-F with ethyl acetate, the remaining disulfides in the aqueous phase are treated with SBD-F at 60°C for 20 min in the presence of tri-n-butylphosphine, a reducing agent. The ABD-thiols and SBD-thiols thus produced are separated by reversed-phase chromatography and detected by fluorimetry (380- nm excitation, 510 - nm emission). SBD-cystein, SBD-homocystein, ABD-homocysteine, ABD-cysteine, SBD-glu- tathione, ABD-homocystein, SBD-N-acetylcystein, ABD-glutatione and ABD-N-acetylcysteine are well separated by linear gradient elution from 0.15 M H3PO4/CH3CN (96:4) to 0.15 M H3PO4/CH3CN (85:15) over 30 min followed by isocratic elution with 0.15 M H3PO4/CH3CN (85:15) fro 10 min. The detection limits for the derivatives are in the range 0.09–0.9 pmol. When the method was applied to the determination of thiols and disulfides in rat tissues, cystein (0.75 μmol g-1) and cystine (0.62 μmol g-1) were obtained in kidney and reduced glutathione (1.4–3.4 μmol g-1) was observed in liver, spleen, heart and testicle.
影响因子：
2.9
作者：
REED, DJ;BABSON, JR;POTTER, DW
通讯作者：
POTTER, DW
耦合因子 1 的 γ 亚基的含半胱氨酰肽的表征。
DOI：
--
发表时间：
1984
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Moroney,JV;Fullmer,CS;McCarty,RE
通讯作者：
McCarty,RE