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GSHandGSSGconcentrationsweredeterminedaccordingtothemethodofAnderson[23].Briefly,tiss...
GSH and GSSG concentrations were determined according to the
method of Anderson [23]. Briefly, tissue samples were homogenized in
5% 5-sulfosalicylic acid and centrifuged at 18 000 3 g for 5 min. For
GSH measurements, the supernatant was added to a reaction cocktail that
was equilibrated at 258C and consisted of stock buffer (143 mM sodium
phosphate, 6.3 mM Na4-EDTA, pH 7.5); NADPH (0.247 mg/ml); and
5,59-dithiobis(2-nitrobenzoic acid) (DTNB) (6 mM). GSSG reductase (266
U/ml) was added to initiate the assay, and the change in absorbance was
read at 412 nm. GSSG was determined by the same reaction, except that
GSH was blocked from reacting with DTNB by a 1-h derivatization at
258C with 2-vinylpyridine. It was determined that GSH assay recording
remained linear down to 0.005 nmol/mg tissue and GSSG to 0.001 nmol/
mg tissue. 展开
method of Anderson [23]. Briefly, tissue samples were homogenized in
5% 5-sulfosalicylic acid and centrifuged at 18 000 3 g for 5 min. For
GSH measurements, the supernatant was added to a reaction cocktail that
was equilibrated at 258C and consisted of stock buffer (143 mM sodium
phosphate, 6.3 mM Na4-EDTA, pH 7.5); NADPH (0.247 mg/ml); and
5,59-dithiobis(2-nitrobenzoic acid) (DTNB) (6 mM). GSSG reductase (266
U/ml) was added to initiate the assay, and the change in absorbance was
read at 412 nm. GSSG was determined by the same reaction, except that
GSH was blocked from reacting with DTNB by a 1-h derivatization at
258C with 2-vinylpyridine. It was determined that GSH assay recording
remained linear down to 0.005 nmol/mg tissue and GSSG to 0.001 nmol/
mg tissue. 展开
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谷胱甘肽和氧化型谷胱甘肽的浓度是依照安德森[23]的方法决定的。简而言之,组织样品在5%的5-磺基水杨酸中成均匀分布微粒并以180003g离心5分钟。对谷胱甘肽的测量,将上浮物质加入反应混合物中,此混合物是在258C下平衡并由储备缓冲剂(143毫摩钠磷酸盐,6.3毫摩乙二胺四乙酸,ph7.5)组成;NADPH(还原性辅酶Ⅱ)(0.247mg/ml);5,59-二硫代双(2-硝基苯甲酸)(DTNB)(6毫摩)。加入氧化型谷胱甘肽还原酶(266U/ml)开始测定,在412纳米处看到吸光度的改变。氧化型谷胱甘肽由同一反应决定,而还原型谷胱甘肽的反应被DTNB的1-h衍生物在258C由2-乙烯基吡啶阻断。这取决于谷胱甘肽的测定记录在下至0.005nmol/mg组织时仍呈线性,氧化型谷胱甘肽则是到0.001nmol/mg组织
终于翻完了,累死了!我是学医的,对GSH有所了解,希望能帮到你。
终于翻完了,累死了!我是学医的,对GSH有所了解,希望能帮到你。
参考资料: 有些部分也不太确切,如离心的单位我只知道r/min,不知文中g是什么意思
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