Green J D, Narahara H T
J Histochem Cytochem. 1980 May;28(5):408-12. doi: 10.1177/28.5.6966645.
An improved spectrophotometric method for measuring succinate dehydrogenase (EC 1.3.99.1) activity with the use of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) is described. The procedure has been evaluated in mitochondrial fractions and homogenates of frog skeletal muscle. For mitochondrial suspensions, extraction of formazan with alcohol was found to be superior to extraction with ethyl acetate. For homogenates, complete extraction of formazan required sequential treatment with alcohol and ethyl acetate; the generally employed procedure of extracting once with ethyl acetate alone led to serious underestimation of the amount of formazan in the tissue. Observations of mitochondrial suspension incubated with various concentrations of INT led to the selection of 0.8 mM INT for optimal results. Higher concentrations, although commonly used, can exert undesirable inhibitory effects on succinate dehydrogenase activity, especially at low concentrations of mitochondria and after longer periods of incubation. The problem of instability of succinate dehydrogenase was solved by the addition of buffer at pH 7.5.
描述了一种改进的分光光度法，该方法使用2-(对碘苯基)-3-(对硝基苯基)-5-苯基四氮唑氯化物（INT）来测量琥珀酸脱氢酶（EC 1.3.99.1）的活性。该方法已在青蛙骨骼肌的线粒体组分和匀浆中进行了评估。对于线粒体悬浮液，发现用乙醇提取甲臜优于用乙酸乙酯提取。对于匀浆，甲臜的完全提取需要依次用乙醇和乙酸乙酯处理；通常仅用乙酸乙酯提取一次的方法会导致严重低估组织中甲臜的量。观察用不同浓度INT孵育的线粒体悬浮液后，选择0.8 mM INT可获得最佳结果。较高浓度虽然常用，但可能对琥珀酸脱氢酶活性产生不良抑制作用，尤其是在低浓度线粒体和较长孵育时间后。通过添加pH 7.5的缓冲液解决了琥珀酸脱氢酶的稳定性问题。
