NADPH氧化酶检测方法的原理

Principle and method：

NADPH-dependent O，- generation was measured using the tetrazolium dye NBT as an electron acceptor.NBT is rapidly converted to monoformazan by two molecules of 02-.This reduction is detected spectrophotometrically （Aminco DW2000 spectrophotometer，SLM Instruments，Urbana，IL）at 530 nm.Monoformazan concentrations （and therefore 0，- concentrations）were calculated using an extinction coefficient of 12.8 mM-l cm-l.The NBT reduction rates are strictly linear with time up to 10 to 15 min and are linearly dependent on the protein concentration （10-50 pg）in the sample.The selective reduction of NBT by O，- was calculated from the difference in the NBT reduction rate in the presence and absence of SOD （50-100 units ml-l，EC 1.15.1.1，Fluka）.The reaction mixture consisted of a Trisbuffer （50 mM Tris-HCI，pH 7.4，250 mM SUCw）ith additions as specified in the text.No NBT reduction with reduced nucleotides （NAD[P]H）was observed in the absence of protein fractions.

Good examples：

1.http://seekspace.resip.ac.cn/bitstream/2239/35034/1/NaCl%E5%AF%B9%E5%B0%8F%E9%BA%A6%E6%A0%B9%E8%B4%A8%E8%86%9CNADPH%E6%B0%A7%E5%8C%96%E9%85%B6%E6%B4%BB%E6%80%A7%E7%9A%84%E5%BD%B1%E5%93%8 D .pdf

2.http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=158513&blobtype=pdf

国外文献中使用较多的NADPH氧化酶活性检测方法，翻译如下：

细胞胰酶酶消化后，在4℃下2，500 g 离心5 分钟，然后以PBS再悬浮，随后加入250 μmol/l NADPH孵育，在λ = 340 nm 下观察5 分钟，通过吸光率的减少来探测NADPH的消耗量。为分析NADPH氧化酶特异的活性，在检测之前30 min加入10 μmol/l DPI ，来检测DPI抑制后的NADPH消耗率（the rate of consumption of NADPH inhibited by DPI）。为了标准化，取等量的细胞加入SDS溶解，浓缩蛋白后通过Lowry solution测定。用来计算NADPH消耗总量的吸收消光系数（absorption extinction coefficient）是6.22 mM-1 cm-1，结果以pmol NADPH / min•mg蛋白表示.