Li F, Lim C K, Peters T J
Division of Clinical Cell Biology, MRC Clinical Research Centre, Harrow, Middx., U.K.
Biochem J. 1987 May 1;243(3):863-6. doi: 10.1042/bj2430863.
An h.p.l.c. method is described for the assay of protoporphyrinogen oxidase activity in rat liver. A relatively pure protoporphyrinogen IX substrate was obtained by selectively removing any protoporphyrin IX unreduced by sodium amalgam on a small disposable cartridge packed with a strong anion-exchanger. The protoporphyrin IX formed was extracted with dimethyl sulphoxide/methanol (3:7, v/v) containing mesoporphyrin as the internal standard for separation and quantification by reversed-phase chromatography. The Km for protoporphyrinogen was 9.5 +/- 1.6 microM, and the enzyme activities were 0.59 +/- 0.11 nmol of protoporphyrin IX produced/min per mg of mitochondrial protein and 33.5 +/- 2.7 nmol protoporphyrin IX produced/min per g of liver tissue homogenate. The method is applicable to the determination of enzyme activity in small amounts of human liver biopsy.
本文描述了一种用于测定大鼠肝脏中原卟啉原氧化酶活性的高效液相色谱法。通过在装有强阴离子交换剂的小型一次性柱上选择性去除未被汞齐钠还原的原卟啉IX，获得了相对纯的原卟啉原IX底物。形成的原卟啉IX用含有中卟啉作为内标的二甲基亚砜/甲醇（3:7，v/v）萃取，通过反相色谱法进行分离和定量。原卟啉原的Km为9.5±1.6微摩尔，酶活性为每毫克线粒体蛋白每分钟产生0.59±0.11纳摩尔原卟啉IX，以及每克肝脏组织匀浆每分钟产生33.5±2.7纳摩尔原卟啉IX。该方法适用于测定少量人肝活检组织中的酶活性。
