Guo R, Lim C K, Peters T J
Division of Clinical Cell Biology, MRC Clinical Research Centre, Harrow, Middlesex, UK.
J Chromatogr. 1991 May 31;566(2):383-96. doi: 10.1016/0378-4347(91)80255-b.
Rapid, sensitive and specific high-performance liquid chromatographic assays are described for protoporphyrinogen oxidase and ferrochelatase in human leucocytes. The enzyme reaction products were separated and quantitated by reversed-phase high-performance liquid chromatography with fluorescence detection. The optimal pH for the protoporphyrinogen oxidase assay was 8.6 and the Michaelis constant for protoporphyrinogen IX was 9.78 +/- 0.96 microM (mean +/- S.D.). The mean (+/- S.D.) activity of protoporphyrinogen oxidase in fourteen apparently healthy subjects was 0.146 +/- 0.023 nmol protoporphyrin IX per min per mg protein. In one patient with variegate porphyria, the activity was 0.028 nmol protoporphyrin IX per min per mg protein. The optimal pH for ferrochelatase was 7.4 and with protoporphyrin and Zn2+ as substrates, the Michaelis constants were 1.49 and 8.33 microM, respectively. The mean activity of ferrochelatase in ten control subjects was 0.24 nM Zn-protoporphyrin or 2.05 nM Zn-mesoporphyrin formed per h per mg protein.
本文描述了用于检测人白细胞中原卟啉原氧化酶和亚铁螯合酶的快速、灵敏且特异的高效液相色谱法。酶反应产物通过带有荧光检测的反相高效液相色谱法进行分离和定量。原卟啉原氧化酶检测的最佳pH值为8.6，原卟啉原IX的米氏常数为9.78±0.96微摩尔（平均值±标准差）。14名表面健康受试者中原卟啉原氧化酶的平均（±标准差）活性为每分钟每毫克蛋白质0.146±0.023纳摩尔原卟啉IX。在一名患有混合型卟啉病的患者中，该活性为每分钟每毫克蛋白质0.028纳摩尔原卟啉IX。亚铁螯合酶的最佳pH值为7.4，以原卟啉和Zn2+作为底物时，米氏常数分别为1.49和8.33微摩尔。10名对照受试者中亚铁螯合酶的平均活性为每小时每毫克蛋白质形成0.24纳摩尔锌原卟啉或2.05纳摩尔锌中卟啉。
