Nunn A V, Norris P, Hawk J L, Cox T M
Department of Haematology, Royal Postgraduate Medical School, Hammersmith Hospital, London, United Kingdom.
Anal Biochem. 1988 Oct;174(1):146-50. doi: 10.1016/0003-2697(88)90529-5.
We describe a fluorometric assay for heme synthetase, the enzyme that is genetically deficient in erythropoietic protoporphyria. The method, which can readily detect activity in 1 microliter of packed human lymphocytes, is based on the formation of zinc protoheme from protoporphyrin IX. That zinc chelatase and ferrochelatase activities reside in the same enzyme was shown by the competitive action of ferrous ions and the inhibitory effects of N-methyl protoporphyrin (a specific inhibitor of heme synthetase) on zinc chelatase. The Km for zinc was 11 micrograms and that for protoporphyrin IX was 6 microM. The Ki fro ferrous ions was 14 microM. Zinc chelatase was reduced to 15.3% of the mean control activity in lymphocytes obtained from patients with protoporphyria, thus confirming the defect of heme biosynthesis in this disorder. The assay should prove to be useful for determining heme synthetase in tissues with low specific activity and to investigate further the enzymatic defect in protoporphyria.
我们描述了一种用于血红素合成酶的荧光测定法，该酶在红细胞生成性原卟啉症中存在基因缺陷。该方法基于原卟啉IX形成锌原卟啉，能够轻松检测1微升人外周血淋巴细胞中的活性。亚铁离子的竞争作用以及N-甲基原卟啉（血红素合成酶的特异性抑制剂）对锌螯合酶的抑制作用表明，锌螯合酶和亚铁螯合酶活性存在于同一种酶中。锌的Km为11微克，原卟啉IX的Km为6微摩尔。亚铁离子的Ki为14微摩尔。在从原卟啉症患者获得的淋巴细胞中，锌螯合酶降至平均对照活性的15.3%，从而证实了这种疾病中血红素生物合成的缺陷。该测定法对于测定低比活性组织中的血红素合成酶以及进一步研究原卟啉症中的酶缺陷应是有用的。
