{"title":"新型灵敏高效液相色谱法测定香豆素7-羟基化。","authors":"P Soucek","doi":"","DOIUrl":null,"url":null,"abstract":"<p><p>In this paper, a novel HPLC-based method with fluorometric detection of coumarin 7-hydroxylase is presented. The described method provides a time-effective, more sensitive and specific alternative to the previously used spectrofluorometric assay. Using the developed method, metabolism of coumarin in 11 samples of human liver microsomes was evaluated and 1790+/-690 pmol/min/nmol cytochrome P450 (CYP) activity was found. Kinetic parameters and linearity of coumarin 7-hydroxylation were studied in a reconstituted system consisting of recombinant CYP2A6 expressed in Escherichia coli, rat NADPH-CYP reductase and usual components. It was found that a 3.5 to 30 min time of incubation is suitable for estimation of coumarin 7-hydroxylase activity. Observed Km and Vmax values in the CYP2A6 reconstituted system were 1.48+/-0.37 microM and 3360+/-180 pmol product/min/nmol CYP, respectively.</p>","PeriodicalId":15425,"journal":{"name":"Journal of chromatography. B, Biomedical sciences and applications","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"1999-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Novel sensitive high-performance liquid chromatographic method for assay of coumarin 7-hydroxylation.\",\"authors\":\"P Soucek\",\"doi\":\"\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>In this paper, a novel HPLC-based method with fluorometric detection of coumarin 7-hydroxylase is presented. The described method provides a time-effective, more sensitive and specific alternative to the previously used spectrofluorometric assay. Using the developed method, metabolism of coumarin in 11 samples of human liver microsomes was evaluated and 1790+/-690 pmol/min/nmol cytochrome P450 (CYP) activity was found. Kinetic parameters and linearity of coumarin 7-hydroxylation were studied in a reconstituted system consisting of recombinant CYP2A6 expressed in Escherichia coli, rat NADPH-CYP reductase and usual components. It was found that a 3.5 to 30 min time of incubation is suitable for estimation of coumarin 7-hydroxylase activity. Observed Km and Vmax values in the CYP2A6 reconstituted system were 1.48+/-0.37 microM and 3360+/-180 pmol product/min/nmol CYP, respectively.</p>\",\"PeriodicalId\":15425,\"journal\":{\"name\":\"Journal of chromatography. B, Biomedical sciences and applications\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":0.0000,\"publicationDate\":\"1999-10-29\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of chromatography. B, Biomedical sciences and applications\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of chromatography. B, Biomedical sciences and applications","FirstCategoryId":"1085","ListUrlMain":"","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
Novel sensitive high-performance liquid chromatographic method for assay of coumarin 7-hydroxylation.
In this paper, a novel HPLC-based method with fluorometric detection of coumarin 7-hydroxylase is presented. The described method provides a time-effective, more sensitive and specific alternative to the previously used spectrofluorometric assay. Using the developed method, metabolism of coumarin in 11 samples of human liver microsomes was evaluated and 1790+/-690 pmol/min/nmol cytochrome P450 (CYP) activity was found. Kinetic parameters and linearity of coumarin 7-hydroxylation were studied in a reconstituted system consisting of recombinant CYP2A6 expressed in Escherichia coli, rat NADPH-CYP reductase and usual components. It was found that a 3.5 to 30 min time of incubation is suitable for estimation of coumarin 7-hydroxylase activity. Observed Km and Vmax values in the CYP2A6 reconstituted system were 1.48+/-0.37 microM and 3360+/-180 pmol product/min/nmol CYP, respectively.
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