{"title":"利用重组蛋白 HUG 对胆红素和胆红素进行联合荧光分析","authors":"Federica Tramer , Paola Sist , Rocio Cardenas-Perez , Ranieri Urbani , Giulia Bortolussi , Sabina Passamonti","doi":"10.1016/j.mex.2024.102979","DOIUrl":null,"url":null,"abstract":"<div><div>Biliverdin is a secondary metabolite of heme catabolism. It is formed by the reaction catalyzed by heme oxygenase, which converts the heme group contained in proteins such as hemoglobin, myoglobin, cytochromes, and catalase into biliverdin, iron (II) and CO in equimolar amounts, consuming NADPH. Biliverdin is then reduced to bilirubin by biliverdin reductase. Biliverdin and bilirubin form a redox couple and are important for the redox homeostasis of cells. Heme oxygenase-1 is an inducible enzyme that is induced by hypoxic conditions, increased availability of heme or proinflammatory mechanisms such as LPS, UV radiation, etc. In addition, both heme oxygenase-1 and biliverdin reductase play roles other than catalysis by modulating specific metabolic pathways at the transcriptional level. There is a need for affordable assays to analyze these bile pigments in biological and clinical samples. Here we present a method for the combined determination of biliverdin and bilirubin that utilizes the specific binding of bilirubin to the fluorescent recombinant fusion protein HUG and the enzymatic conversion of biliverdin to bilirubin.<ul><li><span>•</span><span><div>This method enables the combined measurement of bilirubin and biliverdin in the nM range.</div></span></li><li><span>•</span><span><div>The method does not require solvent extraction or protein precipitation of the samples.</div></span></li></ul></div></div>","PeriodicalId":18446,"journal":{"name":"MethodsX","volume":"13 ","pages":"Article 102979"},"PeriodicalIF":1.6000,"publicationDate":"2024-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Combined fluorometric analysis of biliverdin and bilirubin by the recombinant protein HUG\",\"authors\":\"Federica Tramer , Paola Sist , Rocio Cardenas-Perez , Ranieri Urbani , Giulia Bortolussi , Sabina Passamonti\",\"doi\":\"10.1016/j.mex.2024.102979\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><div>Biliverdin is a secondary metabolite of heme catabolism. It is formed by the reaction catalyzed by heme oxygenase, which converts the heme group contained in proteins such as hemoglobin, myoglobin, cytochromes, and catalase into biliverdin, iron (II) and CO in equimolar amounts, consuming NADPH. Biliverdin is then reduced to bilirubin by biliverdin reductase. Biliverdin and bilirubin form a redox couple and are important for the redox homeostasis of cells. Heme oxygenase-1 is an inducible enzyme that is induced by hypoxic conditions, increased availability of heme or proinflammatory mechanisms such as LPS, UV radiation, etc. In addition, both heme oxygenase-1 and biliverdin reductase play roles other than catalysis by modulating specific metabolic pathways at the transcriptional level. There is a need for affordable assays to analyze these bile pigments in biological and clinical samples. Here we present a method for the combined determination of biliverdin and bilirubin that utilizes the specific binding of bilirubin to the fluorescent recombinant fusion protein HUG and the enzymatic conversion of biliverdin to bilirubin.<ul><li><span>•</span><span><div>This method enables the combined measurement of bilirubin and biliverdin in the nM range.</div></span></li><li><span>•</span><span><div>The method does not require solvent extraction or protein precipitation of the samples.</div></span></li></ul></div></div>\",\"PeriodicalId\":18446,\"journal\":{\"name\":\"MethodsX\",\"volume\":\"13 \",\"pages\":\"Article 102979\"},\"PeriodicalIF\":1.6000,\"publicationDate\":\"2024-09-25\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"MethodsX\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S2215016124004308\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"MULTIDISCIPLINARY SCIENCES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"MethodsX","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2215016124004308","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"MULTIDISCIPLINARY SCIENCES","Score":null,"Total":0}
Combined fluorometric analysis of biliverdin and bilirubin by the recombinant protein HUG
Biliverdin is a secondary metabolite of heme catabolism. It is formed by the reaction catalyzed by heme oxygenase, which converts the heme group contained in proteins such as hemoglobin, myoglobin, cytochromes, and catalase into biliverdin, iron (II) and CO in equimolar amounts, consuming NADPH. Biliverdin is then reduced to bilirubin by biliverdin reductase. Biliverdin and bilirubin form a redox couple and are important for the redox homeostasis of cells. Heme oxygenase-1 is an inducible enzyme that is induced by hypoxic conditions, increased availability of heme or proinflammatory mechanisms such as LPS, UV radiation, etc. In addition, both heme oxygenase-1 and biliverdin reductase play roles other than catalysis by modulating specific metabolic pathways at the transcriptional level. There is a need for affordable assays to analyze these bile pigments in biological and clinical samples. Here we present a method for the combined determination of biliverdin and bilirubin that utilizes the specific binding of bilirubin to the fluorescent recombinant fusion protein HUG and the enzymatic conversion of biliverdin to bilirubin.
•
This method enables the combined measurement of bilirubin and biliverdin in the nM range.
•
The method does not require solvent extraction or protein precipitation of the samples.