Tramer Federica, Sist Paola, Cardenas-Perez Rocio, Urbani Ranieri, Bortolussi Giulia, Passamonti Sabina
Department of Life Sciences, University of Trieste, via Giorgieri 1, I-34127, Trieste, Italy.
Group of Bioengineering in Regeneration and Cancer, Biogipuzkoa Health Research Institute, San Sebastián, Spain.
MethodsX. 2024 Sep 25;13:102979. doi: 10.1016/j.mex.2024.102979. eCollection 2024 Dec.
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.
胆绿素是血红素分解代谢的次级代谢产物。它由血红素加氧酶催化的反应形成，该酶将血红蛋白、肌红蛋白、细胞色素和过氧化氢酶等蛋白质中所含的血红素基团转化为等摩尔量的胆绿素、铁（II）和一氧化碳，消耗还原型辅酶II（NADPH）。然后，胆绿素被胆绿素还原酶还原为胆红素。胆绿素和胆红素形成一个氧化还原对，对细胞的氧化还原稳态很重要。血红素加氧酶-1是一种诱导型酶，可由缺氧条件、血红素可用性增加或诸如脂多糖、紫外线辐射等促炎机制诱导。此外，血红素加氧酶-1和胆绿素还原酶除了催化作用外，还通过在转录水平调节特定代谢途径发挥其他作用。需要有经济实惠的检测方法来分析生物和临床样本中的这些胆汁色素。在此，我们提出一种联合测定胆绿素和胆红素的方法，该方法利用胆红素与荧光重组融合蛋白HUG的特异性结合以及胆绿素向胆红素的酶促转化。•该方法能够在纳摩尔范围内联合测定胆红素和胆绿素。•该方法不需要对样品进行溶剂萃取或蛋白质沉淀。
