School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA, USA.
Parker Petit Institute for Bioengineering and Biosciences, Atlanta, GA, USA.
Methods Mol Biol. 2024;2839:151-194. doi: 10.1007/978-1-0716-4043-2_9.
Heme b (iron protoporphyrin IX) is an essential but potentially cytotoxic cofactor, signaling molecule, and nutritional source of iron. Its importance in cell biology and metabolism is underscored by the fact that numerous diseases, including various cancers, neurodegenerative disorders, infectious diseases, anemias, and porphyrias, are associated with the dysregulation of heme synthesis, degradation, trafficking, and/or transport. Consequently, methods to measure, image, and quantify heme in cells are required to better understand the physiology and pathophysiology of heme. Herein, we describe fluorescence-based protocols to probe heme bioavailability and trafficking dynamics using genetically encoded fluorescent heme sensors in combination with various modalities, such as confocal microscopy, flow cytometry, and microplate readers. Additionally, we describe a protocol for measuring total heme and its precursor protoporphyrin IX using a fluorometric assay that exploits porphyrin fluorescence. Together, the methods described enable the monitoring of total and bioavailable heme to study heme homeostatic mechanisms in virtually any cell type and organism.
血红素 b（铁原卟啉 IX）是一种必需但潜在细胞毒性的辅助因子、信号分子和铁的营养来源。许多疾病，包括各种癌症、神经退行性疾病、传染病、贫血症和卟啉症，都与血红素合成、降解、运输和/或转运的失调有关，这凸显了其在细胞生物学和新陈代谢中的重要性。因此，需要测量、成像和定量细胞内血红素的方法，以更好地理解血红素的生理学和病理生理学。在此，我们描述了使用遗传编码的荧光血红素传感器结合各种方法（如共聚焦显微镜、流式细胞术和微孔板读数器）来探测血红素生物利用度和运输动力学的荧光法方案。此外，我们还描述了一种使用荧光法测定总血红素及其前体原卟啉 IX 的方法，该方法利用卟啉荧光。总之，所描述的方法能够监测总血红素和生物可利用血红素，从而研究几乎任何细胞类型和生物体中的血红素稳态机制。
