Measurement of Heme Concentration

Abstract Heme (iron protoporphyrin IX) is a prosthetic group for a number of hemoproteins in different tissues (e.g., hemoglobin, myoglobin, cytochrome P‐450s, mitochondrial cytochromes, catalases, and peroxidases). Mutations in the biosynthetic pathway can affect the synthesis and/or degradation of heme. Several assays are provided in this unit for quantifying heme: a spectophotometric assay based on the characteristic absorption spectrum of oxidized and reduced form of the hemochrome formed by replacing the nitrogen ligands with pyridine; a fluorescence assay based on removal of the iron by a heated, strong oxalic acid solution to produce fluorescent protoporphyrin; a reversed‐phase HPLC assay to measure heme and intermediates in the synthetic pathway; and a radiometric assay to measure newly synthesized heme in tissue culture cells.