title: Rapid and sensitive quantitation of heme in hemoglobinized cells.
abstract: Rapid and accurate heme quantitation in the research lab has become more desirable as the crucial role that intracellular hemoproteins play in metabolism continues to emerge. Here, the time-honored approaches of pyridine hemochromogen and fluorescence heme assays are compared with direct absorbance-based technologies using the CLARiTY spectrophotometer. All samples tested with these methods were rich in hemoglobin-associated heme, including buffered hemoglobin standards, whole blood from mice, and murine erythroleukemia (MEL) and K562 cells. While the pyridine hemochromogen assay demonstrated the greatest linear range of heme detection, all 3 methods demonstrated similar analytical sensitivities and normalized limits of quantitation of ∼1 µM. Surprisingly, the fluorescence assay was only shown to be distinct in its ability to quantitate extremely small samples. Using the CLARiTY system in combination with pyridine hemochromogen and cell count data, a common hemoglobin extinction coefficient for blood and differentiating MEL and K562 cells of 0.46 µM-1 cm-1 was derived. This value was applied to supplemental experiments designed to measure MEL cell hemoglobinization in response to the addition or removal of factors previously shown to affect heme biosynthesis (e.g., L-glutamine, iron).
authors.affiliation: Department of Biochemistry and Molecular Biology, Biomedical and Health Sciences Institute, University of Georgia, Athens.
authors.forename: Jason R
authors.initials: JR
authors.lastname: Marcero
authors.affiliation: Department of Biochemistry and Molecular Biology, Biomedical and Health Sciences Institute, University of Georgia, Athens.
authors.forename: Robert B
authors.initials: RB
authors.lastname: Piel Iii
authors.affiliation: Department of Biochemistry and Molecular Biology, Biomedical and Health Sciences Institute, University of Georgia, Athens.
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Burch
authors.affiliation: Department of Biochemistry and Molecular Biology, Biomedical and Health Sciences Institute, University of Georgia, Athens.
authors.forename: Harry A
authors.initials: HA
authors.lastname: Dailey
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D004867:Equipment Design; D004909:Erythrocyte Indices; D005260:Female; D006418:Heme / Q000031:analogs & derivatives / Q000032:analysis* / Q000737:chemistry*; D006454:Hemoglobins / Q000032:analysis / Q000737:chemistry*; D006801:Humans; D020014:K562 Cells; D057230:Limit of Detection; D016014:Linear Models; D008297:Male; D051379:Mice; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D006454:Hemoglobins; C023647:pyridine hemochrome; D006418:Heme
keywords: K562 cells; fluorescence heme assay; heme; hemoglobin; in-situ spectroscopy; integrating cavity; murine erythroleukemia cells; pyridine hemochromogen assay