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).

Sideroflexin 4 affects Fe-S cluster biogenesis, iron metabolism, mitochondrial respiration and heme biosynthetic enzymes

“…Germline mutations in SFXN4 cause anemia and defects in hemoglobinization 2 . To test the effects of SFXN4 knockout on hemoglobin synthesis, we induced erythroid differentiation in K562 SFXN4 knockout cells and scrambled controls using sodium butyrate and measured hemoglobin (Hb) levels using non-denaturing electrophoresis 27 . SFXN4 knockdown substantially reduced hemoglobin levels (Fig.…”

From Synthesis to Utilization: The Ins and Outs of Mitochondrial Heme

“…This method allows for differentiating heme b from other heme types, including heme o or heme a [233]. Alternatively, extracted heme or heme from soluble lysates or isolated hemoproteins may be quantified using the pyridine hemochromagen assay, which is based upon the distinct spectral features of the bis-pyridine ferrous heme complex [234][235][236][237]. The development of commercial instrumentation to measure absorbing species in turbid samples, including in cells and tissues, has greatly facilitated the measurement of heme in biological matrices.…”

“…The development of commercial instrumentation to measure absorbing species in turbid samples, including in cells and tissues, has greatly facilitated the measurement of heme in biological matrices. The Olis CLARiTY UV-vis spectrophotometer employs a quartz cuvette within an integrating cavity absorption meter (ICAM) [237]. The quartz cuvette contains a reflective coating that effectively increases the path length, making the instrument much more sensitive to absorption of light in turbid samples [237,238].…”

“…The Olis CLARiTY UV-vis spectrophotometer employs a quartz cuvette within an integrating cavity absorption meter (ICAM) [237]. The quartz cuvette contains a reflective coating that effectively increases the path length, making the instrument much more sensitive to absorption of light in turbid samples [237,238]. Finally, the measurement of porphyrin fluorescence is a very sensitive technique for the quantification of heme, with a limit of detection of 10 −9 M [232,233].…”

Glutamine via α-ketoglutarate dehydrogenase provides succinyl-CoA for heme synthesis during erythropoiesis

“…For cells that are undergoing late erythropoiesis and beginning to hemoglobinize, we measured cellular heme content 41 at days 3 and 6 following removal of cytokines/growth factors other than erythropoietin (EPO). For these cells, we found that AOA inhibited hemoglobinization and that this could be overcome by addition of DMK and ALA, but not DES, to the medium ( Figure 5C; supplemental Figure 3).…”