Tetrapyrroles were extracted from homogenized leaves using 300 µL of acetone:0.2 M NH4OH (9:1), incubated for 30 min at −20°C and centrifuged for 30 min (16,000 × g, 4°C). The supernatant was analyzed by HPLC for (Mg) porphyrins and Chls. Noncovalently bound (ncb) heme was extracted from the remaining pellet by resuspension in 200 µL of AHD (acetone:HCl:DMSO, 10:0.5:2) and incubated for 15 min at RT. After centrifugation for 15 min (16,000 × g, RT), the amount of ncb heme in the supernatant was quantified by HPLC. HPLC analyses were performed on Agilent LC systems following previously described methods (Richter et al. 2019 (link)) and using authentic standards for peak quantification.
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Quantifying Leaf Tetrapyrroles and Heme
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Corresponding Organization : Humboldt-Universität zu Berlin
Other organizations : University of Havana
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Variable analysis
independent variables
- Extraction method (acetone:0.2 M NH4OH (9:1))
- Incubation time (30 min at -20°C)
- Centrifugation (16,000 × g, 4°C)
- Resuspension method (AHD: acetone:HCl:DMSO, 10:0.5:2)
- Incubation time (15 min at RT)
- Quantity of (Mg) porphyrins and Chls
- Quantity of noncovalently bound (ncb) heme
- Homogenized leaf samples
- HPLC analysis
- Positive control: Authentic standards for peak quantification
- Negative control: Not explicitly mentioned
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