This protocol is used to measure intracellular heme concentrations. Note that it measures total heme levels (not free heme - most if not all protein-bound heme is liberated during the assay). The protocol is derived from the assay described by Sassa (reference below).
- 2 M oxalic acid
- 20 mM oxalic acid
- Black 96-well plates
- Hemin standard (~1 g/L in DMSO)
- Grow cells to desired density. Ideally, you want roughly 8 OD*mL of yeast (e.g. 20 mL at OD 0.4). Measure the density of each sample.
- Pellet cells (in a Falcon tube), 5 minutes at 4 °C and 5000 g. Wash with water, transfer to an amber Eppendorf tube, and repellet for 5 minutes at 4 °C and 8000 g.
- Discard supernatant, resuspend in 500 μL of 20 mM oxalic acid. Store in a closed box in the fridge overnight (16-24 hours).
- Add an equal volume (500 μL) of 2 M oxalic acid. Pipet to mix, then transfer one volume (500 μL) to a new Eppendorf tube.
- 2 M oxalic acid is not soluble in water at room temperature. You must heat the oxalic acid to dissolve it before starting the protocol
- Transfer the first amber Eppendorf tube to a heat block at 95-98 °C. Heat for 30 minutes. Meanwhile, store the replicate tube in a closed box at RT.
- If you put the replicate tube at 4 °C, the oxalic acid will crash out of solution again. 1 M oxalic acid is soluble at RT but not 4 °C.
- Spin all tubes for 2 minutes at 16000 g.
- Transfer 200 μL of each sample to a black 96-well plate and measure the fluorescence (excite at 400 nm and measure emission at 620 nm).
- For a standard curve: Prepare extra cell samples as above. Add hemin (diluted into water to the desired concentration) to samples, either before or after lysis (measurements are similar). 100 μL/g is approximately the upper end of the linear range.
- For each boiled sample, subtract the fluorescence of the corresponding unboiled sample to correct for background fluorescence.
- Remember to correct for the effect of variable sample concentrations.
- To estimate intracellular concentrations:
- CSY3 is ~0.53 mg DCW/(OD*mL)
- Hans et al. quote a value of 2.38 mL/g DCW as the intracellular volume of S. cerevisiae (Han et al., 2001).
- We arrive at similar numbers (within a factor of 1.5) by reasoning from first principles (cell number per OD and average cell size).
- For 8 OD*mL, this gives an approximate intracellular volume of 10μL.
- Reproducibility between extracts of a given sample is ~10%, similar to the reproducibility between samples.
- Hans MA, Heinzle E, & Wittmann C (2001) Quantification of intracellular amino acids in batch cultures of Saccharomyces cerevisiae. Applied microbiology and biotechnology 56(5):776-779.
- Sassa S (1976) Sequential induction of heme pathway enzymes during erythroid differentiation of mouse Friend leukemia virus-infected cells. The Journal of experimental medicine 143(2):305-315.