ALAD Activity Assay
AMINOLEVULINIC ACID DEHYRATASE (ALAD)
Mouse liver homogenate
- Weigh out ~100mg liver.
- Add 400 µL 1mM DTT (dithiothreitol) in 50mM KPi (potassium phosphate) pH 6.8.
- Homogenize with ten up-and-down strokes in a 2-mL glass-Teflon (Potter- Elvehjem) tissue homogenizer in an ice bath.
- Store the resulting 20%w/v homogenate at −80 °C until needed.
Cultured cell homogenate
- Wash the cells with phosphate buffered saline (PBS) pH 7.4.
- Resuspend in about three pellet volumes of 50mM KPi pH6.3 for mammalian cells, or 200mM glycine/NaOH pH 9.2 for yeast.
- For mammalian cells, sonicate (homogenize) while in an ice bath at the lowest practicable power setting for 3 cycles x 5 sec at 50% duty (2.5 sec on, 2.5 sec off).
- Sonicate yeast cells at the highest power setting that does not cause significant aerosolization of the mixture for 12 5-sec cycles.
- Store the resulting homogenate at −80 °C until needed.
ALAD Assay
- Prepare two replicate pairs for each sample, two live and two heat-deactivated for 10 min in boiling water (for use as blank).
- Prepare similar solutions of PBG (porphobilinogen, ALAD product) for use as standard curve.
- Add 175µL solution containing 1mM DTT and 1.714mM d-aminolevulinic acid or ALA (for a final 1.5mM in 200µL) to 25µL (500µg protein) sample or standard PBG.
- Incubate at 37˚C for 30 min.
- Add 400µL of 100mM Tris pH 7.6 containing 150µM succinylacetone (SA) and 1 mg/mL recombinant porphobilinogen deaminase (PBGD). (The Tris adjusted the system to near pH 7.6 for optimal PBGD activity, while the SA inhibited further ALAD activity.)
- Incubate at 37˚C for 45 min.
- Add 200µL of 6 M HCl to stop the reaction.
- Expose the mixture to UV light for 30 min or ambient light for 2h to oxidize all porphyrinogen formed.
- Centrifuge at 16000xg for 10 min. Collect the supernatant.
- Quantify the porphyrins by UPLC (ultra performance liquid chromatography).
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