5-aminolevulinic acid, tetrapyrroles, chlorophyll, porphyrins, uroporphyrinogen III, coproporphyrinogen III, protoporphyrinogen IX, protoporphyrin IX, Mg protoporphyrin IX, Mg protoporphyrin IX monomethylester
Tetrapyrroles are essential cofactors and pigments in cyanobacteria, algae and plants. 5-aminolevulinic acid (ALA) is the first committed precursor of tetrapyrroles. Eight molecules of ALA form the cyclic (chlorophyll, heme and siroheme) or linear tetrapyrroles (phytochromobiline and phycobilines). Here, we outline experimental procedures to determine the ALA-forming capacity in tobacco leaves and to quantify the steady-state levels of intermediates of tetrapyrrole biosynthesis within a single plant extract. Since ALA steady-state levels are low, the synthesis rate is determined by inhibition of ALA dehydratase leading to accumulation of ALA. Different metabolites of tetrapyrrole biosynthesis can be extracted from tobacco leaves in a single organic solvent followed by identification and quantification using separation protocols for HPLC.