Abstract
Publisher Summary Porphobilinogen synthase (δ-aminolevulinic acid dehydratase) is the second enzyme in the mammalian heme biosynthetic pathway. The enzyme catalyzes the condensation of two molecules of δ-aminolevulinic acid (ALA) to produce the monopyrrole, porphobilinogen (PBG). In the coupled-enzyme assay of PBG synthase, ALA is converted to the monopyrrole, PBG, by ALA-dehydratase. PBG is then converted to the linear tetrapyrrole, hydroxymethylbilane, by PBG-deaminase. PBG deaminase catalyzes the sequential formation of stable enzyme-substrate intermediates (i.e., mono-, di-, tri-, and tetrapyrroles) in the stepwise synthesis of the linear tetrapyrrole. Thus, to ensure linearity with time in the conversion of PBG to hydroxymethylbilane, PBG-deaminase first must be saturated with these intermediates. Hydroxymethylbilane nonenzymatically cyclizes to uroporphyrinogen, which then can be oxidized quantitatively to the highly fluorescent compound, uroporphyrin I (URO I) by exposure to ultraviolet light at low pH. The amount of URO I produced is measured fluorometrically with excitation and emission wavelengths of 405 and 600 nm, respectively. The chief advantage of this coupled-enzyme assay is its increased sensitivity. The coupled-enzyme assay is able to detect the low activity levels in cultured fibroblasts and amniocytes; these activities were undetectable by the standard colorimetric methods. Thus, the specificity, sensitivity, and rapidity of this fluorometric coupled-enzyme assay make possible the reliable determination of low levels of ALA-dehydratase activity in small amounts of crude tissue homogenates or in cultured cells.
Published Version
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call