Fluorometric coupled-enzyme assay for delta-aminolevulinate synthase.
The rapid and specific determination of picomole quantities of gamma-aminolevulinate has been accomplished by its enzymatic conversion to uroporphyrinogen I and subsequent fluorometric detection of the oxidized uroporphyrin I. The coupled-enzyme assay was linear with time and protein concentration and required less than 3 h for 25 individual determinations. Under the standard assay conditions, 5-100 pmol of uroporphyrin I was reliably quantitated; these values corresponded to a range of gamma-aminolevulinate synthase activities from 0.2 to 15 nmol/h/ml enzyme. The sensitivity of this method was comparable to the more time-consuming radiochemical determinations of gamma-aminolevulinate synthase activity. The gamma-aminolevulinate synthase activity of liver homogenates from uninduced rats was 8.6 U/g liver (37 degrees C).
D000624:5-Aminolevulinate Synthetase / Q000032:analysis*; D000818:Animals; D006168:Guinea Pigs; D006863:Hydrogen-Ion Concentration; D011163:Hydroxymethylbilane Synthase / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008297:Male; D000623:Porphobilinogen Synthase / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013050:Spectrometry, Fluorescence / Q000379:methods; D013389:Succinate-CoA Ligases / Q000378:metabolism
D000624:5-Aminolevulinate Synthetase; D011163:Hydroxymethylbilane Synthase; D000623:Porphobilinogen Synthase; D013389:Succinate-CoA Ligases