Reappraisal of the uroporphyrinogen I synthase assay, and a proposed modified method.
Problems encountered in the uroporphyrinogen I synthase assay were found to be due to the Triton reagent, slit-width and excitation wavelength settings of the spectrophotofluorometer, and leg-phase in the enzyme reaction. Of Triton X-100 from four suppliers, only three were found suitable. Differences in enzyme activity owing to slit-width adjustments were resolved by measuring the fluorescence of the sample (uroporphyrin) at 405 nm and the standard (coproporphyrin) at 401 nm. The lag-phase in the activated-enzyme assay is incorporated in the 30-min preincubation of the blood specimen in our modified method, thus giving higher enzyme activity than found with the original assay. The range for our method is 7.3--15.8 nmol s-1 L-1 (mean = 10.9; SD = 2.07).
D000642:Ammonia-Lyases / Q000097:blood*; D004912:Erythrocytes / Q000201:enzymology; D005260:Female; D006801:Humans; D011163:Hydroxymethylbilane Synthase / Q000097:blood*; D007700:Kinetics; D008297:Male; D017830:Octoxynol; D011092:Polyethylene Glycols; D012016:Reference Values; D013050:Spectrometry, Fluorescence / Q000379:methods
D011092:Polyethylene Glycols; D017830:Octoxynol; D011163:Hydroxymethylbilane Synthase; D000642:Ammonia-Lyases