Assay Report Card

ID: ALA3887281

Type: Binding

Description: Enzymatic assay: The assay is based on the method described by Jordan (Shoolingin-Jordan P M et al. (1997). Methods in enzymology 281: 327-336) with some modifications. 10 uL of porphobilinogen deaminase (PBGD) at a concentration of 150 uM in 48 uL of buffer (20 mM Tris-HCl pH 8.0, NaCl 150 mM) is preheated at 37 C. for two minutes. If the assay is performed in the presence of an inhibitor, the latter is added in dissolved form into the buffer. 25 uL of the porphobilinogen (PBG) substrate are then added, diluted to different concentrations (0.15, 0.3, 0.45, 0.6, 0.9, 1.2, 1.5, 1.8, 2.1, 2.4, 2.7, 3, 3.3, 3.9 mM) in the aforementioned buffer and are incubated at 37 C. for five minutes. A control assay containing a buffer without substrate (PBG) in the same proportions is performed at the same time. The enzymatic reaction is stopped by means of freezing the samples in liquid nitrogen. Then the mixture is incubated again with 6 uL of 25 mM uroporphyrinogen 3 synthase (U3S).