PKA was diluted in 20 mM HEPES, pH 7.4, w/0.05% TX-100 and incubated in a final reaction buffer consisting of 20 mM HEPES, pH 7.4, 0.1 mM cAMP, 1 mM ATP, 5 mM MgCl2, 1 mM DTT with either fluorescein Kemptide (71.25 uM) or Oregon Green Kemptide as the enzymatic substrate at a final concentration of 71.25 or 114.25 μM, respectively, with a final reaction volume of 30 μl, for a reaction time of 1 hour in a 96 well plate format using an opaque white plate. Subsequently, 120 μl of 0.25×Fe(III) working reagent prepared according to Example I was added to the enzymatic reaction mixture and the fluorescence was measured using a 485/538 ex/em filter set. The results are shown in FIGS. 14 and 15.
