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Buffer assay: Human recombinant renin (Proteos) at 100 pM was incubated in the presence or absence of renin inhibitors and 6 lM of Q-FRET substrate 9 DNP-Lys-His-Pro-Phe-His-Leu-Val-Ile-His-D,L-Amp in 50 mM MOPS, 100 mM NaCl, pH 7.4, 0.002% Tween. The reactions take place in a Costar 384 well black plate at 37°C for 3 h. Fluorescence was measured at times 0 and 3 h in a SpectraMax Gemini EM reader with excitation and emission filters at 328 and 388 nm, respectively. Plasma assay: Frozen human EDTA-plasma was rapidly thawed in warm water and centrifuged at 2900g for 15 min at 40°C. The supernatant was collected and recombinant human renin (Proteos) added at 1 nM nominal concentration. The plasma was transferred to Costar black 384 well plates, renin inhibitors added and the mixture pre-incubated at 37°C for 10 min. The renin Q-FRET substrate QXL520-Lys-His-Pro-Phe-His-Leu-Val-Ile-His-Lys-(5-FAM) (Proteos), diluted in 3 M Tris/200 mM EDTA, pH 7.2 was added to the plasma with final concentrations of 342 mM Tris, 23 mM EDTA and 6.8 μM substrate. The plate was incubated at 37°C for 1 h and the plate read in a SpectraMax Gemini EM reader with excitation and emission filters at 490 and 520 nm, respectively, at time 0 and 1 h.
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