title: Kinetic analysis of renin and its inhibitors by detecting double-labelled peptidic substrates with an immunoassay.
abstract: The proteolytic activity of renin is a key element in the regulation of blood pressure and a main target for inhibitor design. Currently, the activity of renin and its inhibitors is mainly analyzed using radioimmunoassays or FRET-substrates, which both have their limitations. Here, a novel kinetic assay is presented that combines the advantages of a homogeneous proteolytic reaction and a robust heterogeneous detection in a sandwich immunoassay format. The proteolysis in solution is not influenced by surface interactions and yields accurate kinetic values, while the specific detection of the cleavage products on a microtiter plate strongly reduces interference by concomitant substances and allows for a self-referenced signal readout. A new enzyme kinetic scheme for the inhibition of renin has been developed and validated by using the model inhibitor pepstatin. This kinetic analysis is amenable to parallelization for large-scale inhibitor screening. Furthermore, it can be easily adapted to inhibitors of other medically important proteases.
authors: Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, D-93040 Regensburg, Germany. Hans-Heiner.Gorris@ur.de
authors: Hans H
authors: HH
authors: Gorris
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D007118:Immunoassay / Q000379:methods; D007700:Kinetics; D051379:Mice; D010436:Pepstatins / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000032:analysis; D012083:Renin / Q000097:blood*; D000092502:Renin Inhibitors
chemical_list: D010436:Pepstatins; D011480:Protease Inhibitors; D012083:Renin; D000092502:Renin Inhibitors; C031375:pepstatin