Issue 7, 2013

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.

Graphical abstract: Kinetic analysis of renin and its inhibitors by detecting double-labelled peptidic substrates with an immunoassay

Supplementary files

Article information

Article type
Paper
Submitted
17 Oct 2012
Accepted
01 Feb 2013
First published
01 Feb 2013

Analyst, 2013,138, 2104-2109

Kinetic analysis of renin and its inhibitors by detecting double-labelled peptidic substrates with an immunoassay

H. H. Gorris, Analyst, 2013, 138, 2104 DOI: 10.1039/C3AN36508E

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