An In Vitro Assay for Measuring Neutrophil Serine Protease Activity Using a Fluorescent Reporter

Take a microplate containing neutrophil serine proteases or NSPs. These soluble enzymes with proteolytic activity, secreted by neutrophils, function in inflammatory responses.

Add fluorescent reporter probes carrying a recognition motif that acts as a cleavage site for NSPs. The probes are conjugated to a donor and an acceptor fluorophore.

Upon excitation by a specific wavelength, the donor fluorophore emits energy. Due to its proximity to the donor, the acceptor fluorophore absorbs the energy and emits fluorescence of a different wavelength.

Detect the donor and acceptor signals separately and compute the donor-to-acceptor ratio to assess NSP activity. A low ratio indicates quenching of donor fluorescence in the absence of probe cleavage.

The NSPs recognize and cleave the recognition motif on the probes, releasing the acceptor.

The distance from the acceptor enables fluorescence emission from the donor fluorophore, increasing the donor-to-acceptor ratio. A high ratio indicates efficient probe cleavage by the NSPs.