This article presents a method for assessing neutrophil serine protease (NSP) activity using fluorescent reporter probes. The technique involves measuring the donor-to-acceptor fluorescence ratio to determine the efficiency of NSP cleavage of the probes.
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.
Thaw enzymes on ice, and set up an enzyme standard curve as described in the text manuscript. In parallel to the standard preparation, dilute sputum samples in activation buffer. On the plate reader, set the excitation wavelength for the NE FRET probe NEmo-1 to 354 nanometers and the emission wavelength to 400 nanometers for donor and 490 nanometers for acceptor.
For the CG FRET probe sSAM, set the excitation wavelength to 405 nanometers, and emission to 485 nanometers for donor, and 580 nanometers for acceptor. Add 40 microliters of samples, standards, or blanks into the wells of a Black 96-well half-area plate and add the master mix.
Start the plate reader, and record the donor-to-acceptor ratio increase after every 60 to 90 seconds for at least 20 minutes or until the increase in the signal reaches a plateau. Export the data and calculate the donor-to-acceptor ratio by dividing the donor RFU by the acceptor RFU for each time point and sample. Then, calculate the donor-to-acceptor ratio mean and standard deviation. Determine the slope within the linear growth of the donor-to-acceptor ratio change.
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