1 research outputs found
Three Wavelength Substrate System of Neutrophil Serine Proteinases
Neutrophil serine proteases, including elastase, proteinase
3,
and cathepsin G, are closely related enzymes stored in similar amounts
in azurophil granules and released at the same time from triggered
neutrophils at inflammatory sites. We have synthesized new fluorescence
resonance energy transfer (FRET) substrates with different fluorescence
donor–acceptor pairs that allow all three proteases to be quantified
at the same time and in the same reaction mixture. This was made possible
because the fluorescence emission spectra of the fluorescence donors
do not overlap and because the values of the specificity constants
were in the same range. Thus, similar activities of proteases can
be measured with the same sensitivity. In addition, these substrates
contain an N-terminal 2-(2-(2-aminoethoxy)ethoxy)acetic acid (PEG)
moiety that makes them cell permeable. Using the mixture of these
selected substrates, we were able to detect the neutrophil serine
protease (NSP) activity on the activated neutrophil membrane and in
the neutrophil lysate in a single measurement. Also, using the substrate
mixture, we were in a position to efficiently determine NSP activity
in human serum of healthy individuals and patients with diagnosed
Wegener disease or <i>microscopic polyangiitis</i>