Activated human polymorphonuclear neutrophils at inflammatory sites release the
chymotrypsin-like protease cathepsin G, together with
elastase and
proteinase 3 (
myeloblastin), from their azurophil granules. The low activity of
cathepsin G on synthetic substrates seriously impairs studies designed to clarify its role in tissue
inflammation. We have solved this problem by producing new
peptide substrates with intramolecularly quenched fluorescence. These substrates were deduced from the sequence of putative
protein targets of
cathepsin G, including the reactive loop sequence of
serpin inhibitors and the N-terminal domain of the
protease-activated receptor of
thrombin, PAR-1. Two substrates were selected, Abz-TPFSGQ-EDDnp and Abz-EPFWEDQ-EDDnp, that are cleaved very efficiently by
cathepsin G but not by
neutrophil elastase or
proteinase 3, with specificity constants (k(cat)/K(m)) in the 10(5) M(-1).s(-1) range. They can be used to measure subnanomolar concentrations of free
enzyme in vitro and at the surface of neutrophils purified from fresh human blood. Purified neutrophils express 0.02-0.7 pg of
cathepsin G/cell (n=15) at their surface. This means that about 10(4) purified cells may be enough to record
cathepsin G activity within minutes. This may be most important for investigating the role of
cathepsin G as an inflammatory agent, especially in bronchoalveolar lavage fluids from patients with pulmonary inflammatory disorders.