Enzymatic Assay of Serine Proteases

Human sputum leukocyte elastase was obtained from Elastin Products Company (Owensville, MO). Human thrombin, factor Xa, factor XIa, and plasmin were obtained from Haematologic Technologies (Essex Junction, VT). Human leukocyte cathepsin G, human neutrophil proteinase 3, N-succinyl-Ala-Ala-Val-p-nitroanilide (S1384), N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (S7388), N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide (M4765), elastin-congo red, and bovine pancreatic DNase (DN25) were obtained from Sigma (St. Louis, MO). Low-binding polypropylene 96-well plates for HNE activity assay were from (Greiner Bio One, Monroe, NC. Spectrozyme TH (H-D-cylohexylalanyl-Ala-Arg-p-nitroanilide) and Spectrozyme factor Xa (methoxycarbonyl-D-cyclohexylglycyl-Gly-Arg-p-nitroanilide) were from American Diagnostica (Greenwich, CT). Factor XIa chromogenic substrate (S-2366, L-PyroGlu-Pro-Arg-p-nitroaniline·HCl) was from Diapharma (West Chester, OH). APTT reagent containing ellagic acid (APTT-LS), thromboplastin-D, and 25 mM CaCl₂ were obtained from Fisher Diagnostics (Middletown, VA). The library of 60 NSGMs were synthesized with >95% purity using reported schemes^{30 (link)–32 (link),39 –41 (link)} and were characterized by NMR and UPLC-MS.