1. The action of trypsin on synthetic chromogenic arginine substrates
O Somorin, S Tokura, N Nishi, J Noguchi J Biochem. 1979 Jan;85(1):157-62. doi: 10.1093/oxfordjournals.jbchem.a132305.
A new arginine derivative, N-benzyloxycarbonyl-L-phenylalanyl-L-valyl-L-arginine-p-nitroanilide hydrochloride (ZPVAPA.HCl) was synthesized by the condensation of N-benzyloxy-carbonyl-L-phenylalanyl-L-valine and L-arginine-p-nitroanilide dihydrochloride using dicyclohexylcarbodiimide as a coupling reagent and 1-hydroxy-benzotriazole as an additive. L-ZPVAPA.HCl was split by trypsin more readily than Na-benzyloxycarbonyl-L-arginine-p-nitroanilide hydrochloride (L-ZAPA, HCl), Na-benzoyl-L-arginine-p-nitroanilide hydrochloride (L-BAPA.HCl), Na-tosyl-L-arginine-p-nitroanilide hdyrochloride (L-TAPA.HCl) and Na-benzoyl-DL-arginine-p-nitroanilide hydrochloride (DL-BAPA.HCl) by factors of 100, 400, 600, and 1,200, respectively. Low concentrations of dimethyl formamide (DMF) enhanced the trypsin-catalyzed hydrolyses of L-ZAPA.HCl and L-TAPA.HCl, contrary to the findings of other authors that DMF has no effect on the tryptic hydrolysis.
2. L-Pyroglutamyl-L-prolyl-L-valine-p-nitroanilide, a highly specific substrate for granulocyte elastase
J A Kramps, C van Twisk, A C van der Linden Scand J Clin Lab Invest. 1983 Sep;43(5):427-32.
L-Pyroglutamyl-L-prolyl-L-valine-p-nitroanilide was found to be a highly specific substrate for human granulocyte elastase. At pH 8.3 and 37 degrees C, its Km = 0.55 mmol/l and the value for kcat was 6 sec-1, whereas with porcine pancreatic elastase these values were approximately 2 mmol/l and less than 0.001 sec-1, respectively. It is not cleaved by trypsin or chymotrypsin. With granulocyte elastase this new substrate is 50 times more sensitive compared to succinyltrialanyl-p-nitroanilide. L-Pyroglutamyl-L-prolyl-L-valine-p-nitroanilide can also be used for the assay of granulocyte elastase inhibitors.
3. A sensitive and specific assay for granulocyte elastase in inflammatory tissue fluid using L-pyroglutamyl-L-prolyl-L-valine-p-nitroanilide
H Tanaka, T Shimazu, H Sugimoto, T Yoshioka, T Sugimoto Clin Chim Acta. 1990 Feb 28;187(2):173-80. doi: 10.1016/0009-8981(90)90344-r.
Granulocyte elastase (GE, EC 3.4.21.37) is a key enzyme in tissue injury. To elucidate the role of GE in tissue injury, a new method of measuring GE activity in various inflammatory tissue fluids was developed using diazotization and the chromogenic synthetic substrate, L-pyroglutamyl-L-prolyl-L-valine-p-nitroanilide (S-2482). GE activity demonstrated first order kinetics in the range from 1.9 to 30 U/l. Other proteases, such as pancreatic elastase, trypsin, and chymotrypsin did not hydrolyze S-2484. This assay permits the determination of GE activity with a coefficient of variance less than 7.8% and 95.6 to 105.4% recovery. With this method, hydrolytic GE activity was found to be increased in bronchoalveolar lavage fluid from patients with ARDS or pneumonia, synovial fluid from patients with rheumatoid arthritis, and blister fluid from burn patients.
