Purification and characterization of a serine protease with fibrinolytic activity from the dung beetles, Catharsius molossus.

Catharsius protease-1 (CPM-1) was isolated from the whole body of the dung beetles, Catharsius molossus, using three purification steps (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and affinity chromatography on DEAE Affi-Gel Blue gel). The purified CPM-1 that has a molecular weight of 27 kDa was assessed homogeneous by SDS-polyacrylamide gel electrophoresis and an isoelectric point of 4.4 was determined by isoelectric focusing. N-terminal amino acid sequence of the protease was composed of Ile-Val-Gly-Gly-Gln-Ala-Val-Glu-Ile-Gly-Asp-Tyr-Pro-Ala-Gln. The enzyme was inactivated by Cu(2+) and Zn(2+) and strongly inhibited by typical serine proteinase inhibitors such as TLCK, soybean trypsin inhibitor, aprotinin, benzamidine and alpha-antitrypsin. However, EDTA, EGTA, cysteine, beta-mercaptoethanol, E64, chymostatin, elastatinal and TPCK did not/less affect activity. Also, antiplasmin and antithrombin III were not sensitive to CPM-1. On the basis of amidolytic activity test, CPM-1 preferably hydrolysed chromogenic protease substrates containing Arg or Lys residues of the P1 position at pH 7.0 and 37 degrees C. CPM-1 preferentially cleaved the oxidized B-chain of insulin between Arg(22) and Gly(23). CPM-1 readily digested Aalpha- and gamma-chains and more slowly Bbeta-chain of fibrinogen. The nonspecific action of the enzyme resulted in extensive hydrolysis, releasing a variety of fibrinopeptides of fibrinogen and fibrin. D-dimer concentration increased on incubation of cross-linked fibrin with CPM-1, indicating that the enzyme has a significant fibrinolytic activity.