Fibrinolytic serine protease from spirodela polyruiza

A serine protease was purified from Chinese herb (Spirodelapolyrhiza). Protease has a molecular mass of 180,000 dalton and 43,000 dalton in gel filtration and SDS-PAGE, respectively, implying it is a trimer. Its optimum pH was 4.5-5.0. Enzyme was stable below 42C0 and after lyophilization. The enzyme activity was inhibited significantly by leupeptin and to less degree by PMSF. The protease hydrolyzed not only fibrin but also fibrinogen, cleaving A and B without affecting chain of fibrinogen. However, no hydrolysis was found with hemoglobin, immunoglobulin, and albumin under the same condition. It cleaved preferentially Arg or Lys residue of synthetic substrates (P' position). The enzyme had an anticoagulatory activity measured with activated partial thromboplastin time and thrombin time test. It delayed APTT and TT two times at the protein concentration of 5.0 and 5.7 ug, respectively and drastically reduced after heat treatment. Introduction Blood clots are formed fiom fibrinogen by thrombin and are lyzed by plasmin, which is activated from plasminogen by tissue plasminogen activator (tPA). The blood clotting and lysis systems are tightly regulated. Its disturbance results in serious cardiovascular disease and cerebral infarction. The factors involved in fibrinolysis and thrombolysis are a promising target for chemotherapy (1 -3). Hemorrhagic toxin from the snake venom of Crotalus atrox (4), fibrinolytic and thrombolytic agents from Lumbricus rubellus ( 5 ) and anticoagulant, hirudin from Hirudo medicinalis (6), fibrinolytic protease fiom Pleurotus ostreatus (7) and FZammuZina velutipes (8) have been reported and characterized. We have purified and characterized a fibrinolytic protease fiom Spirodela polyrhiza. The protease showed anticoagulant activity probably due to its fibrinolytic and thrombolytic activity. This enzyme may be useful in clinical applications for fibrinolysis and thrombolysis. . Results Purification and molecular properties. Protease activity was detected from crude extracts of Spirodela polyrhiza and purified using an assay with Z-Lys-SBzl as a substrate. The enzyme was bound tightly to phenyl Sepharose and eluted with 10 mM Tris-HC1, pH 7.0. It was eluted with arginine through an affinity column of p-aminobezamidine Agarose. Finally purified protease was obtained after FPLC , phenylsuperose column chromatography. The enzyme was elecrophoretically homogeneous. The molecular mass of purified enzyme was determined to be 180,000 dalton in Sephadex G-150 gel filtration column and 43,000 dalton in SDS-PAGE, indicating that the enzyme is a tetramer. The PI of the protein was estimated to bc: pH 8.0 from a chromatofocusing column chromatography. Effect of pH on the enzyme activity was tested with various buffers. Maximum activity was found in the range of 4.5 to 5.0, indicating it is a aicdic protease. Thermal stability of purified protease was determined after treatment at different temperatures for 15 min. Half the activity was lost by incubation at 42OC and totally inactivated at 5OoC. Since lyophilization did not affect enzyme activity, enzyme was stored after lyophilization. 0-7803-5729-9/99/$10.00