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Rapid purification and biochemical characteristics of lumbrokinase III from earthworm for use as a fibrinolytic agent
Article in Biotechnology Letters · February 1998
DOI: 10.1023/A:1005384625974
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Biotechnology Letters, Vol 20, NO 2, February 1998, pp. 169-172
Rapid purification and biochemical characteristics of lumbrokinase III from earthworm for use as a fibrinolytic agent
Yong-Doo Park, Jong-Won Kim, Byong-Goo Min¹, Jeong-Won Seo, and Jong-Moon Jeong²
¹ Department of Biomedical Engineering, College of Medicine, Seoul National University
² Department of Life Science, College of Natural Sciences, The University of Suwon, Hwasung- Gun, Kyonggi-Do, 445-743, Korea, Fax: 82-031-222-9385,
E-mail: [email protected]
A fibrinolytic enzyme was purified from the earthworm (Lumbricus rubellus) by column chromatography and identified as lumbrokinase type III. Affinity chromatography and NH₂-terminal amino acid sequences indicated that this lumbrokinase III-2(34.2 kDa) had additional amino acids at the carboxyl terminus of lumbrokinase III-1(34 kDa). The lumbrokinase III-1 was considerably stable at pH 2 to 11 and at up to 65℃. It had trypsin-like characteristics with high substrate specificity against fibrin, suitable as a fibrinolytic agent. Degradation profiles of fibrinogen by lumbrokinase III-1 and their peptide sequences were also investigated.
Introduction
Various treatments for chrombosis include bypass surgery, angioplasty, thrombectomy, or administration of thrombolytic agents such as streptokinase, urokinase, tissue-type plasminogen activator, and heparin (Yusuf et al., 1985; Wilson and Lampman, 1979).
However, heparin also functions as a catalytic cofactor for an antithrombin III resulting in the inhibition of thrombosis (Maaroufi et al., 1997). Both streptokinase and urokinase can covert plasminogen into active plasmin followed by the uncontrollable acceleration of fibrinolysis (Ferres, 1987; Hollander, 1987).
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Due to the limitation of present thrombolytic agents, a noble thrombolytic agent, which has the ability of direct degradation of fibrinogen and fibrin, has been widely sought after. Using snakes, several fibrinolytic enzymes have been purified from their venoms (Retzios and Markland, 1992; 1994). Most of these enzymes were metalloproteases, which can be inhibited by EDTA (Ahmed et al., 1990). Degradation profiles of fibrin by snake enzymes were usually different from those acted upon by other typical
thrombolytic enzymes, such as plasmin and urokinase. However, it has been observed that some of these enzymes can degrade certain coagulation factors as well (Lollar et at., 1987).
The earthworm has been used as a fibrinolytic agent in East Asia for several thousand years. Fibrinolytic activities by the earthworm have been tested by Mihara et al.
(1983;1991) and named lumbrokinase from generic name Lumbricus rubellus. Recently, Ryu et al. (1994) immobilized the enzyme onto the polyurethane surface, a basic material of the artificial organs, to test the fibrinolytic activity both in vitro and in vivo.
In this study, to assess the suitability of lumbrokinase III-1 as a fibrinolytic enzyme for the treatment of thrombosis, we purified the lumbrokinase containing the highest fibrinolytic activity earthworm homogenate by two column chromatographies and investigated its biochemical characteristics.
Materials and methods
Purification of the fibrinolytic enzymes
Earthworm powder (1kg, Gisam Compost Inc.) was dissolved in 4 volumes of Tris/
saline (TBS; 10mM Tris/HCl, 145mM NaCl, pH 7.4) at room temperature for 4 days and then centrifuged at 12,000g for 30min. The supernatant was heated at 55℃ for 1h and centrifuged again at 12,000g for 30min. Following filtration of the supernatant through a cotton column, ammonium sulfate was added slowly to a final concentration of 60%
(w/v). The pelleted proteins were dissolved in 200ml of 50mM Tris/HCl (pH 8.0) and were dialyzed twice again 2 liters of the same buffer. A protein solution was loaded onto
a DEAE Sephadex A25 column (5cm x 100cm) and eluted with a linear gradient of NaCl (0-500mM). Pools of the fibrinolytic activity obtained from the anion exchange column were loaded onto the Benzamidine Sepharose 6B. After washing with 20mM Tris/HCl (pH 8.0), the adsorbed proteins were eluted with 0.5M arginine in 20mM sodium acetate (pH 5.0). Proteins were determined by the bicinchoninic acid method and their profiles were analyzed by SDS-PAGE.
Measurements of proteolytic and fibrinolytic activity
Each protease (0.1ml) was mixed with the chromogenic protein substrate (azocasein or azoalbumin; 2mg/ml) and incubated at 37℃. Trichloroacetic acid (5%) was added to stop the reaction and the absorbance of the supernatant was measured at 340nm (Sarath et al., 1989). For fibrinolytic activity, the fibrin plate clearance method was employed (Astrup and Mullertz, 1952). After incubation of the fibrin plate at 37℃ for 2h, the diameter of the lysed area around the spotted fibrinolytic enzyme was measured against a turbid background.
Amino and sequencing
Peptides for amino acid sequencing were separated by SDS-PAGE and
electrotransferred to the PVDF (polyvinylidene difluoride) membrane after rinsing with CAPS (3-[cyclohexylamino]-1-propane-sulfonic acid) buffer for 5 min. Then, the membrane was rinsed with deionized water and visualized with Coomassie Blue R250 in methanol. The amino acid sequencing was carried out by the Korea Basic Science Institute.
Results and discussion
Purification of lumbrokinase from earthworm
When the affinity-purified fibrinolytic enzyme from earthworm was analyzed by gel electrophoresis, it was an almost homogeneous protein with a molecular size of
approximately 34kDa. Interestingly, fractions eluted slowly from the ion-exchange column contained a 34.2kDa sized protein corresponding to lumbrokinase type III-2 (Fig.1), indicating that type III-1 lumbrokinase might be derived from type III-2 by a different reading frame of mRNA during protein synthesis or by partial proteolysis. Along with this assumption, NH₂-terminal amino acid sequences for both type III-1 and III-2 were identical (Ile-Val-Gly-Gly-Ile-Glu-Ala-Arg-Pro-Tyr-Glu-Phe-Pro). This means that lumbrokinase III-2 (34.2kDa) may have additional amino acids at the carboxyl terminus of type III-1 (34kDa). However, lumbrokinase type III-2 was recovered at a level of about 1% of type III-1. From 15g of ammonium sulfate-precipitated earthworm proteins, it was possible to purify at least 200mg of lumbrokinase type III-1, meaning that the trypsin-like lumbrokinase was one of the abundant proteins in earthworm extract.
Stability and inhibition of lumbrokinase III-1
Fibrinolytic activities of purified lumbrokinase III-1 at various temperatures and concentrations of hydrogen ion were examined by fibrin plate clearance method. The enzyme maintained almost constant fibrinolytic activity in the range of pH to pH 11 at up to 45℃. Even at 65℃, 90% of the fibrinolytic activity was measured at around pH 8.0, indicating that this enzyme was considerably stable (Fig.2A). Moreover, this type III-1 lumbrokinase had more than 85% of fibrinolytic activity when the enzyme solution was kept at room temperature for 10 months (data not shown). However, the addition of trypsin inhibitor (TLCK, Nα-p-tosyl-L-lysine chloromethyl ketone) inactivated the lumbrokinase III-1 almost completely, whereas neither EDTA nor ε-aminocaproic acid affected its fibrinolytic activity (Fig. 2B). These results and the purification of
lumbrokinase III-1 by benzamidine affinity chromatography indicate that this enzyme is a trypsin-like enzyme, not a metalloprotease or a plasmin-like enzyme.
Substrate specificity
It was examined by the fibrin plate clearance method and proteolytic activity assay using azocasein or azoalbumin if lumbrokinase III-1 had fibrin specificity compared with trypsin, urokinase, or plasmin. First, fibrinolytic activities of lumbrokinase III-1 and other proteases were adjusted to have the same clear area on the fibrin plate (data not shown).
However, each protease had different proteolytic activities when albumin or casein was
used as a substrate (Fig.3). Trypsin-digested casein and albumin are almost three times faster than the lumbrokinase or plasmin. Urokinase showed little proteolysis against casein or albumin with its high substrate specificity. Unlike urokinase or streptokinase, lumbrokinase III-1 did not activate the plasminogen into plasmin, possibly leading to systemic bleeding, unlike urokinase or streptokinase (data not shown). Here, it is tempting to say that lumbrokinase is considerably useful for the treatment of thrombosis in terms of these biochemical characteristics.
Digestion profiles of fibrinogen
When fibrinogen was digested with lumbrokinase, trypsin, or plasmin, its Bβ chain degraded rapidly compared with that of the Aα chain. It seemed that the first digestion product of y-chain was resistant to further digestion by lumbrokinase or trypsin.
Digestion profiles with increased incubation time showed that lumbrokinase had almost the same pattern with trypsin, but not with plasmin (Fig.4). In addition, NH₂-terminal amino acid sequences (Asp-Asn-Glu-Asn-Val for peptide 1 and His-Gln-Leu-Tyr-X-Asp for peptide 2 in Fig. 4A) of fibrinogen degradation products by lumbrokinase III-1
showed that the enzyme hydrolysed fibrinogen right after lysine (133rd and 148th amino acids of Bβ chain; Watt et al., 1978). Both results indicate its order trypsin-like
properties.
Acknowledgements
We wish to thank Gisam Compost Inc. for the supply of earthworm powder. This work was aided by a grant (`95 Biotech 2000 program project) from the Korea Science and Engineering Foundation.
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