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ISSN 0582-9879                                          ACTA BIOCHIMICA et BIOPHYSICA SINICA 2003, 35(8): 689–694                                    CN 31-1300/Q

Jerdonase, a Novel Serine Protease with Kinin-releasing and Fibrinogenolytic Activity from Trimeresurus jerdonii Venom

JIA Yong-Hong1,2, JIN Yang1, LŰ Qiu-Min1, LI Dong-Sheng1, WANG Wan-Yu1, XIONG Yu-Liang1*

( 1 Department of Animal Toxicology, Kunming Institute of Zoology, the Chinese Academy of Sciences, Kunming 650223, China; 2 the Graduate School of the Chinese Academy of Sciences, Beijing 100039, China )

 

Abstract        A novel kinin-releasing and fibrin(ogen)olytic enzyme termed jerdonase was purified to homogeneity from the venom of Trimeresurus jerdonii by DEAE Sephadex A-50 anion exchange, Sephadex G-100 (superfine) gel filtration and reverse-phase high performance liquid chromatography (RP-HPLC). Jerdonase migrated as a single band with an approximate molecular weight of 55 kD under the reduced conditions and 53 kD under the non-reduced conditions. The enzyme was a glycoprotein containing 35.8% neutral carbohydrate. The N-terminal amino acid sequence of jerdonase was determined to be IIGGDECNINEHPFLVALYDA, which showed high sequence identity to other snake venom serine proteases. Jerdonase catalyzed the hydrolysis of BAEE, S-2238 and S-2302, which was inhibited by phenylmethylsulfonyl fluoride (PMSF), but not affected by ethylenediaminetetraacetic acid (EDTA). Jerdonase preferentially cleaved the Aα-chain of human fibrinogen with lower activity towards Bβ-chain. Moreover, the enzyme hydrolyzed bovine low-molecular-mass kininogen and releasing bradykinin. In conclusion, all results indicated that jerdonase was a multifunctional venom serine protease.

 

Key words     fibrinogenase; jerdonase; kinin-releasing enzyme; venom

 

Snake venoms, especially from Crotalidae and Viperidae families, are abundant in proteolytic enzymes[1,2]. According to the difference of the enzymatic active site, these proteinases can be divided into two groups: serine protease and metalloproteinase, both of them are known to affect the haemostatic system by a variety of mechanisms[3]. Among these venom proteinases, some hydrolyze N-terminal end of fibrinogen releasing fibrinopeptide A or B or both resulting in the formation of fibrin[4], this activity exhibition resembles that of thrombin, which results in the term thrombin-like enzyme (TLE)[1,5]; some degrade the Aα-, Bβ- or both chains of fibrinogen at the C-terminus making it unclottable by thrombin[6], which is called fibrinogenase[1]; many proteinases can also cleave kininogen releasing bradykinin or kallidin, which leads to the name kinin-releasing enzyme or kininogenase[7]; what’s more, there exist a lot of venom components interacting with plasminogen, protein C or some blood factors[8]. Generally each of these proteinases exhibits only one specific enzymatic activity. However, up to now to our knowledge, five multifunctional proteinases which possess double enzymatic activities have been reported, including cratalase from Crotalus adamanteus venom[9], KN-BJ from Bothrops jararaca venom[10], flavovilase from Trimeresurus flavoviridis (habu) venom[11], β-fibrinogenase from Trimeresurus mucrosquamatus venom[12], and halystase from Agkistrodon halys blomhoffii venom[13]. The former three were kinin-releasing and fibrinogen-clotting enzymes, and other two were kinin-releasing and fibrin(ogen)olytic enzymes. The catalytic mechanisms of these multifunctional proteinases have not yet been clarified. Fortunately, from the venom of Trimererusus jerdonii distributed in the southwest region of Yunnan Province, we also isolated a novel proteinase of multifunction.

In this paper, we studied the biochemical and enzymatic properties of this enzyme, the result showed that the enzyme, designated as jerdonase, was a serine protease with kinin-releasing and fibrin(ogen)olytic activities.

 

1    Materials and Methods

1.1   Materials

The lyophilized T. jerdonii crude venom was from the stock of Kunming Institute of Zoology, the Chinese Academy of Sciences. DEAE Sephadex A-50 and Sephadex G-100 (superfine) were from Amersham Bioscience (London, UK). RP-HPLC C4 and C18 columns were obtained from Waters (Milford, USA). Low molecular weight markers and reagents for SDS-polyacrylamide gel electrophoresis (SDS-PAGE), human fibrinogen (plasminogen-free), human thrombin, low-molecular-mass bovine kininogen, kallidin (Lys-bradykinin), trypsin, Nα-benzoyl-L-arginine ethyl ester hydrochloride (BAEE), phenylmenthylsulfonyl fluoride (PMSF), ethylenediaminetetraacetic acid (EDTA), 1-4-dithio-L-threitol (DTT), soybean trypsin inhibitor and L-cysteine were from Sigma (Colorado, USA). Synthetic chromogenic substrates S-2238 (H-D-Phe-Pi-Arg-pNA), S-2302 (H-D-Pro-Phe-Arg-pNA) and S-2251 (H-D-Val-Leu-Lys-pNA) were from Kabi Vitrim (Stockholm, Sweden). Other reagents used were of analytic grade from commercial sources.

1.2   Enzyme purification

The lyophilized crude venom of T. jerdonii (1 g) was dissolved in 5 mL Tris-HCl (50 mmol/L, pH 8.9), and the insoluble material was removed by centrifugation (5000 g for 10 min). The supernatant was applied to a DEAE Sephadex A-50 (3.2 cm × 80 cm) column pre-equilibrated with the same buffer; elution was achieved with a 0－0.5 mol/L NaCl gradient. The fractions containing jerdonase were collected, concentrated, and then loaded to a Sephadex G-100 (superfine) gel filtration (3.2 cm × 120 cm) column pre-equilibrated with Tris-HCl (25 mmol/L, pH 7.8), and eluted with the same buffer containing 0.15 mol/L NaCl. The active fractions were further chromatographied on an RP-HPLC C4 column with a gradient solution B (acetonitrile, containing 0.1% TFA) of 0%－20%, 20%－60%, and 60%－100% at a flow rate of 0.7 mL/min. Each fraction collected was about 3 mL, and was monitored spectrophotometrically at 280 nm. The enzymatic activity of jerdonase was assayed with kinin-releasing and fibrin(ogen)olytic methods.

1.3   SDS-PAGE and glycoprotein assay

12.5% SDS-PAGE was performed following the methods of Laemmli[14], the gel was stained with Coomassie brilliant blue R-250. Phosphorylases b (94 kD), bovine serum albumin (67 kD), ovalbumin (43 kD), carbonic anhydrase (30 kD), soybean trypsin inhibitor (20.1 kD) and α-lactalbumin (14.4 kD) were used as molecular mass standards. For glycoprotein detection, the SDS-PAGE gel was stained with periodate/Schiff (PAS) according to the procedure of Zacharius et al.[15]. Neutral sugars were determined by the phenol-sulfuric acid method[16].

1.4   N-terminal amino acid sequence

N-terminal amino acid sequence of jerdonase was determined with Model 476A protein sequencer (Applied Biosystem, USA). The homology of the enzyme was analyzed using Vector NTI Suite 6.0.

1.5   BAEE activity and inhibitor assay

The BAEE activity of jerdonase was determined according to the method of Glazer[17]. The inhibitor assay was performed as followings: different inhibitors were incubated with jerdonase (2.0×10－3 mg) in 0.5 mL with reaction solution 50 mmol/L Tris-HCl (pH 7.8) at room temperature for 30 min, then the residual BAEE activity of jerdonase was examined with the same method[17]. Thus, the inhibition ratio of different inhibitors was calculated. The inhibitors were phenylmethylsulfonyl fluoride (PMSF) in dimenthylsulfoxide(DMSO), EDTA, soybean trybean inhibitor, L-cysteine, and DTT were in 50 mmol/L Tris-HCl (pH 7.8, containing 0.1 mol/L NaCl). The protein concentration was determined by the method of Lowry et al.[18].

1.6   Chromogenic assay of amidolytic activity using synthetic substrate

The amidolytic activity of the enzyme was measured with a spectrophotometer (UV 4060, Pharmacia Fine Chemicals, Uppsala, Sweden) in 1 cm path-length plastic cuvettes, following the method of Zhang et al.[19]. Assays were performed in 0.5 mL reaction solution with 50 mmol/L Tris-HCl (pH 7.8) and 0.01% Tween-80. The reactions were initiated by addition of jerdonase, and the formation of p-nitroanilide(pNA) was monitored continuously at 405 nm. The amount of substrates hydrolyzed was calculated from the absorbance at 405 nm using a molar extinction coefficient of 10 000 (mol/L)－1 ·cm－1 for free pNA. Appropriate amounts of the enzyme were incubated with different concentrations of substrates, ranging from 2.0×10－3 mmol/L to 4 mmol/L. The enzyme reaction was plotted in a Lineweaver-Burk manner to obtain the Michaelis constant Km and the catalytic rate constant kcat.

1.7   Fibrin(ogen)olytic and fibrinogen clotting activity

The fibrinogenolytic activity was determined by the method of Ou-Yang and Teng[20] with small modifications, 0.2 mL of the human fibrinogen solution (0.4% human fibrinogen in 50 mmol/L Tris-HCl buffer, pH 7.6, containing 0.15 mol/L NaCl ) was mixed with 2.0×10－3 mg jerdonase and incubated at 37 ℃ for 0, 5, 15, 30, 60 min, or 2, 4, 12, 24 h, respectively, an aliquot of 0.02 mL reaction mixture was drawn at different time and analyzed by reduced SDS-PAGE (12.5% gels).

The fibrinolytic activity was assayed on the fibrin plates according to the method of Graham et al.[21].

Fibrinogen clotting was determined by the method of Serrano et al.[10] and Jin et al.[22], 5.0×10－3 mg of jerdonase was incubated with 0.2 mL 0.4% human fibrinogen solution at 37 ℃ for 30 min observing if fibrin clots  form. After that, the mixture was heated for 3 min in boiling water to stop the reaction and centrifuged at 5000 g for 10 min to precipitate the soluble protein. The supernatant was analyzed by RP-HPLC C18 with a gradient solution B (acetonitrile, containing 0.1% TFA) of 0%－30%, 30%－40%, and 40%－50%, and the releasing products were monitored at 215 nm.

1.8   Kinin-releasing and identification assay

The kinin-releasing activity was assayed according to the method of Matsui et al.[13] and Wang et al.[23], and the released kinin was identified according to the method of Fiedler and Geiger[24]. 2.5×10－3 mg bovine low-molecular-weight plasma kininogen was incubated with 2.0×10－3 mg jerdonase in 50 mmol/L Tris-HCl (pH 7.8) in a total volume of 1 mL at 37 ℃ for about 30 min, the released kinin was identified by RP-HPLC C18 with solution B (acetonitrile, containing 0.1% TFA) with the gradient of 0%－30%, 30%－100% at a flow rate of 0.7 mL/min. Meanwhile, aminopeptidase activity of jerdonase was identified as following: 1.0×10－3 mg jerdonase was incubated with 5.0×10－3 mg kallidin (Lys-bradykinin) in 50 mmol/L Tris-HCl (pH 7.8) in a total volume of 1 mL under the same conditions, the hydrolyzed products were identified by the same methods.

 

2    Results

2.1   Chromatography process

Jerdonase was obtained by three chromatography steps including anion exchange on DEAE Sephadex A-50, gel filtration on Sephadex G-100 (superfine) and RP-HPLC C4. The fractions of the tube number from 233-254 of DEAE Sephadex A-50 exhibited strong kinin-releasing and α-fibrin(ogen)olytic activities [Fig.1(A)], these fractions were pooled, concentrated, and then loaded on a Sephadex G-100 (superfine) gel filtration column, fractions from 52 to 68 of tube number displayed double activities [Fig.1(B)], which were collected and further purified by RP-HPLC C4 column [Fig.1(C)]. Finally, the homogeneity protein termed jerdonase was achieved.

 

Fig.1       Purification schemes of jerdonase from the lyophilized venom of Trimeresurus jerdonii

(A) The lyophilized T. jerdonii venom (1 g) was chromatographied on a DEAE Sephadex A-50 column previously equilibrated with 50 mmol/L Tris-HCl buffer (pH 8.9). The column (3.2 cm × 80 cm) was eluted with 0－0.5 mol/L NaCl in the same buffer. The fractions of tube number from 233－254 (indicated by an arrow) were pooled and concentrated. (B) The collected and concentrated solution of DEAE Sephadex A-50 was rechromatographied on gel filtration Sephadex G-100 (superfine) column previously equilibrated with 25 mmol/L Tris-HCl (pH 7.8). The column (3.2 cm × 120 cm) was eluted with 0.15 mol/L NaCl in the same buffer. The fractions from 52－68 (indicated by an arrow) of tube number were collected. (C) The collected solution of Sephadex G-100 (superfine) was further purified by RP-HPLC C4 chromatography. Jerdonase was found in peak 3 (indicated by an arrow).

 

2.2   SDS-PAGE

SDS-PAGE showed the molecular weight of Jerdonase was 55 kD under the reduced conditions and 53 kD under the non-reduced conditions (Fig.2). The enzyme was a glycoprotein with 35.8% neutral sugar (data not shown).

Fig.2       SDS-PAGE analysis of Jerdonase

1, Jerdonase performed under the reduced conditions; 2, the protein molecular weight markers; 3, jerdonase performed under non-reduced conditions. From top to bottom, the protein markers and their molecular weight are: phosphorylase b (94 kD), bovine serum albumin (67 kD), ovalbumin (43 kD), carbonic anhydrase (30 kD), soybean trypsin inhibitor (20.1 kD), α-lactalbumin (14.4 kD).

 

2.3   Sequence analysis

The N-terminal sequence of jerdonase was IIGGDECNINEHPFLVALYDA, which showed high identity to other venom proteases (Table 1).

Table 1   The N-terminal amino acid sequence of jerdonase and alignment with other venom serine proteases

Enzyme

N-terminal sequence

Identity

Reference

Jerdonase

IIGGDECNINEHPFLVALYDA

 

This work

KN-BJ

IVGGDECNINEHRSLVVLK--

66.7%

Serrano et al., 1998 [10]

Halystase

IIGGDECNINEHRFLVALYTP

87.5%

Matsui et al., 1998 [12]

TM-VIG

VIGGDECNINEHPFLVLVYYD

76.2%

Hung et al., 1994 [25]

Batroxobin

VIGGDECNINEHPFLAFMYYS

66.7%

Itoh et al., 1987 [26]

Jerdofibrase

VIGGDECNINEHPFLVLVYYD

76.2%

Jin et al., 2001 [22]

Jerdonobin

IVEGQDAEVGLSPWQVMLFRK

81.0%

Lu et al., 2000 [27]

Kallikrein-like protease

VVGGYNCEMNSQPWQVAVYYF

38.1%

Bjarmason et al., 1983 [28]

 

2.4   Chromogenic effect and inhibition

Amidolytic activity showed that jerdonase most effectively hydrolyzed S-2302, a substrate for plasma kallikrein, with Km of 6.25×10－3 mmol/L and kcat/Km of 194.4×10－3 (mmol/L)－1·min－1, respectively. The enzyme also catalyzed S-2238, a thrombin substrate, with Km of 41.6×10－3 mmol/L and kcat/Km of 24.5×10－3 (mmol/L)－1·min－1, respectively. However, the enzyme showed no activity to S-2251, a plasminogen activator substrate (Table 2).

Table 2   Amidolytic activity and kinetic parameters of jerdonase on three chromogenic substrates

Substrate

Km

(10-3 mmol/L)

kcat

(min-1)

kcat/Km

(10–3 mmol/L-1min–1)

H-D-Phe-Pip-Arg-pNA (S-2238)

41.67×10–3

1021

24.5×10–3

H-D-Pro-Phe-Arg-pNA (S-2302)

6.25×10–3

1215

194.4×10–3

H-D-Val-Leu-Lys-pNA (S-2251)

–

–

–

－, the activity was not detectable under the assay conditions.

 

Hydrolyzing activity of jerdonase on BAEE can be inhibited by several inhibitors (Table 3).

Table 3   Effects of inhibitors on BAEE activity of jerdonase

Inhibitor

Concentration

(mmol/L)

Residual activity

(%)

Control

–

100

PMSF

2

0

EDTA

5

95 ± 5

Soybean trypsin inhibitor

2

0

L-cysteine

5

20 ± 3

DTT

5

36 ± 3

 

 

2.5   Fibrin(ogen)olytic activity and fibrin clotting

Jerdonase hydrolyzed Aα-chain of fibrinogen  preferentially, degraded Bβ-chain with lower activity; but had little effect on digesting γ-chain (Fig.3). The enzyme also showed fibrinolytic activity when applied to fibrin plate (data not shown).

Fig.3       SDS-PAGE analysis of the digestion of jerdonase to human fibrinogen

0.2 mL human fibrinogen solution (0.4% human fibrinogen in 50 mmol/L Tris-HCl buffer, pH 7.6, containing 0.15 mol/L NaCl ) was incubated with 2.0×10－3 mg jerdonase at 37 ℃ for 0, 5, 15, 30, 60 min, or 2, 4, 12, 24 h (lane 1－9), respectively.

 

No fibrin clot was observed when jerdonase was incubated with human fibrinogen solution, moreover, the supernatant was isolated without the releasing of fibrinpeptide A (FpA) or fibrinpeptide B (FpB) (Fig.4).

Fig.4       Analysis of fibrinpeptides of jerdonase hydrolyzing to human fibrinogen

The mixture of jerdonase and human fibrinogen solution incubated at 37 ℃ for 30 min was centrifuged, and then the supernatant was analyzed by RP-HPLC C18 with with solution B (acetonitrile, containing 0.1% TFA) with gradient of 0%－30%, 30%－40% and 40%－50%, and the releasing products were monitored at 215 nm.

 

The released kinin of bovine low-molecular-weight kininogen hydrolyzed by jerdonase was separated through RP-HPLC C18 spectrophoto-metrically monitored at 215 nm. Its pharmacological activity on guinea-pig ileum contraction in vitro and the peptide sequence determination identified this kinin as bradykinin (Fig.5). Furthermore, jerdonase had no aminopeptidase activity and couldn’t convert kallidin to bradykinin when assayed with the same method.

Fig.5       Identification of the product of bovine low-molecular-weight plasma kininogen hydrolyzed by jerdonase

The released kinin of bovine low-molecular-weight plasma kininogen hydrolyzed by jerdonase was separated by RP-HPLC C18 with gradient solution B (acetonitrile, containing 0.1% TFA) of 0%－30%, 30%－100% at a flow rate of 0.7 mL/min monitored at 215 nm, bradykinin was identified (indicated by arrow).

 

3    Discussion

Jerdonase, a multifunctional serine protease with kinin-releasing and fibrin(ogen)olytic activities, was isolated from T. jerdonii venom. The molecular weight of this enzyme was approximately 55 kD under the reduced conditions and 53 kD under the non-reduced conditions, it also showed strong hydrolyzing activity on Aα-chain of human fibrinogen. Generally, the venom serine proteases preferentially exhibit degradation activity on Bβ-chain of human fibrinogen with the molecular weight ranging from 22.9 kD to 26 kD[1], such as, a β-fibrinogenase (22.9 kD) from Crotalus atrox[29] and protease III (24 kD) from Crotalus atrox[30]. While venom metalloproteinases usually preferentially hydrolyze Aα-chain of human fibrinogen[1] with the molecular weight ranging from 21.5 kD to 58 kD[1], including atroxase (23.5 kD) from Crotalus atrox[31], fibrolase (22.891 kD) from Agkistrodon contortrix contortrix[32]. However, jerdonase was an exception, it exhibited its specific characterizations in not only size but also fibrinogen hydrolyzing activity. Compared with other venom serine proteases, it has high content of carbohydrate chain that may be one of main reasons for its high molecular weight. For instance, β-fibrinogenase from Vipera lebetina containing 23% neutral carbohydrate has a molecular weight of 52.5 kD[33], the molecular weight of  bothrops protease A from Bothrops jararaca with a carbohydrate content higher than 40% is 65 kD[34]. The interesting question is why so large amount of carbohydrate existed in these enzymes and what is the function? But, little is known till now. Lochnit et al.[35] thought that the carbohydrate was characterized as a pattern of structure element when he studied batroxobin, a thrombin-like enzyme from Bothrops moojeni venom.

Jerdonase could catalyze hydrolysis of S-2238 and S-2302, but had no activity on S-2251. The kinetic specificity constants showed that the Km (affinity capacity) value of jerdonase on S-2238 was approximately six-fold higher than on S-2302, while the kcat/Km (the catalytic efficiency) value of the enzyme on S-2302 was about eight-fold to that on S-2238, indicating that S-2302 was the better substrate for jerdonase (Table 2). On the other hand, S-2238, a substrate for thrombin, was the better substrate to jerdonase than S-2302, a substrate for kallikrein, but the enzyme could not clot human fibrinogen, moreover, the enzyme was a kinin-releasing and fibrin(ogen)olytic enzyme. These results imply that there are no direct relationships between the chromogenic activity exhibition and the enzymatic activity exhibition.

The N-terminal amino acid sequence of jerdonase showed high homology to other venom serine proteases; however, their enzymatic behaviors were greatly different from each other. It is a pity that we have not found the protein clone; otherwise, we can apply multifunctional alignment and expect to reveal the relationship between structure and function of these multifunctional enzymes. Perhaps, the structure statue nearby the enzymatic active site tends to change because of some amino acid mutations, which might lead to the activity change of these proteinases. The great homology of amino acid sequence of venom serine proteases may imply that they may evolve from the common precursor protein and possibly adapt to digest different target protein of the snake’s prey[12]. Wang et al.[36] pointed out through the analysis of phylogenetic tree that there existed three major subtypes of venom serine proteases with the independent evolution: the coagulating enzymes (CL), the plasminogen activator (PA), and the kininogenases (KN). In the course of this parallel evolution, probably, random mutation of serine protease’s ancestral gene resulted in the diverse genes, therefore expressed different proteins including the multifunctional proteins, which might be selected by natural environment.

 

References

1     Ou-Yang C, Teng CM, Huang TF. Characterization of snake venom components acting on blood coagulation and platelet function. Toxicon, 1992, 30: 945－966

2     Jennings BR, Spearman CW, Kirsch RE, Shephard EG. A novel high molecular weight fibrinogenase from the venom of Bitis arietans. Biochim Biophys Acta, 1999, 1427(1): 82－91

3     Matsui T, Fujimura Y, Titani K. Snake venom proteases affecting hemostasis and thrombosis. Biochim Biophys Acta, 2000, 1477(1－2): 146－156

4     Herrick S, Blanc-Brude O, Gray A, Laurent G. Fibrinogen. Int J Biochem Cell Biol, 1999, 31: 741－746

5     Hutton RA, Warrell DA. Action of snake venom components on the haemostatic system. Blood Rev, 1993, 7(3): 176－189

6     Samel M, Subbi J, Siigur J, Siigur E. Biochemical characterization of fibrinogenolytic serine proteinases from Vipera lebetina snake venom. Toxicon, 2002, 40: 51－54

7     Rocha e Silva M, Beraldo WT, Rosenfeld G. Bradykinin, a hypotensive and smooth muscle stimulating factor released from plasma globulin by snake venoms and trypsin. Am J Physiol, 1949, 156: 261－273

8     Markland FS. Snake venoms and the hamostatic system. Toxicon, 1998, 36: 1749－1800

9     Markland FS, Kettner C, Schiffman S, Shaw E, Bajwa SS, Reddy KN, Kirakossian H et al. Kallikrein-like activity of crotalase, a snake venom enzyme that clots fibrinogen. Proc Natl Acad Sci USA, 1982, 79: 1688－1692

10    Serrano SM, Hagiwara Y, Murayama N, Higuchi S, Mentele R, Sampaio CA, Camargo AC et al. Purification and characterization of a kinin-releasing and fibrinogen-clotting serine proteinase (KN-BJ) from the venom of Bothrops jararaca, and molecular cloning and sequence analysis of its cDNA. Eur J Biochem, 1998, 251: 845－853

11    Komori Y, Tatematsu R, Tanida S, Nikai T. Thrombin-like enzyme, flavovilase, with kinin-releasing activity from Trimeresurus flavoviridis (habu) venom. J Nat Toxins, 2001, 10(3): 239－248

12    Hung CC, Chiou SH. Fibrinogenolytic proteases isolated from the snake venom of Taiwan habu: Serine proteases with kallikrein-like and angiotensin-degrading activities. Biochem Biophys Res Commun, 2001, 281: 1012－1018

13    Matsui T, Sakurai Y, Fujimura Y, Hayashi I, Oh-Ishi S, Suzuki M, Hamako J et al. Purification and amino acid sequence of halystase from snake venom of Agkistrodon halys blomhoffii, a serine protease that cleaves specifically fibrinogen and kininogen. Eur J Biochem, 1998, 252: 569－575

14    Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970, 227: 680－685

15    Zacharius RM, Zell TE, Morrison JH, Woodlock JJ. Glycoprotein staining following electrophoresis on acrylamide gels. Anal Biochem, 1969, 30: 148－152

16    Dubois M, Gilles KA, Hamilton KK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substrates. Anal Chem, 1956, 28: 350－356

17    Glazer AN. Esteratic reactions catalyzed by subtilisins. J Biol Chem, 1967, 242(3): 433－436

18    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem, 1951, 193: 265－275

19    Zhang Y, Wisner A, Xiong Y, Bon C. A novel plasminogen activator from snake venom, purification, characterization and molecular cloning. J Biol Chem, 1995, 270: 10246－10255

20    Ou-Yang C, Teng CM. Fibrinogenolytic enzymes of Trimeresurus mucrosquamatus venom. Biochim Biophys Acta, 1976, 420: 298－308

21    Graham SB, Tu AT, Sapru ZZ. A fibrinolytic enzyme from the venom of the Western diamondback rattlesnake (Crotalus atrox). In: Pirkle H, Markland FS eds., Hemostasis and Animal Venom: Hematology, 1988, Vol. 7, New York: Marcel Dekker, 203－212

22    Jin Y, Lu QM, Wei JF, Li DS, Wang WY, Xiong YL. Purification and characterization of jerdofibrase, a serine protease from the venom of Trimeresurus jerdonii snake. Toxicon, 2001, 39: 1203－1210

23    Wang XM, Chi CW. Purification and characterization of kallikrein I from the venom of Agkistrodon halys pallas. Acta Biochim Biophys Sin, 1984, 16(1): 15－25

24    Fiedler F, Geiger R. Separation of kinins by high-performance liquid chromatography. Methods Enzymol, 1988, 163: 257－262

25    Hung CC, Huang KF, Chiou SH. Characterization of one novel venom protease with beta-fibrinogenase activity from the Taiwan habu (Trimeresurus mucrosquamatus): Purification and cDNA sequence analysis. Biochem Biophys Res Commun, 1994, 205(3): 1707－1715

26    Itoh N, Tanaka N, Mihashi S, Yamashina I. Molecular cloning and sequence analysis of cDNA for batroxobin, a thrombin-like snake venom enzyme. J Biol Chem, 1987, 262(7): 3132－3135

27    Lu QM, Jin Y, Li DS, Wang WY, Xiong XL. Characterization of a thrombin-like enzyme from the venom of Trimeresurus jerdonii. Toxicon, 2000, 38: 1225－1236

28    Bjarnason JB, Barish A, Direnzo GS, Campbell R, Fox JW. Kallikrein-like enzymes from Crotalus atrox venom. J Biol Chem, 1983, 258: 12566－12573

29    Sapru ZZ, Tu AT, Bailey GS. Purification and characterization of a fibrinogenase from the venom of Western diamondback rattlesnake (Crotalus atrox). Biochim Biophys Acta, 1983, 747: 225－231

30   Pandya BV, Budzynski AZ. Anticoagulant proteases from Western diamondback rattlesnake (Crotalus atrox) venom. Biochemistry, 1984, 23: 460－470

31    Baker BJ, Wongvibulsin S, Nyborg J, Tu AT. Nucleotide sequence encoding the snake venom fibrinolytic enzyme atroxase obtained from a Crotalus atrox venom gland cDNA library. Arch Biochem Biophys, 1995, 317(2): 357－364

32    Randolph A, Chamberlain SH, Chu HL, Retzios AD, Markland FS Jr, Masiarz FR. Amino acid sequence of fibrolase, a direct-acting fibrinolytic enzyme from Agkistrodon contortrix contortrix venom. Protein Sci, 1992, 1(5): 590－600

33    Siigur E, Siigur J. Purification and characterization of lebetase, a fibrinolytic enzyme from Vipera lebetina (snake) venom. Biochim Biophys Acta, 1991, 1074, 223－229

34    Reichl AP, Assakura MT, Mandelbaum FR. Biophysical properties and amino acid composition of Bothrops protease A, a proteolytic enzyme isolated from the venom of the snake Bothrops jararaca (jararaca). Toxicon, 1983, 21: 421－427

35   Lochnit G, Geyer R. Carbohydrate structure analysis of batroxobin, a thrombin-like serine protease from Bothrops moojeni venom. Eur J Biochem, 1995, 228: 80－816

36    Wang YM, Wang SR, Tsai IH. Serine protease isoforms of Deinagkistrodon acutus venom: Cloning, sequencing and phylogenetic analysis. Biochem J, 2001, 354: 161－168

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Received: April 15, 2003        Accepted: May 27, 2003

*Corresponding author: Tel, 86-871-5192476; Fax, 86-871-5191823; e-mail, [email protected] or [email protected]