Purification and partial characterization of a thrombin-like enzyme from the venom of the bushmaster snake, Lachesis muta noctiv aga

Toxka~. Vol . 19, No . 2, pp. 279-294. 1981

0041-0101/81/020279-IS 502 .OOA ® 1981 Pergamon Preis Ltd .

Printed in Great Britain

PURIFICATION AND PARTIAL CHARACTERIZATION OF A THROMBIN-LIKE ENZYME FROM THE VENOM OF THE BUSHMASTER SNAKE, LACHESIS MUTA NOCTIVAGA* ARINOS MAGALHAES, GILSON JOSÉ DE OLIVEIRAt and CARLOS R. DINIZ Departamento de Bioquimica, Insdtuto de Ciéncias Biohigicas, Universidade Federal de Minas Gerais, 30000 Helo Horizonte, C .P . 2486, Brasil

(Acceptedjor publication 22 September 1980) A . MAGALHAES, G . J . DE OLIVEIRA end C . R. DINIZ Purification and partial characterization of a thrombin-like enzyme from the venom of the bttsllmaster snake Lachesis mists noctivaga. Toxicon 19, 279-294, 1981 .-A thrombin-like enzyme has been purified from the venom of Lachesis mists noctivaga (41-fold purification and 43 ~ yield). The steps included : gel filtration with Sephadex G100, hydroxylapatite and DEAFcellulose chromatography, and finally Sephadex G-150 filtration (twice) . The material was homogeneous in polyacrylamide electrophoresis and gel filtration on Sephadex G-150. The enzyme is a glycoprotein of molecular weight 36,300 as determined by gel filtration. The thrombin-like enzyme hydrolyses tosyl-L-arginine methyl ester (Km = 1 ~45 x 10 - ` M, Vm = 353 pmole/min ~ mg, Kcat = 212 sec - `) with an optimum pH of 8 30. The enzyme also hydrolyses tx-N-benzoyl-DL-arginine p-niuoanilide and is competitively inhibited by ben7amidine, ßnaphtamidine and phenylgttanidine. Clotting and amidase activities are inhibited by düsopropylfluorophosphate. The enzyme molarity was determined by active site titration with p-nitrophenyl-p_ guanidine benzoate (92 ~ pure) . Injected in dogs 2 kg of the purified enzyme reduce, in 30 min, the plasma fibrinogen concentration to values less than 15 ~ of the original level. In vitro the activity for human fibrinogen-fibrin conversion was equivalent to 1650 f 12NIH thrombin traits/mg protein enzyme.

INTRODUCTION

THROMBIN-LIKE enzyme is defined as a proteinase which catalyses the cleavage of internal peptide bonds of plasma fibrinogen, similarly to thrombin. The substrate undergoes a limited proteolysis by the enzyme and converts to fibrin (BLOMSAC1c,1958). Thrombin-like activity is widely distributed in venoms of the Crotalidae subfamily of snakes (COPLEY et al., 1973 ; GAFFNEY, 1977). As with thrombin, the enzymes from snake venom hydrolyses synthetic substrates typical of trypsin (peptides, amides and esters at bonds involving the carbonyl group of basic amino acids) and are inhibited by düsopropylfluorophosphate (iPr Z P-F). However, they are not inhibited by heparin or hirudin, well known thrombin inhibitors . DEtrISCH and DINIZ (1955) studied the proteolysic activity of 15 North and South American crotalid venoms . They used different substrates including bovine fibrinogen and the synthetic substrate a-N-benzoyl-l.-arginine ethyl ester (Bz-1.-Arg-OEt) . The venom of Lachesis mists,

' Part of the data in this paper was presented at the X International Congress of Biochemistry at Hamburg, Germany, 25-31 July 1976 and at the VI International Symposium on Animal, Plant and Bacterial Toxins, Uppeala, Sweden, 23-27 July 1979. t Present address : Depto . de Bioquimica, Institute de Ciências Biolbgicas e Geociências, Universidade Federal de Juiz de Fora, MG, Brasil . 279

280

ARINOS MAGALHAES, GILSON JOSÉ DE OLIVEIRA and CARLOS R. DINIZ

the bushmaster ofthe American Continent, displayed the highest thrombin-like activity ofall venoms tested . It was considered of interest to purify the coagulant principle in L. muta venom and to study some of its chemical and enzymatic properties in view ofthe biochemical and clinical significance of the coagulant action of snake venoms. In the present paper we describe a procedure for theisolation ofa thrombin-like enzyme from L. muta noctivaga in an apparently homogeneous form and report some of its physical, chemical and enzymatic properties. MATERIALS AND METHODS The crude venom of L mots noctivaga, a subspecies collected in the Atlantic forest of Brazil, was obtained from Laboratories Pinheiros, Säo Paulo, Brazil . Sephadex G-100, G-150 and blue dextran were obtained from Pharmacia, Uppsala, Sweden. DEAE cellulose (DE-52 Inicrogranular) was purchased from H . R. Angel, London, U.K ., and hydroxylapatite (Bio-Gel HTP) was obtained from Bio-Rad Laboratories, Richmond, CA, U.S.A . ; tosylL-arginine methyl ester (Tos-L-Arg-OMe~ düsopropylfluorophosphate (iPr,P-F) and lysozyme were purchased from Sigma Chemical Co., St. Louis, MO, U .SA. ; a-N-benzoyl-DL-arginine lrnitroanilide (Bz-DL-Arg-NHNp) and L-1-chloro-3-tosylamido-7-amino-heptanone (Tos-LysCH~CI) were purchased from Calbiochem, San Diego, CA, U.S .A ., and trypsinogen from Worthington Biochemical Corp., Freehold, NJ, U.S.A. Benzamidine was obtained from Aldrich Chemical Co, Milwaukee, WI, U .S .A. Phenylguanidine was prepared as described by MARES-Guu and SHAW (1%5) and ß-naphtamidine was synthesized as described by MARES-GuIA (1%Ba) . p-Nitrophenyl-p'guanidino benzoate (NPGB) was supplied by Professor Elliot Shaw, Brookhaven National Laboratories. Thrombin and human fibrinogen were purified and supplied by Professor J. R . Giglio, Ribeiräo Preto, Säo Paulo. All other reagent chemicals were reagent grade purchased from Merck, Darmstadt, Federal Republic of Germany . Absorbance was measured with a Beckman Du Mode12400 Spectrophotometer or a Coleman Junior Mode16-D . The titrations were carried out with a Radiometer TTT-1C titrator, equipped wiW an Auto-burette ABU-12, a SBR-

L,5

A ~ 280 nm _.o.. . o -_ A~10 nm

E c W V 2

W

a m C O O

m a

u 2

a m

K O N

1,5

m a

0 VOLUME

( ml )

FIG. 1. GEL FILTRATION OVER SEPHADEX G-100. Crude venom (420 mg) was added to two columns in series (22 x % cm each). Elution : 0-05 M acetate buffer, 0~3 M in NaCI, containing methiolate at 1 : 50,000, at pH 7~3 ;flow rate was 17 ml/hr, with 6~3 ml fractions being collected at 4°C. Activity was measured with Bz-DL-Arg-NHNp. Fractions pooled are indicated by the solid bar.

Thrombin-like Enzyme from L. muta

28 1

2 recorder, G-202 glass electrodeand Kfi05 calomel electrode at 25 t 1°C. All column operations were performed in the cold (3-5°C) and fractions were collected with the aid of an automatic fractions collector (GME-Model VL). Amino acid analyses were carried out with a Beckman Model 1200 analyser. Protein was determined according to the prooodun of LOwRY d al. (1951) uaiag bovine serum albumin as a standard or from the abeorbance at 280nm with a cell of 1 cm light path. Fibrinogen was determined according to the method of RATNOFF and MENZIE (1951) . Amidaseactivity toward Bz-DL-Arg-NHNp was measured essentially as described by ERUNGERet al. (1961). The esterase activities on Tas-L-Arg-OMewen measured in theRadiometer titrator. The total volume at the specified pH was 10 ml and the reaction mixture was 0" 10 M in KCI, temperature 25°C . Sodium hydroxide (0 " 702mM) was the usual titrant, delivered from a 2~Sml syringe in the ABU-12 unit. Humid nitrogen was continuously passed over the solution in the titratar. Exact substrate concentrations wen obtained from total hydrolysis carried out with trypsin . Clotting activity of the enzyme was assayed with human fibrinogen essentially according to the method Of WARE and SEEGERS (1949). One unit of coagulant activity is approximately equivalent to one NIH thrombin unit Specific coagulant activity is defined as the number of units contained in 1 mg of enzyme . The values of Km and Ym for Tos-L-Arg-OMe hydrolysis were determined according to the procedure of LINEWFAVER and BURK (1934) using the variance of Y as statistical weight (waKINSON,1961). Thecak:ulations wen carried out with the help of a Fortran program called KMVM (MARES-GutA, 1972). The disaociadon constants for benzamidine, ß-naphtamidine and phenylguanidine inhibitions of Bz-UL-Arg-NHNp hydrolysis were determined according to themethod of MARES-Guu and SttAw (1965) and MARES-GuIA (196ßa)and thecalculation wascarried out with the help of a program called INCON (MARES-GurA (19686). An IBM-1130 computer was used for these calculations.

RESULTS

Purification : Sephadex G-100 chromatography

Four samples of 40(1`500 mg of crude venom totalling 1635 mg protein were used. Each sample was dissolved in 15 ml of 005 M ammonium acetate buffer (pH 7~3, 0~3 Min NaCI) and centrifuged to remove any insoluble residue. The solution was applied to two columns in series (2~2 x 96 cm each), the gel being previously equilibrated with the same buffer subsequently used for elution. The flow rate was 17 ml/hr and the Gactions 6~3 ml each . The active fractions were identified with Bz-nL-Arg-NHNp (Fig. 1). From preliminary experiments we observed that the coagulant action of the venom was restricted to the Bz-DL-ArgNHNp peak ofhydrolysis . The active fractionswere pooled, dialysed against deionized water at 4°C and lyophilized (Step 1). The lyophilized material from the filtration of the four TAHLE 1. PURIFICATION OF A THROMBIN-LIKE ENZYME FROM L. muta noctiaaga VENOM

Step 1 2

3

4

Procedure Crude venom Sephadex G-100 chromatography Hydroxylapatite chromatography P1 P~ DEAF-Cellulose chromatography of P~ Pß " 1 Pß " 2 Sephadex G-150 chromatography of P= " 1 (twice)

Total protein (mg)

Specific activity' (Eemole/(mg " min)

Total amldase activity (amok/min)

1635

0"075

123

1

100

Purification factor

Yield (;°)

108

0"68

73

9

59

30 46

1 "03 1 " 27

31 SS

14 17

25 47

26 . 17

1 "33

23

18

19

17

3 " 10

53

41

43

'Specific activity was determined using Bz-UL-Arg-NHNp as a substrate.

28 2

ARINOS MAGALHAES, GILSON JOSÉ DE OLIVEIRA and CARLOS R, DINIZ

W u z a m rc 0 m a

VOLUME 1 ml I FIG . 2. CHROMATOGRAPHY OF' THE MATERIAL FROM FIG. 1 . (solid bar) ON HYDROXYLAPATITECOLUMN (2 " 2 X 40 cm) . Twenty-six milligrams was applied and eluted with 340 ml of 0-005 M potassium phosphate buffer, 0 "3 M in KCI, at pH 6 "8 ; followed by 145 ml of 0 " 1 M potassium phosphate buffer, containing 0 " 3 M KCI, pH 6 "8. Finally, 200 ml of 0 " 2 M phosphate buffer 0 ß M in KCI at pH 6 " 8 was passed through the column (arrow indicates the position of buffer application) . Flow rate was 19m1/hr, with 3 " 2 ml fractions being collected at 4°C. Activity was measured with Bz-Dt: Arg-NHNp. Fractions pooled are indicated by the solid bar.

W u z a m a 0 N m a

VOLUME 1 ml) F1G . 3 . DEAE-CELLULOSE CHROMATOGRAPHY . Forty-six milligrams of material from the previous step (pool 2) was applied to a 22 x 21 cm column, equilibrated with 0-05 M Tris buffer, 0 " 1 M in sucrose, pH 8~5 . Elution took place with the same buffer. A linear ionic strength gradient from 0 to 0 " 25 M NaCI in 0 " 05 M Tris (pH 8-05) was started after the collection of 215 ml ofelutee. The flow rate was 36 ml/hr, and fractions of 3 ~2 ml were collected at 4°C ; activity was determined with Bz-DL-Arg-NHNp . Fractions pooled are indicated by the solid bar.

Thrombin-like Enzyme from L. muta

VOLUME

1

28 3

ml 1

FIG . 4. GEL FILTRATION OVER SEPHADEX G-ISO. (A) Twenty-six-milligram samples of material from the preceding step (pool 2.1) were applied to a 2~2 x 83 cm column, equilibrated with O~OS M ammonium acetate buffer, pH 7~3, 0~3 M in NaCI . Elution took place with the same buffer, at a How rate of 18 ml/hr, and fractions of 2~7 ml were collected at 4°C ; activity was determined with Bz-Dt-Arg-NHNp ; (B) The material obtained in (A) was applied to the same column and eluted underidentical conditions. Fractions pooled areindicated by the solid bar.

samples was combined (108 mg) and used for further purification . A purification of 9~0-fold was obtained up to this point (Table 1). Hydroxylapatite chromatography The material from the preceding step was divided into four portions (25-35 mg each) separately applied in a 2~2 x 40 cm cohunn of hydroxylapatite and eluted with 340ml of 0005 M potassium phosphate buffer, 0~3 M in KCI, at pH 6~8, followed by 145 ml of 0~1 M potassium phosphate buffer containing 0~3 M KCI, pH 6~8 . Finally 200 ml of 0~2 M phosphate buffer, 0~3 M in KCI, at pH 6~8 was passed through the column. The most active component was eluted with the first buffer. The fractions were pooled as indicated in Fig . 2 (pool 1 and 2), dialysed against deionized water and lyophilized (step 2). Pool 2 contained the highest specific activity. They were combined and used in the subsequent step. A 1 ~9-fold purification was obtained in relation to the previous step. DEAE-Cellulose chromatography Active material from the previous step containing 46 mg protein dissolved in about 5 ml of 005 M Tris (pH 8~5) was applied to a 2~2 x 21 cm column of DEAE-cellulose previously equilibrated with the same buffer, which was subsequently used for elution . A linear ionic strength gradient, from 0 to 025 M NaCI in 005 M Tris (pH 8~5), was started after the collection of215 ml ofeluate. The flow rate was 36 ml/hr ; the fraction volume was 3~2 ml. The protein was eluted with the ionic strength gradient. The active fractions were pooled as indicated in Fig. 3 (pools 2.1 and 2.2, Fig . 3), dialysed against deionized water at 4°C and

284

ARINOS MAGALHAES, GILSON JOSÉ DE OLIVEIRA and CARLOS R. DINIZ

lyophilized (step 3). The poo12.1 displayed the highest specific activity (Table 1) andwas used in the final step of purification. Sephadex G-150 chromatography

The enzyme concentrated in the poo12.1(26 mg protein) in the previous step was dissolved in 5 ml of 005 M ammonium acetate buffer, 0~3 M in NaCI, at pH 7~3. The solution was applied to a G-150 Sephadex column, 2~2 x 835 cm, previously equilibrated with the same buffer, which was also used for elution. The flow rate was 18 ml/hr and the fractions 2 ~7 ml (Fig . 4A). The active fractions were pooled, dialysed against deionized water at 4°C and lyophilized. The dried material was dissolved in 5 ml of the same buffer and refiltered in the same column under the same conditions. The pattern of rechromatography is shown in Fig. 4B. A protein peak of constant specific activity was obtained. The fractions were pooled as indicated, dialysed against deionized water and lyophilized. The amount of active protein recovered was 17 mg. A purification flow sheet is shown inTabk 1. The overall yield was43 and the purification factor was 41. This material was used for characterization and kinetic studies are presented in this paper. Pools Pt and Pz .z from steps 2 and 3 were combined and filtered over Sephadex G-150 as described above. Inactive material was removed ; however, impurities were still detected by polyacrylamide electrophoresis. Physicochemical characterization : electrophoresis

The fraction obtained by Sephadex G-150 rechromatography showed a single band after TABLE

2.

AMINO ACID COMPOSITION OF L THROMBIN-LIKE ENZYME

Amino acid Lys His Arg Asp Thr Ser Glu Pro Gly Ala Hall-Cys Val Met Ile Leu Tyr Phe Trp Mol. wt

muta noctivoga

Amino acid residue

Nearest integral No. (mole/mole)

21 ~0 7-0 189 392 161 224 31 ~5 196 35-0 154 140' 19 ~6 42* 154 222

21 7 19 39 16 ~ 22 31 20 35 15 14 20 4 15 22

133 63t -

13 6 36,456$

los

lo

* Methionine and cysteine were determined as methionine sulfone and cysteic acid, respectively, according to HIRS (1967). tTryptophan was apectrophotometrically determined by the method Of SPANDE Snd wrrxOP (1967) . $Themolecular weight wasdetermined by gelSltration on Sephadez G-150.

Thrombin-like Enzyme from L. muta

FIC . S . PUL.YACRYLAMIDF CFI . FLECI'RUPHURFSIS.

One hundred micrograms of protein was added to each tube and developed for 60 min, at 5 mA per tube in 7~5 °, ;; acrylamide gels, pH 8~2 . Staining was with Amido Alack IOB . (A) Crude venom ; (B) after chromatography on Sephadex G-150 (Table 1, Step 4).

28 5

Thrombin-like Enzyme from L. awls

287

electrophoresis in 7 ~5 ~ polyacrylamide for 60 min in Tris-glycine at pH 8 ~2 (Fig. 5). The runs were made at 4mA/column according to the method of DAVIS (1964) and the protein (0 ~ 1 mg) was stained with Amido Black lOB in 7 % acetic acid. The glycoprotein nature of the enzyme was observed using cellogel electrophoresis [0~ 1 M borate buffer, pH 9~0, according to the method Of WYBENGA (1974)]. After staining by Schiff's periodic acid (PAS) technique 0~1 mg of the purified enzyme showed one single band after 90 min electrophoresis at 200 V, 1 mA/cm. Molecular weight determination

The purified enzyme was passed through a Sephadzx G-150 column calibrated with lysozyme, trypsinogen, ovalbumin and serum albumin, all proteins of known molecular weight. The method Of ANDREWS (1965) was used to estimate the molecular weight of the purified enzyme . The void volume was determined with blue dextran. The enzyme gave an elution volume corresponding to a molecular weight of 36,300.

0

N

O

5 N ~O

i 3

M

0 .5

2 1 0

10

20 S

FIG . Ô. LINEWEAVER-BURK PLOT FOR

L.

t 10

30 3

Illuta IIOCt1tMAa ENZYME-CATALYSED TOS-L-Arg-OMe HYDROLYSIS.

Conditions : pH 8-0, 25°C ; O~SUg of enzyme/lOml of incubation mixture ; solvent was 010 M KCI containing the substrate. Ordinate, reciprocal ofrate, in min ~ mg/Eunole ; abscissa, reciprocal of molar concentration of substrate. Inset, pH dependence of rate, measured as above, with 1 025 mM substrate.

288

ARINOS MAGALHAES, GILSON JOSÉ DE OLIVEIRA and CARLOS R. DINIZ

75

2S

0

0.5

L0

L5

2 .0

~ 10 4 FIG. 7. LINEWEAYER-BURK PLOT FOR L . muta rioctiuaga ENZYME-CATALYSED Bz-DI:Arg-NHNp HYDROLYSIS . Conditions : pH 8~1, 37°C ; 1~25pg of enzyme/2 ml of incubation mixture; solvent was O~IOM Tris containing the substrate . Ordinate, reciprocal of rate, in sec/M ; abscissa, reciprocal of molar concentration of substrate.

Titration with p-nitrophenyl-p' guanidino6enzoate (NPGB) The enzyme was titrated by NPGB, an active center titrant of trypsin described by CHnse and SHnw (1967). Calculations of the molarity indicated that the enzyme was 92 % pure in active centers. For this calculation, the molecular weight was taken as 36,300 (see above) and protein was determined by the method of LOWRY et al. (1951), using bovine serum albumin as a standard. Amino acid analysis One milligram samples of the enzyme were hydrolysed in ampoules with constant boiling HCI, at 110 ± 2°C for 20 and 40 hr. After drying the hydrolysate in a desiccator with moist NaOH pellets the residues were dissolved in 0~5 ml of02 N citrate buffer (pH 2'2) (SPACKMAN et a1.,1958). The analysis was carried out in the short column (resin PA-35) for basic amino acids and in the long column (resin AA-15) for neutral and acid amino acids. Methionine and cysteine were determined as methionine sulfone and cysteic acid, respectively, after performic acid oxidation according to the method of H1RS (1967) . Tryptophan was determined spectrophotometrically, according to the procedure of SrnNne and WITKOP (1967). The maximum number of residues obtained in the two hydrolysis times used were taken for the calculation of the amino acid composition . A summary of the results is shown in Table 2. A minimum molecular weight of 5208 per histidine residue was obtained. The presence of TABLE 3. KINETIC PARAMETERS OF LaCitCSiS mots riOCtillaga THROMBIN-LIKE ENZYME Substrate Tos-L-Arg-OMe' Bz-DL-Arg-NHNpt$

Vm

Km

(M)

(sec - ')

(sec''~M-`)

353'0 1~5

1 ~5 x 10 -` 2~7 x 10 -~

212 090

146 x 104 033 x 10 4

(~cmole/min~mg)

pH 8-0, 25°C . tpH 8~1, 37°C. $Apparent parameters calculated from Fig. 7.

Kcat

Kcat/Km

Thrombin-life Enzyme from L. muta

289

sullhydryl groups was investigated by reaction of the venom enzyme with 5,5' (dithiobis) nitrobenzoic acid in the presence of 6 M guanidine-HCI. No reaction was observed, indicating the absence of any turntable sulfhydryl groups. Amino acid analysis, following performic acid oxidation and hydrolysis of the venom enzyme, gave 14 half-cystine residues, which suggests the presence of seven disulfide bridges. The carbohydrate composition ofthe enzyme is now under investigation. Kinetic characterization The Lachesis muta fibrinogenase displayed esterase and amidase activities toward synthetic substrates. Kinetic parameters for Tos-r.-Arg-OMe and Bz-DL-Arg-NHNp calculated from Lineweaver-Burk plots (Figs. 6 and 7), with the help ofa Fortran program called KMVM, are presented in Table 3. The maximal rate of esterase activity was near pH 8~3 (inset of Fig. 6). The amidase activity of L. muta enzyme toward Bz-nL-Arg-NHNp was inhibited by ßnaphtamidine HCI, benzamidine-HCI and phenylguanidine sulfate with the inhibition being competitive (Fig . 8). Ki values calculated according to MARES-GUIA and SHAW (1965), compared with the effects of these inhibitors upon trypsin, are shown in Table 4.

30

n 'o

15

n 'o .

20

10

10

1 ,

S

0

1

2

3

ß- NAPHTHA111DINE,

'P

-6

M" tO S

0

S

8EN2A111DINE,

10

15

AI " 10S

15

M

14

6

-2

-1

0

1

2

PHENYLGUANIDINIUM,

FIG. 8 . COMPETITIVE INHIBI770N OF

Lachesis

3

i

5

M " 10~

ENZYME BY AMIDINES AND GUANIDINE .

Ordinate : reciprocal of the rate in sec/M ;abscissa, molar concentration of inhibitor. Concentration of Bz-DL-Arg-NHNp 405 x 10 -` M (lower lines) and 275 x 10 - ` M (upper lines) . Enzyme concentration was 25 ßg/2 ml of incubation mixture. Experimentsconducted in Tris (010 M~ pH 8~ 1 and 37°C. Incubation was for 60min. Each point represents duplicate determinations .

290

ARINOS MAGALHAES, GILSON JOSÉ DE OLIVEIRA and CARLOS R. DINIZ

TABLE 4. COMPETITIVE INHIBITORSOF L. mtltü IüX'tivü(Jü TFIROMBIN-LIKE ENZYME AND TRYPSIN 10 5 'Kî (M) Inhibitors

Lachesis enzyme'

Trypsint

0'66 6'87 20'90

l'46 l'84 7'25

ß-Naphtamidine ~ HCI Benzamidine ' HCI Phenylguanidine ~ O'SHZSO4

Determined according to procedure of MARES-GUIA and SF1AW (1965) ; all the Ki values were calculated as described in Materials and Methods. 'Tris 0~1 M. pH 8'1, 37°C. tTris 0'1 M . pH R'15, 15°C.

Irreversible inhibition The venom enzyme was incubated with 4~2 mM iPr Z P-F in 005 M veronal buffer, pH 8~0, at room temperature . Amidase and clotting activities were simultaneously and irreversibly reduced to approximately 5 % of the activity ofthe controls, after a 3 hr period of incubation (Fig. 9). Preliminary experiments showed that an excess of Tos-Lys-CH Z Cl elicited a 25 inhibition of amidase activity after a pre-incubation of 4 hr. Biological characterization Employing bovine fibrinogen as substrate, the clotting activity of the thrombin-like

T t >_

T _>

O O~ C_

r _o U

O

_v E Q

Time,

hr

EFFECT OF DIISOPROPYLFLUOROPHOSPHATE ON BZ-DI:A[g-NHNp AMIDASE AND CLOTTING ACTIVITIES OF THE VENOM ENZYME . The enzyme (47 ug) was incubated with 4~2mM iPrZ F-P in a final volume of 0'30 ml containing 005 M veronal buffer, pH 8~0. Aliquots were removed after various times of incubation at room temperature and assayed as described under Materials and Methods. All determinations were performed in duplicate.

FIG.9.

Thrombin-Gke Enzyme from L . muta

29 1

enzyme from L. muta noctivaga obtained by the procedure described in this work was 1650 f 12 NIH thrombin units/mg protein enzyme. Intravenous injection of the purified enzyme caused fibrinogen depletion in dogs ; 2leg of the enzyme per kg animal (two experiments) caused an 86 ~ reduction of the fibrinogen content of plasma in 30 min. No effect was observed on the blood pressure . None of the dogs died during the experiments and no bleeding was observed from the skin, urinary tract or mucosa . DISCUSSION

Gel filtration, ion exchange and adsorption chromatography have been used by different laboratories to isolate thrombin-like enzymes from snake venoms (HENRIQuES et al., 1960 ; ESNOUF and TUNNAH, 1967 ; OUYANG 8t al., 1971 ; MARKLAND ând DAMUS, 1971 ; ANDERSON, 1972 ; HATTON, 1973 ; OUYANG ând YANG, 1974 ; STOCKER and BARLOW, 1975 ; BONILLA, 1975 ; MARSH and WHALER (1974) . These procedures have also been employed to separate the coagulant enzyme from L. muta noctivaga. In the eluates the peak of activity upon Bz-DL-ArgNHNp coincided with the coagulant action of the enzymes upon purified bovine fibrinogen. The amidase assay simplified considerably the routine of isolation of the thrombin-like enzyme by column chromatography . The amidase and coagulant activities were inhibited to the same degree by iPr2 P-F (Fig. 9) from which it can be concluded that the two activities involve the active site of the same serine proteinase . The Tos-L-Arg-OMe esterase activity by contrast is associated, besides the amidase and clotting activities, with kinin releasing and a non-proteolytic esterase also present in venom from Lachesis muta noctivaga (MAGALIIAES et al., 1978). In some of the steps of purification (Table 1) an excess of enzyme activity was observed when compared with the previous step. This finding was found in all five experiments of purification done in this work, and is interpreted as being due to the removal of inhibitors of amidase activity. Activation of unknown origin was also observed by MARKLAND and DAMUS (1971) in the purification of a similar enzyme in the venom of C. adamanteus. The thrombin-like enzyme from L. muta noctivaga was obtained in a high degree of homogeneity at the end of the purification procedure described in this paper. Upon rechromatography on Sephadex G-150 a single peak of enzyme of constant specific activity was obtained (Fig . 4B). The titration of its active center with NPGB, related to the molecular weight of 36,300 (found by gel filtration), gave 924 ~ purity . The amidolytic enzyme also produced single bands on cellogel electrophoresis at pH 9~0 with identical mobilities, revealed either by Amino Black lOB or PAS. A single band of protein was also obtained after polyacrylamide gel electrophoresis at pH 8'2 (Fig . 5). The amino acid analysis gave data that needed minimum rounding-off when the minimum molecular weight was multiplied by seven to give a molecular weight close to that found by Sephadex G-150 chromatography ("fable 2). The chemical, physicochemical and enzymatic properties described in this work for the purified thrombin-like enzyme from L. muta noctivaga are also shared by similar enzymes obtained from A. rhodostoma (Arvin, ESNOUF and TUNNAH, 1967), B. moojeni (Defibrase, Batroxobin, STOCKER and BARLOW, 1975), Î. gramineus (OUYANG and YANG, 1974), C. adamanteus (MARKLAND ând DAMLJS, 1971), A. aCtliuS (OUYANG Ci al., 1971), i'. okinavensis (ANDERSON, 1972), C. h. horridus (BONILLA, 1975). They are all glycoproteins with molecular weights in the 32,000 range. The amino acid composition of the thrombin-like enzyme from L. muta noctivaga compares well with that of other thrombin-like enzymes and thrombin, although there are some distinct differences. Amino acid analysis of the Lachesis enzyme following performic acid oxidation and hydrolysis gave 14 half-cystine residues. No

29 2

ARINOS MAGALHAES, GILSON JOSÉ DE OLIVEIRA and CARLOS R. DINIZ

reaction was observed with 5,5' (dithiobis) 2-nitrobenzoic acid in the presence of 6 M guanidine-HCI, indicating the absence of any titratable sullhydryl group which suggests the presence of seven disulfide bridges in the enzyme . MARKLAND and DAMUS (1971) observed similar results with their thrombin-like enzyme from the venom of C. adamanteus. Preliminary experiments by cellogel electrophoresis have shown a pI value of 5~1 for the Lachesis enzyme . The serine proteinase nature of the thrombin-like enzyme from snake venoms originally shown for Arvin by ESNOUF and TUNNAH (1967) is further demonstrated for Lachesis enzyme by the iPrZ P-F inhibition curve in Fig. 9. THOMPSON (1970) has reported a similar result by using bovine thrombin in the presence of Sarin, an inhibitor of the organophosphate type . The thrombin-like enzyme from L. muta noctivaga, as thrombin and similar enzymes from other snake venoms, displayed the same specificity towards the aryl esters ofthe basic amino acids (trypsin-like enzymes) . The catalytic efficiency upon synthetic substrate is higher in Lachesis enzyme as compared to other coagulating enzymes isolated from snake venoms and was only one order of magnitude below pure ß-trypsin. The KcatlKm values, using Tos-LArg-OMe as substrate, is 1 ~80 x 10'sec- t M- ' for ß-trypsin and 1 ~46 x 106 sec- ' ~ M - t for the purified enzyme . The pH curve ofrate ofhydrolysis when using thrombin-like enzymes from other snake venoms was also in the range ofpH 4~5-9 ~0 with a pH optimum around 8~3, suggesting the participation of histidine in the catalytic process (inset of Fig. 6). This was substantiated by the finding that Tos-LysCH Z CI inhibited the venom enzyme, although at a substantially slower rate than either thrombin (SEEGERS, et al. 1974) or trypsin (SHAW et al., 1965). This rate difference probably reflects structural differences around the reactive histidine residue of the venom enzyme ; nevertheless, the inhibitory effect ofthe chloromethyl ketone of tosyl-L-lysine suggests the involvement of this' amino acid in both the amidase and clotting activities of the venom enzyme . The demonstration that ß-naphtamidine, benzamidine and phenylguanidine are competitive inhibitors of the Lachesis enzyme reinforce the view that the active center of this enzyme is similar to that of trypsin, proposed to consist of an anionic and a hydrophobic binding site responsible for the binding oflysyl or arginyl residues in substrates or the carbon side chains of inhibitors such as those used in this work (MARES-GUIA and SHAW, 1965). However, when the Ki values of these inhibitors are compared in the trypsin and Lachesis enzyme (Table 4), differences are evident. The specific clotting activity of the Iachesis enzyme is in the range of, or higher than, similar purified enzymes ofother Crotalidae. The specific activity ofLachesis thrombin-like enzyme was 1600 NIH unit/mg protein, Arvin, 1200 (EsxouF and T[JNNAH, 1967), Repulses, 70 (EGSERG et al., 1971), Crotalase, 223 (MARKLAND and DAMUS, 1971). A purified bovine thrombin was found to have 7770 NIH unit/mg dry weight (SEEGERS et al., 1974). In vitro, with human plasma, the Lachesis enzyme did not induce platelet aggregation (CHAMONE, 1976). Observations ofthe effect of the purified Lachesis enzyme on dogs have been extended by CHAMONE et a1.,1979). They found that following i.v. administration of 2 F~g/kg weight of the L. muta noctivaga enzyme to 20 mongrel dogs the fibrinogen level decreased in 1 hr from 335±24 (~C f SEM) mg's to 12-6 f 7~1. Three and six hours after the enzyme administration,when the fibrinogenwas completely depleted, no difference was observed in the levels of factors II, V, VII, VIII and X ; the platelet count decreased moderately (ca. 20%). Acknowledgements-We are indebted t0 Dr. NEUZA MARIA DE MAGALHÄES ROCHA fOr help Wlth the klnetiC measurements and to Professor ARMANDO A . NEVes and Cdssto M . COSTA for the amino acid analysis. Thanks are also due to Professor IBRAIN F. HENEINE, who carried out the sulphydryl group analysis. This work was supported

Thrombin-like Enzyme from L. muta

293

by grants from the Conselho National de Desenvolvimento Cientffico e Tecnol8gico-CNPq and Financiadora de Projetos Especisis (FINEP). REFERENCES ANDERSON, L (1972) Isolation of thrombin-like activity from the venom of Trimeresurus okinrmensis. Haemostasis 1, 31 . ANDREWS, P. (1965) The gel-filtration behaviour of proteins related to their molecular wtights over a wide range. Biochem. J. 96, 595. BLOMHACK, B. (1958) Studies on theaction of the thrombic enzyme on bovine fibrinogen as measured by N-terminal analysis . Arkiv. Kemi 12, 321. BONILLA, C.A. (1975) Defibtinatiagenzyme from Timber rattlesnake (Crotales h. horridus) venom : a potential agent for therapeutic defibrination . I. Purification and properties . Thrombus. Res. 6, 157. CHAMONE, D.A. F. (1976) Clotasedemonstraçào da ausencia de açäo agregante de plaquetas in vitro. Cümc. Cult . 28, 589. CHAMONE, D. A. F., MAGALHxES, A., DtNtz, C. R., Mtt.o, L. T. V. and JAMRA, M. (1979) The effect of Lachesis muta venom (Clotase) on blood coagulation factors . Thrombus. Haemos . 42, 95 . CHASE, T., JR. and SHAW, E. (1967) p-Nitrophenyl-p =guanidinobenzoate. HCI : a new active site titrant for trypsin . Bicehem. Biophys. Res . Common. 29, 508. COPLEY, A. L., BARNFJEE, S. and DEVt, A. (1973) Studies of snake venons on blood coagulation. I. The thrombo serpentin (thrombin-like) enzyme in the venons. Thrombus . Res. 2, 487. DAMS B. J. (1964) Disc electrophoresis-II-Method and application to human serum protein. Ann. N.Y. Acad. Sci. 121, 404. DEUTSCH, H. F. and Dtxtz, C. R. (1955) Some proteolytic activities of snake venons. J. biol . Chem . 216, 17. EGBERG, N., HLOMBACK, M., YOHANSSON, B., ABILGAARD, V., BLOMBACK, B., DIENER, G., EKESTROM, S., CORANSSON, L., YOHANSSON, S. A., MCDONAGH, R., NILSON, S. F., NORDSTROON, S., OLSON, P. and WiMAN, B. (1971) Clinical and experimental studies on reptilase. In : Thrombolytic Therapy. Transactions of the Nineteenth Annual Symposium on Blood, p. 379. (MAMMEN, E. F., ANDERSON, G. F. ând GARNHART, M. L, Eds.) . Stuttgart : F. K. Schaflauer. ERLINGER, H. E., KOtcowstcv, N. and COHEN, W. (1961) The preparation and properties of two new chromogenic substrates of trypsin . Archs Biochem. Biophys. 95, 271. EsNOUe, M. P. and TUNNAH, G. W. (1967) The isolation and properties of the thrombin-like activity from Agkistrodon rhodostoma venom. Br . J. Haemat. 13, 581. GAFFTiEY, P. J. (1977) Report of the task force on standards for thrombin and thrombin-like enzyme . Thrombus . Haemostas. (Stuttg.) 38, 562. HATTON, M. W. C. (1973) Studies on the coagulant enzyme from Agkistrodon rhodostnma venom. Isolation and some properties of the enzyme . Biochem. J. 131, 799. HENRiQUES, O. H., FICHMAN, M. and HENRtQues, S. B. (1960) Partial purification and some properties of the bloodclotting factor from the venom of Bothrops jararaca. Biochem . J. 75, 551 . HIRS, C. H. W. (1967) Determination of cystine as cysttic acid. In : Methodsin Enzymology, Vol. 11, p. 59 (HIRS, C. H. W., Ed .) . New York : Academic Press. LINEWSAVER, H. and HuRK, D. (1934) The determination ofenzyme dissociation constants. J. Am . them . Soc. 56,658. LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L. and RANDALt, R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem . 193, 265. MAGALHAES, A, OLtvetRA, G. L. and DINIZ, C. R. (1973) Pretenses de serpentes brasileiras . I. Separaçào da enzima coaguhutte (clotase) de veneno de Lachesis meta. Ciénc. Cult . 25, 872. MAOALHÂFS, A., SILVA, L. M. and DtNtz, C. R. (1978) Isolation and characterization of arginil esterases from the venom of Lachesis muta (CrotaGdae). Agent Actions 8, 156. MARES-GUIA, M. (1968a) Hydrophobic interactions in the trypsin active center. The sensitivity of the hydrophobic binding site to side chain modifications in competitive inhibitors of the amidinium type . Archs Biochem. Biophys. 127, 317. MARES-GutA, M. (19686) The calculation of the enzyme-inhibitor dissociation constant, Ki, using a digital computer. An. Acad. Brasil . Ciênc. 40, 51 . MARES-GutA, M. (1972) A computer program for the preparation of numerical exercises on enzyme kinetics of the Michaelis-Menten type. Ciênc. Cult. 24, 607. MARES-GutA, M. and SHAW, E. (1965) Studies on the active center of trypsin . The binding of amidines and guanidines as models of the substrate side chain. J. btol . Chem. 240, 1579. MARKLIND, F. S. and DAMS, P. S. (1971) Purification andproperties of a thrombin-Gke enzyme from the venom of Crotales adomanteus (eastern diamondback rattlesnake) . J. biol. Chem . 246, 6460. MARSH, N. A. Snd WHALER, B. C. (1974) Separation and partial characterization of a coagulant enzyme from Bitis gabonica venom. Br. J. Haemat. 26, 295. OUYANG, C., HONG, J. S. and TENG, C. M. (1971) Purification and properties of the thrombin-like principle of Agkistrodon acutes venom and its comparison with bovine thrombin. Thrombus. Diath. haemorrh. (Stuttg. ) 26, 224.

294

ARINOS MAGALHÂES, GILSON JOSÉ DE OLIVEIRA and CARLOS R . DINIZ

OUYANG, C. and YANG, F. J. (1974) Purification and properties of the thrombin-like enzyme from Trimesurus gramineus venom. Biochim. biophys. Acts 351, 354 . RATNOFF, O. and MENZ~E, C. A. (1951) A new method for the determination of fibrinogen in small samples of plasma . J. Lab. clin. Med. 37, 316. SEEGERS, W. H., WAtz, D. A., REUTERBY, J. and MCCov, L. E. (1974) Isolation and some properties of thrombin E and others of prothrombin derivation . Thrombus . Res. 4, 829. SHAW, E., MARES-GUtA, M. and CoHeN, W. (1965) Evidence foran active center histidine in trypain through use of a spocific reagent, l-chloro-3-tosylamido-7-amino-2-heptanone, the chloromethul ketone derivedfrom N°-tosyl-Llysine. Biochemistry 4, 2219. SPACICMAN, D. H., STEIN, W. H. and MooeE, S. (1958) Automatic recording apparatus in the chromatography of amino acids. Analyt. Chem . 30, 1190. SPANDE, T. F. and W~TKOP, B. (1967) Determination of the tryptophan content of proteins with Nbromosuccinimide . In : Methods in Enzymology, Vol. ll, p. 498 (HERS, C. H. W., Ed .). New York : Academic Press. STOCKER, K. F. and BARLOW, G. H. (1975) Characterization ofdefibrase. in : Deftbrinierung mit Thrombin i{hnlichen Schlagengijt Enzymes. Aktuelle Problem in der Angiologie . Vol. 26, pp. 45-62 (MARTIN, M. and SCHOOP, W., Eds.) . Bern : Hans Huber. THOMPSON, A. S. (1970) Inhibition of thrombin by saris. Biochim. biophys. Acts 198, 392. WARE, A. G. and SEEGERS, W. H. (1949) Two stage procedure for the quantitative determination of prothrombin concentration . Am . J. clip . Path. 19, 471. WILKINSON, G. N. (1961) Statistical estimations in enzyme kinetics. Biochem. J. 80, 324. WvsEIVOA, D. R. (1974) Electrophoresis. In : Clinical Chemistry-Principles and Technics, p. 98 (HENRY, R. J., CANNON, D. C. and WINKELMAN, J. W., Ed.) . NeW York : Harper BG ROW .