A kinin-releasing enzyme was isolated from Bitis arietans (puff adder) venom by Sephadex G-100 and DEAE-cellulose column chromatographies. The kinin-releasing enzyme was shown to be homogeneous as demonstrated by a single band on acrylamide gel electrophoresis, isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodiffusion. Its molecular mass is approximately 45 kDa with an isoelectric point of 6.5. Kinin-releasing enzyme possesses proteolytic activity which hydrolyzes the Leu6-Cys 7, Hisl°-Leu u and Ala14-Leu Is bonds of the B chain of oxidized insulin and the Aa and Bfl chain of fibrinogen. Kinin-releasing and benzoyI-L-arginine ethyl ester hydrolytic activities of this enzyme were inhibited by diisopropyi fluorophosphate, suggesting that the serine hydroxyl group is involved in enzymatic activities.
Introduction
Hypotension and shock are symptoms which have tong been associated with Viperidae and Crotalidae venom poisoning. Bradykinin is a strong hypotensive agent because of its vasodila- tor action, and also a potent substance for increas- ing capillary permeability. Release of bradykinin from animal intestines, uterus and smooth muscle by Bothrops jararaca venom was first observed by Rocha e Silva et al. [1]. The bradykinin-releasing activity of B. jararaca venom is not related to its proteolytic activity on casein [2], but does corre- spond to its arginine ester hydrolytic activity [3]. Recent investigations on the purified kinin-releas- ing enzyme isolated from the venom of Crotalus atrox [4], Trimeresurus mucrosquamatus [5,6],
Abbreviation: Z-, benzoyloxycarbonyl-.
Correspondence address: Dr. H. Sugihara, Department of Mi- crobiology, Faculty of Pharmacy, Meijo University, Tenpaku- ku, Nagoya, Japan.
Agkistrodon halys blomhoffii [7], Echis coloratus [8] and Vipera ammodytes ammodytes [9] showed that these enzymes contain the benzoyl-L-arginine ethyl ester or tosyl-L-arginine methyl ester hydrolytic activity.
The present paper reports the isolation of a kinin-releasing enzyme from the venom of Bitis arietans, and the chemical and biological proper- ties of this enzyme are discussed.
Materials and Methods
Lyophilized crude venom was purchased from the Japan Snake Institute, Gunma, Japan. Seph- adex G-100 and molecular weight protein stan- dard kits were from Pharmacia, Uppsala, Sweden. Ampholyte (pH 3.5-10.5) was from LKB-Pro- dukter, Stockholm, Sweden. DEAE-cellulose was from Whatman Biochemicals, Ltd. The B chain of oxidized bovine insulin was from SERVA Fein- biochemica, Heidelberg, F.R.G. Benzoyl-L- arginine ethyl ester, tosyl-L-arginine methyl ester, tosyl-L-lysine methyl ester, acetyl-L-tyrosine ethyl ester, benzoyl-L-arginine amide, tosyl-L-arginine
amide, Z-Phe-Arg 4-methylcoumaryl-7-amide and Pro-Phe-Arg 4-methylcoumaryl-7-amide were from the Peptide Institute Inc., Osaka, Japan. Bovine fibrinogen was from Calbiochem, Behring Corp. Other chemicals used were of analytical grade from commercial sources.
Assay for arginine ester hydrolase Arginine ester hydrolase activity was assayed
using benzoyl-L-arginine ethyl ester as the sub- strate. The reaction mixture contained 0.1 ml en- zyme, 0.8 ml 0.4 M Tris-HC1 buffer (pH 8.5) and 10 /~mol benzoyl-L-arginine ethyl ester in a total volume of 1.0 ml. This was incubated for 15 min at 37°C. The amount of benzoyl-L-arginine ethyl ester hydrolyzed was determined by the hydrox- amate method of Roberts [10]. One unit of ben- zoyl-L-arginine ethyl ester-hydrolase activity was defined as the amount of enzyme which hydro- lyzed 1 #mol of substrate per min.
Assay for capillary permeability-increasing activity Using the method described by Miles and
Wilhelm [11], the dorsal hair on white rabbits weighing 2-2.5 kg was depilated by applying a 20% barium sulfide solution followed by a thor- ough cleansing with warm water. A 1% solution of Evans Blue was injected intravenously. The puri- fied enzyme (30/~g) was then injected intraderm- ally in the depilated area. The rabbit was killed after 60 min and the skin was removed for inspect- ion. By the procedure of Miles and Wilhelm an increase in capillary permeability allows the Evans Blue to move out of the capillaries into the sur- rounding tissues, causing a blue spot to appear.
Assay for kinin-releasing activity The assay for kinin-releasing activity was origi-
nally developed by Rocha e Silva et al. [1] using the ileum (small intestine), but was later modified by Erspamer and Erspamer [12] and Trautschold [13] who used the rat uterus. A piece of rat uterus 1-1.5 cm long was suspended in a bath containing De Jalon's solution (NaC1, 9 g; KC1, 0.4 g; CaClz, 0.06 g; NaHCO 3, 0.15 g; glucose, 1 g/liter). 1 ml of the purified high molecular weight kininogen (1 mg/ml) was incubated for 5 rain at 37"C with kinin-releasing enzyme. A linear dose-response curve was obtained with synthetic bradykinin by
503
plotting the height of contraction against the loga- rithm of bradykinin concentration.
Determination of molecular weight The molecular weight was determined by poly-
acrylamide gel electrophoresis using the method of Weber and Osborn [14]. Protein standards used were phopshorylase b (94000), bovine serum al- bumin (67000), ovalbumin (43000), carbonic anhydrase (30000), soybean trypsin inhibitor (20100) and lactalbumin (14400). Samples and standards were treated with 0.2% sodium dodecyl sulfate and reduced with 3% fl-mercaptoethanol for 3 min at 100 ° C and run on a polyacrylamide gel.
Amino acid composition The amino acid composition of the carboxya-
mide-methylated kinin-releasing enzyme was de- termined with a Hitachi high speed automatic analyzer, Model 835. Samples were hydrolyzed with constant boiling HC1 at l l0°C. A minimum of three analyses each from 24~ 48 and 72 h hydrolysates were used. Tryptophan content was determined by the method of Edelhoch [15].
4-Methylcoumaryl-7-amide- substrate hydro- lytic activity was assayed by the method of Morita et al. [16]. The substrate was dissolved in dimethyl sulfoxide and the solution was diluted to a final concentration of 0.1 mM, using 50 mM Tris-HC1 buffer (pH 8.0) containing 100 mM NaC1 and 10 mM CaC12. The reaction was started by the ad- dition of 2/~g of enzyme in a total volume of 2.5 ml and the fluorescence of 7-amino-4-methyl- coumarin was monitored using a Hitachi fluores- cence spectrophotometer, Model 650-60. Measure- ments were carried out by excitation at 380 nm with emission at 460 nm. The instrument was standardarized so that a 10 /tM solution of 7- amino-4-methylcoumarin in 0.1% dimethyl sulfox- ide gave a 1.0 relative fluorescence unit. The hy- drolysis value is expressed as /~mol per rain per mg protein.
Fibrinogenase activity Fibrinogenase activity was measured by the
504
method of Ouyang and Teng [17]. Kinin-releasing enzyme (4.2 #g) in 5 mM Tris-HC1 buffer (pH 7.5) containing 10 mM NaCI was mixed and in- cubated with the same amount of fibrinogen at 37°C for various time intervals. Subsequently, 0.5 ml of the above incubation mixture was withdrawn for assay of clottable fibrinogen. Meanwhile, 0.1 ml of the incubation solution was pipetted into a small test tube and 0.1 ml of the solvent contain- ing 10 M urea, 4% sodium dodecyl sulfate and 4% fl-mercaptoethanol was added. This solution was incubated at 37°C overnight and then electro- phoresed on a 7.5% sodium dodecyl sulfate-poly- acrylamide gel.
Isoelectric point The isoelectric point of kinin-releasing enzyme
was estimated by isoelectric focusing/polyacryl- amide gel electrophoresis. The ampholyte con- centration was 4% (w/v) with a pH range of 3.5-10.5. Protein standards used were acetylated cytochrome c, p I 10.6, 9.7, 8.3, 6.4, 4.9 and 4.1. Isoelectric focusing was carried out using a con- stant potential of 200 V at 4°C for 4 h.
Substrate specificity The B chain of oxidized insulin, 1.0 mg in 1.0
ml 0.01 M ammonium acetate buffer (pH 9.0), was incubated with 84.0 #g of kinin-releasing enzyme at 37°C for 2 h. The digestion was stopped by heating at 100°C for 2 min. The sample was injected into a Develosil 300 ODS-7 column equi- librated with 0.1% trifluoroacetic acid. The col- umn was eluted with a gradient from 0 to 100% acetonitrile in 20 min at a flow rate of 1 .0ml /min using a Gilson HPLC System. Peaks were pooled and evaporated to dryness. Fractions were then hydrolyzed in 6 M HC1 for 24 h at 110°C and analyzed for amino acids.
Other methods Proteinase activity determination was carried
out according to the method of Murata et al. [18] using casein as the substrate. Alkaline phos- phatase activities were determined by the method of Suzuki and Iwanaga [19] and Suzuki et al. [20]. High molecular weight kininogen was purified according to the method of Yano et al. [21].
Results
Isolation procedure 500 mg of crude venom from B. arietans was
dissolved at a concentration of 20% in 0.04 M Tris-HC1 buffer (pH 7.2) containing 10 mM NaC1 and 5 mM CaC12 and the insoluble material was removed by centrifugation (2000 × g) for 10 min at 4 ° C. The supematant was applied to a Seph- adex G-100 column (Fig. 1, 1st step). Benzoyl-L- arginine ethyl ester hydrolytic activity was found in fractions 2 and 3. Fraction 3 (53.0 mg) was further fractionated as follows. Fraction 3 was equilibrated by dialysis with 10 mM Tris-HC1 buffer (pH 7.2) containing 5 mM CaC12 and loaded on a DEAE-cellulose column. The column was eluted with a 600 ml linear gradient from 0 to 0.3 M NaC1 in buffer (Fig. 1, 2nd step). Benzoyl- L-arginine ethyl ester hydrolytic activity was found in fractions 3 and 4. Fraction 3 from the DEAE- cellulose chromatography was dialyzed against 10 mM Tris-HC1 buffer (pH 7.2) containing 10 mM NaC1 and 5 mM CaC12. The dialysis residue con- taining 11.4 mg of protein was subjected to col- umn chromatography on DEAE-cellulose. The column was eluted with a 600-ml linear gradient from 0.01 to 0.5 M NaC1 in buffer (Fig. 1, 3rd step). The final preparation demonstrated ben- zoyl-L-arginine ethyl ester hydrolytic (48.8 uni t s / mg), tosyl-L-arginine methyl ester hydrolytic (16.8 uni ts /mg) and kinin-releasing activities but no caseinolytic, clotting, acetyl-L-tyrosine ethyl ester hydrolytic, tosyl-L-lysine methyl ester hydrolytic, benzoyl-L-arginine amide hydrolytic, tosyl-L- arginine amide hydrolytic, 5'-nucleotidase, phos- phodiesterase or ATPase activities were observed. The sample was not lethal to mice at a concentra- tion of 2.61 ~tg/g. The extent of purification and the yield at each step are summarized in Table I.
Homogeneity As shown in Fig. 1, kinin-releasing enzyme is
electrophoretically homogeneous in Tris-glycinate and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of this en- zyme was found to be 6.5. Immunodiffusion shows one band, this being further confirmation of the homogeneity of kinin-releasing enzyme.
Properties The molecular mass of kinin-releasing e n z y m e
was determined to be 45 k D a by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid compos i t ion of the reduced and
carboxymethylated purified preparation is shown in Table II. Based on a molecular mass of 45 kDa, kinin-releasing enzyme consists of 407 amino acid residues: Kinin-releasing activity of the purified enzyme was shown to be 3.9/~g/mg enzyme per 5
TABLE I
SUMMARY OF PURIFICATION OF KININ-RELEASING ENZYME
Step Protein Yield Benzoyl-L-arginine (rag) (%) ethyl ester hydrolytic
Asp 45 Thr ~ 22 Ser ~ 29 Glu 28 Gly 31 Ala 29 Cys/2 21 Val 20 Met 10 Ile 24 Leu 28 Tyr 16 Phe 12 Lys 22 His 16 Arg 12 Pro 37 Trp b 5
Total 407
a The hydrolysis values for threonine and serine were extrapo- lated to time zero.
b Tryptophan content was determined by the spectroscopic method of Edelhoch [15].
A~
B~
Y
MW k i!i!!U
94 ~0
Incubation Time (nun)
5 i0 20 30 60 90 120 180
Fig. 2. Results of time-dependent digestion of fibrinogen by kinin-releasing enzyme, as shown by sodium dodecyl sulfate- polyacrylamide gel electrophoresis.
min . Benzoy l -L-a rg in ine e thyl es ter hyd ro ly t i c and
k in in - r e l ea s ing ac t iv i t ies were s tab le to hea t t reat -
men t , r e t a in ing a p p r o x i m a t e l y 50.8% of thei r ac-
t iv i ty a f te r hea t ing at 9 6 ° C for l 0 rain, p H 7.2.
T h e ef fec ts of va r ious reagen ts (10 min, 3 7 ° C ) on
the benzoy l -L-a rg in ine e thyl es ter hydro ly t i c and
k in in - r e l ea s ing ac t iv i t ies (Ma te r i a l s and M e t h o d s )
o f the f inal p r e p a r a t i o n (28.2 / ~ g / m l ) were
e x a m i n e d . Benzoy l -L-a rg in ine e thyl es ter hyd ro -
lyt ic and k in in - re l eas ing act iv i t ies were inh ib i t ed
b y benzamid ine hydrochlor ide , d i th iothre i to l , L- cys te ine or d i i sop ropy l f luorophospha te , bu t unaf- fected b y soybean t ryps in inh ib i to r or e thylene- d iamine te t raace t i c acid (Table III) . F o r the de- t e rmina t ion of c a rbohyd ra t e content , the an- t h rone -H2SO 4 reac t ion of Koeh le r [22] was used.
507
The results showed c a rbohyd ra t e account ing for a pp rox ima te ly 3% of the total weight, ind ica t ing tha t k in in-re leas ing enzyme is a g lycoprote in . W h e n the pur i f ied enzyme was in jec ted in t r ade rm- ally, an increase in capi l la ry pe rmeab i l i ty was observed.
a)
E . c -
tO
D
B
A
C
E
~ 'L I , ,,],, I
' 5 10 15
Time (rain)
b) 03. °3"20 I 5 I0 15 P h e - V a ~ - A s n - G ~ n - H i s - L e u ~ C y s - G l y - S e r - H i s [ L e u - V a l - G l u ~ A l a ~ L e u - T y r - L e u - V a l - C y s - G l y - G ~ u - A r g - G l y - P h e - P h e - T y r - T h r - ~ r ~ - L y s - A ~ a
, i l J i , A ; _~
: D :B
Fig. 3. (a) HPLC peptide map of kinin-releasing enzyme. Digestion of the B chain of oxidized insulin by kin.in-releasing enzyme (see text for conditions) as analyzed by HPLC on a Develosil 300 ODS-7 column (0.4)<25 cm). Buffer system used was: A, 0.1~ trifluoroacetic acid in H20; B, 0.1% trifluoroacetic acid in CNCH 3. The column was eluted with a linear gradient from 0 to 100% acetonitrile in 20 rain at a flow rate of 1.0 ml/min. Peak F is kinin-releasing enzyme. (b) Cleavage points of the B chain of oxidized insulin,by kinin-releasing enzyme and assignment of peptide fragment.
508
p H stabifity The enzyme at a concentration of 28.2 /~g/ml
was incubated at 37°C for 60 min at various pH values and the enzymatic activity on benzoyl-L- arginine ethyl ester was determined. The Britton- Robinson buffer [23] at a final concentration of 0.02 M was used to dissolve samples. The enzyme was stable in the pH range 3-12.
Determination of Michaelis constant (Kin) and in- hibition constant (Ki)
The Michaelis constant (Km) for benzoyl-L- arginine ethyl ester of kinin-releasing enzyme at pH 8.5 was 5 .10 -2 M. The inhibition constant (Ki) of benzamidine hydrochloride (10 raM) for this enzyme was determined by measuring the initial rate of hydrolysis of benzoyl-g-arginine ethyl ester at pH 8.5. The inhibition of benzamidine hydrochloride was competitive and an inhibition constant of 1 - 10-2 M was obtained.
Fibrinogenase activity As shown by the control run on sodium dode-
cyl sulfate-polyacrylamide gel electrophoresis, re- duced fibrinogen was separated into Aa, B/3 and ~, chains (Fig. 2). When fibrinogen was incubated with kinin-releasing enzyme (4.2 ttg/ml), Aa chain was most sensitive to proteolytic digestion. Pro- longed incubation caused the hydrolysis of the B/3 chain also; however, the "/ chain remained unaf- fected. Degraded products can be seen in the lower region of the electrophoretic gels in Fig. 2.
Proteolytic specificity The B chain of oxidized insulin has been used
as a substrate for proteolytic assays by a number of investigators [24-27]. This substrate was also used for the proteolytic specificity assay of kinin- releasing enzyme. Digested fragments were sep- arated by HPLC (Fig. 3a). A total of three peptide bonds were cleaved by this enzyme; they are Leu 6- Cys 7, Hisl°-Leu n, and Alaa4-Leu 15. Their cleavage points are shown in Fig. 3b. Z-Phe-Arg 4-methyl- coumaryl-7-amide and Pro-Phe-Arg 4-methyl- coumaryl-7-amide were synthesized as possible substrates for plasma, pancreatic and urinary kal- likrein. The enzyme is active on these kallikrein substrates. Purified kinin-releasing enzyme from B. arietans venom hydrolyzed Pro-Phe-Arg 4-
methylcoumaryl-7-amide (1638.'9 // 'mol/min per mg) more readily than Z-Phe-Arg 4-methyl- coumaryl-7-amide (305.6/~mol/min per rag).
Discussion
Kinin-releasing enzyme from B. arietans ve- nom migrates as a single band on both ordinary and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by isoelectric focusing, indi- cating the homogeneity of the purified prepara- tion. It is a glycoprotein composed of approxi- mately 3% carbohydrate and possessing an isoelec- tric point of 6.5. Recently a number of kinin-re- leasing enzyme have been isolated from numerous snake venoms [4-9]. These enzymes have molecu- lar masses of around 30 kDa. The value of 45 kDa reported in this paper is not in agreement with the values reported for arginine-ester hydrolase from other venom sources. It has been known for some time that bradykinin-releasing enzyme also hydro- lyzes synthetic arginine ester [3]. Sato et al. [7] obtained three fractions from snake venom which had arginine esterase activity. The first fraction showed only bradykinin-releasing activity, the sec- ond caused only an increase in capillary permea- bility and the third showed only blood-clotting activity. This investigation indicates that the puri- fied arginine esterase from B. arietans has kinin- releasing activity and capillary-permeability-in- creasing activity. The Aa chain of fibrinogen was cleaved first and the Bfl chain was cleaved later. The purified enzyme shows no fibrinogen-clotting, or lethal activities. The purified enzyme is highly specific for arginine esters, and only benzoyl-L- arginine ethyl ester (48.8 units/mg) and tosyl-L- arginine methyl ester (16.8 units/mg) are hydro- lyzed for synthetic substrates. Kinin-releasing en- zyme, free of caseinolytic activity and inhibitable by diisopropyl fluorophosphate and dithiothreitol, has been prepared from the venom of B. arietans, suggesting that the serine and disulfide bonds are essential for biological activity. The purified en- zyme did hydrolyze purified high molecular weight kininogen, fibrinogen and the B chain of oxidized insulin but no caseinolytic activity was found. Apparently there are diverse types of proteolytic enzymes in snake venom. More extensive research is required to isolate each and identify their chem-
ical and biological properties in order to fully understand the function of proteolytic enzymes in snake venoms.
Acknowledgement
This research was supported by Scientific Re- search Fund of the Ministry of Education, Japan, No. 773-6023-60571063 (H.S.).
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