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Chemical studies on protease A of Bitis arietans (puff adder) venom
Toadcon, Vol. 24, No . 3, pp . 247-257, 1986 . Printed in Great Britain .
0041-0101/96$3 .00+ .00 © 1986 Pergamon Pros Ltd.
CHEMICAL STUDIES ON PROTEASE A OF BITIS ARIETANS (PUFF ADDER) VENOM DANIEL J. STRYDOM,* FRANCOIS J. JOUBERTt and NEVILLE L . HOWARD* National Chemical Research Laboratory, Council for Scientific and Industrial Research, P O Box 395, Pretoria, 0001, Republic of South Africa (Accepted for publication 8 August 1985)
D.
J . STRYDom, F. J . JouBERT and N. L. HOWARD . Chemical studies on protease A of Bitis arietans (puff adder) venom. Toxicon 24, 247 - 257, 1986 . - Protease Aof Bitis arietans venom is
probably a metalloprotease, since it is inhibited by o-phenanthroline and contains 0.77 moles of zinc per mole protein. The enzyme comprises 213 amino acids, including 9 methionine residues and one free sulphydryl group. It contains one polypeptide chain, which is terminated at the carboxyl end by serine . The amino terminal sequence of protease A is : Arg-Ser-Ser-Asp-Pro-AsnLys-Tyr-Phe-Asn-Val-Ile-Val-Val-Val-Asp-Asn-Arg-Met- Val-Asn-Tyr-Tyr-Lys-Gly- Glu -LeuAsn-Lys-Ile-Thr- . Despite diffficulties with 'insoluble peptide core' formation, a number of peptides were purified from peptic and Cryptic digests of S-derivatized protease A.
INTRODUCTION
PROTEASES are among the most ubiquitous
enzymes found in living organisms. They play a fundamental role in many biological processes, ranging from digestion of food through metabolic turnover at the cellular level to control of many processes through proteolytic activation or deactivation of enzymes, hormones and other molecular messengers . Proteases are classified according to certain common features of their chemistry and mechanism of action . The currently accepted classes are the serine proteases, acid proteases, sulphydryl proteases, metalloproteases (WALSH, 1975) and low molecular mass proteases (ZWILLING and DORSAM, 1981). Among these, the metalloproteases are relatively unknown at the protein structural level . Only thermolysin, a bacterial metalloprotease, has been studied extensively and sequenced (LATT et al ., 1969; TITANI et al., 1972). Very little is known about the structures of vertebrate metalloproteases, or even about any eucaryotic metalloproteases, although such activities are well known (e.g. BANDA and WERB, 1981 ; SELLER et al ., 1978; CAWSTON and MURPHY, 1981) and play an important role in metabolism . The main problem in studying these proteins seems to be their low levels in tissues and the concomittant problems in procuring adequate quantities of such proteins . Snake venoms, which are rich sources of many enzymes and proteins, do contain appreciable quantities of proteases (MEBS, 1968, and references therein) . A growing, seemingly disparate, list of proteases, blood protein inactivators, fibrinogenases, hemorrhagins, etc., have been isolated from crotalid and viperid venoms. They have the *Present address: Center for Biochemical and Biophysical Sciences and Medicine, Brigham and Women's Hospital, Boston, MA 02115, U.S.A. "To whom reprint requests should be addressed. *Present address: Wheat Board, P O Box 908, Pretoria, Republic of South Africa. 247
248
D. J. STRYDOM et al.
properties in common of possible metalloproteinase character and molecular weights near 24,000 (KRESS and PAROSKI, 1978; ZWILLING and PFLEIDERER, 1967; PFLEIDERER and SUMYK, 1961 ; BJARNASON and TU, 1978 ; WAGNER et al., 1968; SPIEKERMANN et al., 1973; TAKAHASHI and OHSAKA, 1970; OUYANG et al., 1977 ; VAN DER WALT and JOUBERT, 1971 ; NIKAI et al., 1977 ; MORAN and GEREN, 1981 ; LAWRENCE and MORRIS, 1981 ; XU et al., 1981 ; KURECKI and LASKOWSKI, 1978 ; PANDYA and BUDZYNSKI, 1984). The size of these proteins make them readily amenable to sequence studies, which could enable a comparative study of the metalloprotein characteristics of these vertebrate proteases with bacterial metalloproteases such as thermolysin (LATT et al., 1969; TITANI et al., 1972). One of the proteases from the venom of the puff adder, Bitis arietans, named protease A, is easily purified in large quantities (VAN DER WALT and JOUBERT, 1971). This paper describes an investigation into the chemical structure of protease A. MATERIAL AND METHODS Materials Bitis arietans venom was obtained with the cooperation of D. J. Muller, Professional Snake Catcher (Pty) Ltd., 99 Cedar Road, Northcliff, Johannesburg, Republic of South Africa . The venom was collected in containers held at - 80°C and then lyophilized. Trypsin was supplied by Miles Laboratories (Republic of South Africa) as a twice crystallized diphenylcarbamyl chloride-treated, salt-free preparation and pepsin (twice crystallized) was supplied by Worthington Biochemical Corp . (U .S.A .). Diethylaminoethyl cellulose (DEAEcellulose) was a microgranular preparation from Whatman (U .K .) and Sephadex G-50 (fine) was obtained from Pharmacia (Sweden) . Chromatography Sephadex G-50 and DEAE-cellulose columns were prepared as recommended by the manufacturers and the eluates were monitored at 230 nm or 280 nm with a Beckman spectophotometer . Pur(/ication ofprotease A Protease A was prepared from lyophilized Bitis arietans venom according to the procedure of VAN DER WALT and JouBERT (1971), with a few minor alterations. Concentration of protein between chromatography steps was effected by precipitation with ammonium sulfate, followed by dialysis against the appropriate starting buffer . The elution buffer in the final purification on Sephadex G-50 (instead of Sephadex G-75) was0.2 M ammonium acetate, 0.005 M calcium chloride at pH 5.6 . The intial separation and rechromatography steps were done with ammonium acetate instead of sodium acetate buffer, with a slight change in elution pattern. Assay ofprotease A activity Caseinase activity was measured as described by VAN DER WALT and JOUBERT (1971) . Activity against globin was measured by substituting globin for casein in the above assay. The globin was prepared from bovine haemoglobin by themethod of FANELLI et al. (1958) . The resultant globin wasdialyzed against o-phenanthroline (0.001 M) and then repeatedly against water before lyophilization . Assessment of effectors was done by incubating 100 M1 of a solution of protease A in an appropriate buffer with 10 pl of a 10 mM solution of effector for 15 min, and then assaying for residual proteinase activity. Reduction and S-carboxymethylation Reduction of protease A was performed in 1 M Tris, pH 8.6, containing 6 M guanidinium hydrochloride and 0.1 M EDTA, using a 10-fold molar excess of dithiothreitol over cystine-content of the enzyme. Reduced enzyme was S-carboxymethylated with iodoac etic acid, in quantities equimolar to the free sulfhydryl groups in the reaction mixture. Both reduction and alkylation were carried out under anaerobic conditions . The final product was dialyzed against distilled water and lyophilized. S-suftolysis of protease A Protease A was S-sulfitolysed according to the procedure of PECHERE et al. (1958) . Lyophilized protease A (430 mg) was dissolved in 9.2 ml of 0.005 M HCI and diluted with 15 ml 0.001 M HCI. Urea (29.5 g) was added in small portions, with the pH being kept at 3 with 1 N HCI. Sodium sulfite (6 ml, 1.5 M) was added and the pH adjusted to 10 .2 with ammonia, followed by addition of cupric nitrate solution (1 .2 ml, 2 M), the pH being kept at 10.2 . The volume was adjusted to 60 ml and after 1 hr, dialysed with a DOW-beaker dialyzer with hollow
Protease A of Bitis arietans Venom
249
cellulose fibres against 30 liters of 0.01 M ammonium hydrogencarbonate solution . This solution was used directly for a tryptic digest .
Digestion with proteolytic enzymes
S-sulfo protease A (430 mg) in 100 m10.01 M NH,HCO, was digested with two lots of 4.3 mg of trypsin for 4 hr at room temperature. The digestion was stopped by acidification to pH 3 with acetic acid and the digest was lyophilized. Reduced and S-carboxymethylated protease A (420 mg) was dissolved in 2.1 ml formic acid (984`) and diluted with 37 .9 ml water . Pepsin (4 .2 mg) was added and the mixture incubated at 37*C for 2 hr . The mixture was neutralized and lyophilized.
Fractionation ofpeptides
The peptic digest (soluble portion) was fractionated by gel filtration on Sephadex G-50 with 1 % acetic acid as eluant. The fractions were further purified by chromatography on DEAE-cellulose columns using a linear gradient from 0.05 to 0.5 M NH,HCO, . The tryptic digest (soluble portion) of S-sulfocysteinyl protease A was fractionated on DEAE-cellulose columns using a linear gradient from 0.01 to 0.6 M NH,HCO,. Peptide fractions were tested for purity and further purified, using paper chromatography or paper electrophoresis as detailed previously (BOTEs et al., 1971).
Chemical methods
Amino acid analyses were performed with an automatic Beckman amino acid analyser . Samples were hydrolysed with 6 N HCI for 24 hr in sealed tubes and phenol was added to prevent the destruction of tyrosine (SANGER and THOMPSON, 1963). For the determination of tryptophan the samples were hydrolysed with 3 M p-toluene sulphonic acid (Llu and CHANG, 1971). Free sulphydryl groups, in 6 M guanidinium chloride, were assayed in intact protease A samples according to the method of ELLMAN (1959) . Metal analysis was done by atomic absorption on a Varian Techtron AA-5 spectrophotometer .
Carboxy terminal analyses
The C-terminal amino acid of protease A was determined by Amberlite CG-50 catalysed hydrazinolysis as described by BRAUN and SCHROEDER (1967) .
Sequence determination
The N-terminal sequence of reduced and S-carboxymethylated protease A was determined with the Beckman sequencer using the quadrol programme (Beckmann No . 12294 mod) . A large peptide was also sequenced with the sequencer using the peptide program (No. 111374) with N,N-dimethylamino propyne (BRAUNITZER and SCHRANK, 1970) buffer. For small peptides the semi-micro Edman degradation procedure of PEDERSON et al. (1972) was used for manual sequence studies. The phenylthiohydantoin derivatives of amino acids were identified by gas chromatography on an SP-400 column (PIsANo and BRONZERT, 1969) and thin layer chromatography on silica gel (BRENNER et al., 1961 ; CHERBULIEz et al ., 1963). In many cases back hydrolysis of the phenylthiohydantoin to free amino acids with HI (SMrrHiEs et al., 1971) was used as an aid in identification. RESULTS
Inhibition of protease A activity
The effect on caseinase activity at pH 8 of pre-incubation at pH 5.5 of protease A with 0.9 mM concentrations of various metal ions was tested and the results are summarized TABLE 1 . THE EFFECT OF VARIOUS METAL IONS ON CASEINASE ACTIVITY OF PROTEASE A Ion Af. Ba 2" Cd2* Cs* Co'. Cu" Fe'* Hg
Residual activity (%)
Ion
Residual activity (4ti)
96 95 94 95 92 100 97 69
Mg.. Mn'* Ni2* Pb» Sr'* Zn'* Nd3* Water
95 97 100 97 99 98 104 100
250
D . J . STRYDOM et ai.
in Table 1 . Apart from Hg=*, which inhibited the enzyme by 31 %, the effects were negligible . Under the same conditions, o-phenanthroline caused an inhibition of 57% of the activity . Using globin at pH 8 as substrate, protease A activity was tested after 15 min pre-incubation at pH 5.5 or 8 with 10-' M o-phenanthroline. Inhibition was found with o-phenanthroline at both pH values to the extent of 28 and 40% inhibition at pH 5 .5 and 8, respectively, whereas a control experiment with m-phenanthroline did not show any inhibition . Chemical and amino terminal studies
Atomic absorption analysis showed that protease A contains 0.77 moles of Zn2 * per mole (based on a molecular weight of 24,800). Table 2 reports the average results of eleven independent analyses of amino acid composition of protease A. It contains 213 amino acids, including one free sulphydryl group. The carboxyl terminal amino acid for protease A was serine . The N-terminal amino acid sequence of the reduced and Scarboxymethylated protease A is given in Table 3 . Peptic and tryptic peptides ofprotease A
The lyophilized peptic digest was extracted with 5% acetic acid. The acetic acid soluble peptides were dried and extracted with 0.05 M NH,HCO,. These soluble peptides were fractionated by gel filtration on Sephadex G-50 in I% acetic acid (Fig. 1) . Fraction S1 was dried, dissolved in 0.05 M NH4HCO, and further fractionated on a DEAE-cellulose column . A linear gradient of ammonium bicarbonate eluted 18 fractions (P1-P18) (Fig . 2). Fractions P1, P2, P3, P4, P5, P6, P9 and PI I were fractionated further on Sephadex G-25 . The major fractions of P1, P2, P3, P4, P5, P6 and Pll, as well as fraction P10, TABLE 2. AMINO ACID COMPOSITION OF PROTEASE A (GIVEN AS MOLE OF RESIDUE PER MOLE PROTEIN, BASED ON A MOLECULAR WEIGHT OF 24,800)
Amino acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine -Cystine* Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophant Free sulphydryl* Total
Analysis 32 .24 (32) 9 .08 (9) 13 .01 (13) 15 .68 (16) 8 .18 (8) 10 .62 (11) 12 .08 (12) 7 .74 (8) 14 .02 (14) 8 .53 (9) 15 .12 (15) 9 .09 (9) 12 .17 (12) 9 .25 (9) 9 .34 (9) 8 .93 (9) 12.01 (12) 5 .40 (5) 0.76 (1) 213
*Determined as 3-carboxymethyl cysteine . DDteermined as described by Llu and CHANG (1971) . *Determined as described by ELLMAN (1959) .
Protease A of Bibs arierans Venom
251
TABLE 3 . N-TERMINAL SEQUENCE OF REDUCED AND S-CARBOXYMEfHYLATED PROTEASE SEQUENCER (280 lanole was loaded)
Step 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
GC analysis*
Normal
Silylated
Cys/Ser Cys/Ser
Cys/Ser Cys/Ser Asp Pro Asn
Pro Tyr Phe Asp Val Ile/Leu Val Val Val Asn
Tyr Phe Asp Val Ile Val Val Val Asp Asn
Met Val
Met Val
Others Identification Arg Ser Ser Asp Pro Asn Lys Tyr Phe Asn Val Ile Val Val Val Asp Asn Arg Met Val Asn
Step
Arg Ser Ser Asp Pro Asn Lys Tyr Phe Asn Val Ile Val Val Val Asp Asn Arg Met Val Asn
22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41
A USING THE
AUTOMATIC
GC analysis' Normal Silylated Other' Identification Tyr
Tyr Tyr
Thr
Gly Glu Leu Leu Lys Gln Thr
Tyr Tyr Lys Gly Glu Leu Asn Lys Ile Thr
Tyr Tyr Lys Gly Glu Leu Asn Lys Ile Thr
Gly
Val
Val
Val
Val
Tyr Phe
Tyr Phe
Gln
Ile/Leu Ile/Leu
Ile Ile
Gln Tyr Phe Asn Ile Leu
Ile/Leu Ile/Leu
Phe Asn Ile Leu
*Gas chromatography analysis . 'Analysis by thin layer chromatography, back hydrolysis to amino acids, phenanthrenequinone colour reaction for arginine, Pauli-reaction for histidine. *Not identified . The quadrol programme (Beckman No . 122974 mod) was used . The yield of proline at step 5 was 50%. The repetitive yield was 91 .5°10 .
0.4
04l
n
0 .5 ,
0.75 'L ELUATE
VOLUME
I
1
1 .0
(liter)
FIG . 1 . GEL FILTRATION OF THE PEPTIC DIGEST (SOLUBLE PORTION) OF PROTEASE A. Reduced and S-carboxymethylated protease A (420 mg) was digested with pepsin and the soluble portion loaded on a Sephadex G-SO column (3 x 1 .9 x 150 cm) and eluted with 1 % acetic acid . The flow rate was 20 ml/hr, the column temperature was 20°C and the elutee was monitored at 280 nm .
252
D. J . STRYDOM et al.
P3
0 M N 4
P9 0.5 P7
PIO
1P Il
P9 j
i
PI6
P15
P 17
P2 j Pj3 PI4
Pis
1 û ELUATE
VOLUME
(liter)
FIG. 2. CHROMATOGRAPHY OF FRACTION S, ON DEAE-CELLULOSE . Peak S, (Fig . 1) was lyophilized, dissolved in 0.05 M NH,HCO, and loaded on a DEAE-cellulose column (0 .9 x 150 cm) and eluted by a linear gradient of 0.05 -0.5 M NH,HCO, over 2 litres at a flow rate of 50 ml/hr. The column temperature was 20°C and the eluate was monitored at 230 nm .
TABLE 4. AMINOACID COMPOSITION OF THE PEPTIC PEPTIDES OF REDUCED AND S-CARRO:
P1 .1 .1
P1 .1 .2
P1 .1 .3
P2.1
Asp
1.35(1) 2.04(2) 2.06(2)
3.16(3) 0.40 1.01(1) 2.13(2)
3.10(3)
Glu
1.34(1) 1.93(2) 1.51(2)
0.92(1) 2.94(3)
Gly
1.05(1)
1.07(1)
CYs(CM) Thr Ser Pro
Ala Val Met Ile
Leu Tyr
Phe Lys His
Arg Trp
1.00(1)
0.88(1)
1.05(1) 1.00(1) 1.70(2) 1.28(1) 2.82(3) 0.90(1)
0.97(1) 0.38 2.95(3) 1.21(1) 1.13(1) 2.13(2) 2.49(2) 0.81(1) 0.94(1) 2 .80(3)
16 Total Yield (%) 1 .7
2.6
12
3.4
16
P2.3
P3 .2 .2
P4.1 .3 1 .25(2) 3.18(3) 1 .04(1) 1 .90(2) 1 .08(1) 2.05(2)
0.37 0.20 0.13
3.05(3) 0.99(1)
1 .97(2)
1.31(1) 0.2 0.34 0.21
3.02(3)
1.92(2) 1.06(1) 0.94(1)
0.50(1)
0.95(1)
1.00(1) 1 .07(1) 1 .01(1)
0.99(1)
1 .77(2)
1 .94(2) 0.15 1.00(1) 2.91(3) 1.28(1) (1)
57
19
33
6
4.5
12
P5 .4 .1
P5 .4 .2
P5 .4.3
0.92(1)
0.96(1)
0.45(1)
0.94(1)
P4 .2 .1
P6 .3 .1 0.2 0.91(1)
0.94(1) 1 .06(1)
2.05(2) 0.95(1)
1 .11(1)
1.14(1) 0.86(1)
1.1(1) 0.86(1) 1.00(1) 1.20(1) 0.28 0.92(1)
0.95(1) 0.99(1) 10
15
1.9
3
1.4
2
0.9
3
3.0
2
4.3
6
253
Protease A of Bitis arietans Venom
0.10 T8
0 N a
1
Q05
T18 T20T21
r T. 1 T2
TI6
TI5
v
v
n
~
~
i
T19
T24
T22
~
T23
i
w
2
4
ELUATE FIG .
3. CHROMATOGRAPHY
VOLUME
(liter)
THE TRYPTIC DIGEST (SOLUBLE PORTION) OF PROTEASE A ON DEAE-CELLULOSE. S-sulfocysteinyl protease A (430 mg) was digested with trypsin and the soluble portion loaded on a DEAE-cellulose column (1 .9 x 150 cm) and eluted by a linear gradient of 0.01-0.6 M NH 4HCO, over 8 litres at a flow rate of 50 ml/hr . The column temperature was 20°C and the eluate was monitored at 230 nm . OF
hMETHYLATED PROTEASE A (SOLUBLE PORTION) (GIVEN
P6 .3 .2
P6 .3 .3
0.26 1.08(1)
3 .34(3)
0.99(1)
0.43
2.45(3) 0.14
0.4 0.22
0.57(1) 0 .91(1)
1 .80(2) 0.87(1) 0.24
1.01(1) 0.2
6.8
P8 .1
1 .04(1) 1 .10(1)
1 .25(1) 0.22 0.13
0.89(1)
1 .5
8
6.5
5
MOLE
OF
RESIDUE PER MOLE PEPTIDE)
P9 .1 .1
P9.1 .2
P9.1 .3
0.87(1) 1 .10(1)
0.97(1) 1 .07(1) 0.92(1) 0.98(1)
0.84(1) 1 .31(1) 1 .07(1) 1 .06(1)
1 .02(1) 1 .00(1) 1.06(1) 1.94(2) 0.97(1)
1.12(l) 1.04(1) 1.96(2) 1.85(2) 0.75(1) 0.93(1)
0.98(1) 0.99(1) 1 .11(1) 1 .09(1) 0.52(1)
1 .06(1) 0.90(1)
0.88(1) 8
AS
0.86(l) 0.97(1) 0.88(1)
2.1
9
1.03(1) 1.18(1)
2.9
12
0.98(1) 0.90(1)
1 .7
15
P9.1 .4 1 .08(1) 1 .89(2) 2.97(3) 0.92(1) 1.11(1) 1.00(1) 0.97(1)
P9.2 0.85(1)
1 .03(1) 1.97(2) 0.64(1) 1 .00(1)
P10.2 1 .12(1) 1 .96(2) 3.02(3) 1 .03(1) 1.03(1) 0.95(2) 1.03(1)
P11.1 .1
P15
0.98(1)
0.92(1) 2.44(2) 1 .04(1) 1 .04(1) 1 .27(l)
1 .01(1)
0.44
0.72(1) 2.01(2)
1 .87(2) 1.04(1)
0.99(1) 0.93(1)
4.7
11
0.18
14
0.85(1)
6
7.5
12
0.96(l) (1) 3.1
5
5
11
D . J . STRYDOM
25 4
et aL
A
TABLE 5 . AMINO ACID COMPOSITION OF THE TRYPTIC PEPTIDES OF S-SULFOCYSTEINYL PROTEASE PORTION) (GIVEN AS MOLE OF RESIDUE PER MOLE PEPTIDE) T2 .4
Cys(CM) Asp Thr Ser Glu Pro Gly Ala Val Met lie Leu Tyr Phe Lys His Arg Trp
Total Yield (%)
1 .54(2)
T2 .7
T7 .2
1 .26(1)
1 .19(1)
1 .7(1)
2 .07(2)
1 .33(1) 1 .02(1)
0.5 1 .18(1) 0.16
1 .09(1)
0.32
1 .09(1) 0.15
0.31 0.76(1)
1.00(1)
25 .7
4
1 .7(2)
1 .83(2) 0.91(1)
0 .7
4
0.27 0.24 1 .86(2)
7 .2
7
T7 .4
T7 .6
0 .84(1)
1 .09(1)
1 .00(1) 0 .13
4 .0
5
10 .4
4
T8 .1 T9 .1
T10 .1
T11 .9
T17 .1
T21 .2
0 .26
1 .06(1)
0 .62(1) 5 .45(5)
2 .29(3) 4 .86(5)
0 .17 1 .17(1)
0 .17 1 .00(1)
3 .11(3)
1 .38(1) 3 .16(3)
0.13 1 .02(1) 0 .15
0 .34 3 .91(4) 1 .02(1) 0 .64(1) 1 .00(1) 0 .12
0 .32 0 .26 0 .12 0 .14
1 .66(2)
0 .20
1 .16(1) 0 .96(1)
0 .81(1) 0 .14
0.54(2) 1 .12(1) 1 .10(1) 0.97(1) 1 .05(1) 0.97(1) 0.17 0.24
12 12 .1
T8 .6
(SOLUBLE
1 .04(1)
0 .96(1)
8 .9
2
3 .1
9
3 .25(3) 0 .14 0 .93(1) 1 .07(1) 0 .94(1)
0 .12
0 .94(1) 0 .26
1 .85(2) 0 .96(1) 0 .90(1) 1 .00(1)
4 .25(4) 3 .50(4) 3 .46(4) 1 .57(2) 0 .96(1) 0 .80(1) 0 .91(1) 3 .06(3)
(1)
2 .5
3
4 .0
21
TABLE 6 . AMINO ACID SEQUENCES OF THE PEPTIC (P) AND TRYPTIC (T) PEPTIDES OF PROTEASE
2 .2
32
A
Peptide
Sequence results P2 .1 Ala- Glu-Trp-Arg- Glu-Arg-Asp-Leu-Leu-Ser-Arg-Lys-(His,Asx,Lys,Asx,Leu,Ala,Glu) P2 .3 Tyr-Ile-Arg-Pro-(Ile,Leu) P4 .1 .3 Ile-Ser-Thr-His-Asn-Pro-Gln-Cys-Phe-Cys-(Asx,,Ser,Pro,Arg) Val-Met p5 .4 .3 Ile-Ala-Tyr-Val-Gly-Thr P6 .3 .1 P6 .3 .2 Ile-(Ser)-Asn-(Pro,,Met,Tyr) P8 .1 Tyr- Lys-Gly-(GIu,Leu) P9 .1 .1 Met-Cys-Asp-Pro-Lys-His-(Ser, Val,Ala) Val -Gly-Thr-Met-Cys-Asp-Pro-(Lys,His,Ser, Va1,Ala) P9 .1 .2 P9 .1 .3 Ile-Ala-Tyr-Val-Gly-Thr-Met-(Cys)-Asp-(Pro,Lys,His,Ser,Val,Ala) P10 .2 Val-Thr-Gin-Ser-Ala-Ser-Asp-Thr-(Ser,Leu,His,Phe) P11 .1 .1 Val-Gly-Ile-(GIu,Ile) P15 Ile-Glu-Ile-(Trp,Ser,Asx,Arg,Asx,Leu,Cys,Thr) T2 .7 Lys-His-(Asx,Lys) T7 .2 Glu-Arg-Asp-Leu-(Leu,Ser,Arg) T7 .4 Gly- Glu-Leu-(Asn,Lys) T7 .6 Asp-Asn-Pro-Ser The manual semi-micro Edman degradation procedure of PETERSON et al. (1972) was used . 'The sequencer was used with NN-dimethylamino propyne buffer .
were chromatographed on paper, using BPAW as the solvent system, while the major fraction of P9, as well as fractions P7, P8 and P10, were separated by paper electrophoresis at pH 1 .9. The soluble portion of the tryptic digest of S-sulfocysteinyl protease A was fractionated on DEAE-cellulose using a linear gradient of ammonium bicarbonate and yielded 24
Protease A of Bitis arietans Venom
25 5
fractions (Fig . 3). Fractions T2, T7, T8, T9, T10, T11, T17 and T22 were fractionated further by paper chromatography using solvent system BPAW, while pH 1 .9 electrophoresis served to fractionate fraction 21 . Tables 4 and S record the amino acid compositions of some peptic and tryptic peptides of protease A. Sequence studies on some of these peptides are summarized in Table 6. DISCUSSION
Together with earlier data (VAN DER WALT and JOUBERT, 1971), our present results suggest that protease A of Bitis arietans venoms is a metalloprotease with one zinc atom per mole of protein. The pertinent clues are: the inhibition of protease A by the general metal chelator EDTA, as well as by o-phenanthroline, a transition metal chelator ; the failure of m-phenathroline, a non-chelating homologue of o-phenanthroline, to inhibit; the failure of inhibition by serine protease inhibitors (VAN DER WALT and JOUBERT, 1971); the inhibition by Hg (pointing also to a possible sulfhydryl protease activity); the finding of a nearly stoichiometric amount of zinc in the protein molecule . Confirmation of this will have to await definitive studies of the possible metal-binding properties of protease A . The presence of a cysteinyl residue opens the additional possibility that protease A could be a sulphydryl protease, although earlier tests (VAN DER WALT and JOUBERT, 1971) showed that neither phenylmercuric acetate nor N-ethylmaleimide, both sulphydryl protease inhibitors, inhibited protease A. The molecular weight of protease A was found by VAN DER WALT and JOUBERT (1971) to be 21,400 or 24,000, using ultracentrifugation procedures . The amino acid composition is more compatible with the higher molecular weight . Together with this molecular weight, the results we obtained show clearly that protease A consists of a single protein chain of about 213 amino acid residues, terminated on the carboxyl end by serine . The amino-terminal sequence starts with arginine and is given in Table 3. All of the proteolytic digests that were attempted (also including chymotryptic, thermolysin and Staphylococcus aureus V8 protease digests) produced copious quantities of `insoluble core' peptides, which decreased the potential number of peptides that could be isolated. The peptic and tryptic digests have, however, given information on a few isolated areas of protease A, apparently encompassing the majority of methionine residues in protease A, and thereby pave the way for overlapping of cyanogen bromide derived peptides . The existence of a class of metalloproteases of about 24,000 molecular weight in snake venoms is now becoming obvious (Table 7) . Such proteases have now been found in the genera Crotalus, Agkistrodon, Trimeresurus and Bitis. These genera are from crotalids and viperids, both groups being part of the proteroglyphs . A few general studies have suggested the presence of metalloproteases in these families, and their virtual absence in elapid venoms (BERNICK and SIMPSON, 1976 ; KRESS and PAROSKI, 1978). Comparing the information on protease A of Bitis arietans with the other proteases, fibrinogenases and hemorrhagins in Table 7, it is clear that protease A can be considered a representative member of this class of protease . It contains one zinc atom per mole, is inhibited by metal chelating agents, needs calcium for stability and has a molecular weight of about 24,800 . Thus far no information has turned up to suggest homology of protease A with the sequence and structure of any other known protease from any source but snake venom . The amino acid sequence of protease A will therefore be useful in the understanding of the chemistry, biology and evolution of this little known group of proteases.
25 6
D. J. STRYDOM
et al.
TABLE 7. PROPERTIES OF SOME SNAKE VENOM PROTEASES Venom
Protease
Mol. wt
Metal
Inhibitors
Crotalus atrox
a-Protease and two others
23000
Crotalux atrox
4 Hemorrhagins
25000
1 Zn
o-Phenanthroline
Crotales atrox
Protease I
20000
1 Zn
EDTA
Crotalus adamanteus Agkistrodon piscivoris leucostoma
Proteinase 1 Proteinase II
24600 23700
Agkistrodon acutus Agkistrodon acutus
Ac-1 proteinase
24500
Agkistrodon contortrix mokasen Trimerosurus Jlavoviridis Trimeresurus mucrosgwmatus Bitis arietans
Fibrinogenase
22900
Protease H 2
24000
Bitfis arietans
Protease A
Bitfis arietans
Metalloproteinase
Leucostoma peptidase
Hemorrhagins 1, II and 111
25000
EDTA,CN, Hg
1 Zn, 2 Ca
Ca, Mg
MEBs (1968), PFEIDERER and SumYK (1961)
BJARNASON and Tu (1978)
Zn, Ca
1 Zn
KuRECKI and LAsKowsKI (1978)
et
et al. (1977) Xu et ai. (1981) NIKAI
EDTA, cysteine
MoRAN and GEREN (1981)
Cd, Ni, EDTA, cysteine
TAKAHASHI and OHsAKA (1970)
EDTA, cysteine EDTA
1 Zn
PANDYA and BuDzyNSKI (1984)
WAGNER et al . (1968) . SPIEKERMANN al. (1973)
EDTA, o-phenanthroline
EDTA, cysteine
24300
74000
References
EDTA, cysteine
22000
a-Fibrinogenase 21500-23400 Protease A
EDTA, Zn, Ca, cysteine
Activators
o-Phenanthroline, Hg EDTA
Ca
OuYANG (1977)
et al.
VAN DER WALT and JOUBERT (1971) This paper
LAWRENCE and MORRIS (1981)
Acknowledgements -
We thank Mrs A. RUELLE and Dr T. HAYLETT for their assistance with sequencer analyses . Mr N. TALJAARD and Mrs Y. COPPERTHWAITE ably performed the amino acid analyses . We thank Mr T. N. VAN DER WALT of the Department of Analytical chemistry for the atomic absorption analyses . REFERENCES BANDA, M. J. and WERB, Z. (1981) Mouse macrophage elastase . Purification and characterization as a metalloproteinase . Biochem. J. 193, 589. BERNICK, J. J. and SIMPSON, J. W. (1976) Distribution of elastase-like enzyme activity among snake venoms . Comp. Biochem. Physiol. 54B, 51 . BJARNASON, J. B. and Tu, A. T. (1978) Hemorrhagic toxins from Western diamond-back rattlesnake (Crotales atrox) venom: Isolation and characterization of five toxins and the role of zinc in hemorrhagic toxins . Biochemistry 17, 3395 . BOTES, D. P., STRYDom, D. J., ANDERSON, C. G. and CHRISTENSEN, P. A. (1971) Purification and properties of three toxins from Naja nivea (LinnaeusXCape Cobra) venom and the amino acid sequence of toxin d. J. biol. Chem . 246, 3132 . BRAUN, V. and SCHROEDER, W. A. (1967) A reinvestigation of the hydrazinolytic procedure for the determination of C-terminal amino acids. Archs Biochem. Biophys. 118, 241 . BRAUNITZER, G. and SCHRANx, B. (1970) 3-Dimethylamino-l-propin als Puffersubstanz. Hoppe-Seyler's Z. physiol. Chem . 351, 417. BRENNER, M., NDEDERWIEZER, A. and PATAKI, G. (1961) Dünnschicht-chromatographie von aminosäure derivaten aufKieselgel G. N-(2,4-dinitrophenyl)-aminosäuren und 3-phenyl-2-thiohydantoine . Experientia 17, 145 . CAwsToN, T. E. and MURPHY, G. (1981) Mammaliam collagenases . Meth. En4ym. 80, 711 . CHERBuLIEz, E., BAEHLER, B. R., MAnczAr EcK, J., SUSSMAN, A. K. and RABINOWITZ, J. (1963) Etude de structures peptidiques a'l'aide de phenylisothiocyanate . V Sur la formation des phenylthiohydantoines-f°Si et sur leur chromatographie . Helv . chien. Acta 46, 2446.
Protease A of
Bitis arietans
Venom
257
ELLMAN, G. L. (1959) Tissue sulfhydryl groups . Archs Biochem. Biophys. 82, 77 . FANELLI, A. R., ANTONINI, E. and CAPuTo, A. (1958) Studies on the structure of hemoglobin. I. Physicochemical properties of human globin . Biochim. biophys. Acta 30, 608. KRESS, L. F. and PAROSKI, E. A. (1978) Enzymatic inactivation of human serum proteinase inhibitors by snake venom proteinases. Biochem. biophys. Res. Commun . 83, 649. KURECKI, T., LASKOWSKI, M. SR and KRESS, L. F. (1978) Purification and some properties of two proteinases from Crotalus adamanteus venom that inactivate human a,-proteinase inhibitor . J. biol. Chem. 253, 8340 . LATT, S. A., HoLMQuisT, B. and VALLEE, B. L. (1969) Thermolysin: A zinc metalloenzyme. Biochem . biophys. Res. Commun. 37, 333. LAWRENCE, C. H. and MORRIS, B. J. (1981) Mechanism of activation of inactive renin in human plasma by puff adder venom. Biochim. biophys. Acta 657, 13 . LIu, T. Y. and CHANG, Y. H. (1971) The hydrolysis of proteins with p-toluenesulfonic acid. J. biol. Chem. 246, 2842 . MERS, D. (1968) Vergleichende Enzymuntersuchungen an Schlangengiften unter besonderer Berûchsichtigung ihrer Casein-spalten den Proteasen. Hoppe-Seyler's Z. physiol. Chem . 349, 1115 . MORAN, J. B. and GEREN, C. R. (1981) Characterization of a fibronogenase from northern Copperhead (Agkistrodon contortrix mokasen) venom. Biochim . biophys. Acta 659, 161 . NIKAI, T., SUGIHAVA, H. and TANAKA, T. (1977) Enzymochemical studies on snake venoms II Purification of lethal protein Acl-proteinase in the venom of Agkistrodon acutus. J. pharm. Soc. Jpn 97, 507. OUYANG, C., TENG, C. M. and CHEN, Y. C. (1977) Physicochemical properties of a- and P-fibrinogenases of Trimeresurus mucrosquamatus venom. Biochim. biophys. Acta 481, 622. PANDYA, B. V. and BUDZYNSKI, A. Z . (1984) Anticoagulant proteases from Western diamond rattlesnake (Crotalus atrox) venom. Biochemistry 23, 460. PECHERE, J . F., DIXON, G. H., MAYBuRY, R. H. and NEURATH, H . (1958) Cleavage of disulfide bonds in trypsinogen and a-chymotrypsinogen . J. biol. Chem . 233, 1364 . PETERSON, J . D., NEHRLICH, S., OYER, P. E. and STEINER, D. F. (1972) Determination of the amino acid sequence of the monkey, sheep, and dog proinsulin C-peptides by semi-micro Edman degradation procedure. J. biol. Chem . 247, 4866 . PFLEIDERER, G. and SUMYK, G. (1961) Investigation of snake venom enzymes. I. Separation of rattlesnake venom proteinases by cellulose ion-exchange chromatography . Biochim. biophys. Acta 51, 482. PISANO, J. J. and BRONZERT, T. J. (1969) Analysis of amino acid phenylthiohydantoins by gas chromatography . J. biol. Chem . 244, 5597 . SANGER, F. and THOMPSON, E. O. P. (1963) Halogenation of tyrosine during acid hydrolysis . Biochim. biophys. Acta 71, 468. SELLERS, A., REYNOLDS, J. J. and MEIKLE, M. C. (1978) Neutral metallo-proteins of rabbit bone . Separation in latent forms of distinct enzymes that when activated degrade collagen, gelatin and proteoglycans. Biochem. J. 171, 493. SMITHIES, O., GIBSON, D., FANNING, E. M. GOODFLIESH, R. M. GILMAN, J . G. and BALLANTYNE, D. L. (1971) Quantitative procedures for use with Edman-Begg sequenator . Partial sequences of two unusual immunoglobulin light chains Rzf and Sac. Biochemistry 10, 4912 . SPtEKERMANN, A. M., FREDERICKS, K. K., WAGNER, F. W. and PRESCOTT, J. M. (1973) Leucostoma peptidase A: A metalloprotease from snake venom. Biochim. biophys. Acta 293, 464. TAKAHASHI, T. and OHSAKA, A. (1970) Purification and characterization of a proteinase in the venom of Trimeresurusflavoviridus. Complete separation of the enzyme from hemorrhagic activity. Biochim. biophys. Acta 198, 293. TITAN[, K., HERMODSON, M. A., ERICSSON, L. N., WALSH, K. A. and NEURATH, H. (1972) Amino-acid sequence of thermolysin. Nature, New Biol. 238, 35 . VAN DER WALT, S. J. and JouBERT, F. J. (1971) Studies on puff adder (Bitis arietans) venom. I. Purification of properties of protease A. Toxicon 9, 153. WAGNER, F. W., SPIEKERMANN, A. M. and PRESCOTT, J. M. (1968) Leucostoma peptidase A. Isolation and physical properties . J. biol. Chem. 243, 4486 . WALSH, K. A. (1975) Unifying concepts among proteases. Cold Spring Harbor Conference on Cell Proliferation
2 (Protease Biol. Control), p.l.
Xu, X., WANG, C., LIu, J. and Lu, Z. (1981) Purification and characterization of hemorrhagic components from Agkistrodon acutus (hundred pace snake) venom. Toxicon 19, 633. ZWILLING, R. DoRSAM, H., ToRFF, H. F. and R6DL, J. (1981) Low molecular mass protease: evidence for a new family of proteolytic enzymes. FEBS Lett. 127, 75 . ZWILLING, R. and PFLEIDERER, G. (1967) Zur evolution der Endopeptidasen, II Eigenschaften der a-Protease aus dem Gift von Crotalus atrox. Hoppe-Seyler's Z. physiol. Chem . 348, 519.
0041-0101/96$3 .00+ .00 © 1986 Pergamon Pros Ltd.
CHEMICAL STUDIES ON PROTEASE A OF BITIS ARIETANS (PUFF ADDER) VENOM DANIEL J. STRYDOM,* FRANCOIS J. JOUBERTt and NEVILLE L . HOWARD* National Chemical Research Laboratory, Council for Scientific and Industrial Research, P O Box 395, Pretoria, 0001, Republic of South Africa (Accepted for publication 8 August 1985)
D.
J . STRYDom, F. J . JouBERT and N. L. HOWARD . Chemical studies on protease A of Bitis arietans (puff adder) venom. Toxicon 24, 247 - 257, 1986 . - Protease Aof Bitis arietans venom is
probably a metalloprotease, since it is inhibited by o-phenanthroline and contains 0.77 moles of zinc per mole protein. The enzyme comprises 213 amino acids, including 9 methionine residues and one free sulphydryl group. It contains one polypeptide chain, which is terminated at the carboxyl end by serine . The amino terminal sequence of protease A is : Arg-Ser-Ser-Asp-Pro-AsnLys-Tyr-Phe-Asn-Val-Ile-Val-Val-Val-Asp-Asn-Arg-Met- Val-Asn-Tyr-Tyr-Lys-Gly- Glu -LeuAsn-Lys-Ile-Thr- . Despite diffficulties with 'insoluble peptide core' formation, a number of peptides were purified from peptic and Cryptic digests of S-derivatized protease A.
INTRODUCTION
PROTEASES are among the most ubiquitous
enzymes found in living organisms. They play a fundamental role in many biological processes, ranging from digestion of food through metabolic turnover at the cellular level to control of many processes through proteolytic activation or deactivation of enzymes, hormones and other molecular messengers . Proteases are classified according to certain common features of their chemistry and mechanism of action . The currently accepted classes are the serine proteases, acid proteases, sulphydryl proteases, metalloproteases (WALSH, 1975) and low molecular mass proteases (ZWILLING and DORSAM, 1981). Among these, the metalloproteases are relatively unknown at the protein structural level . Only thermolysin, a bacterial metalloprotease, has been studied extensively and sequenced (LATT et al ., 1969; TITANI et al., 1972). Very little is known about the structures of vertebrate metalloproteases, or even about any eucaryotic metalloproteases, although such activities are well known (e.g. BANDA and WERB, 1981 ; SELLER et al ., 1978; CAWSTON and MURPHY, 1981) and play an important role in metabolism . The main problem in studying these proteins seems to be their low levels in tissues and the concomittant problems in procuring adequate quantities of such proteins . Snake venoms, which are rich sources of many enzymes and proteins, do contain appreciable quantities of proteases (MEBS, 1968, and references therein) . A growing, seemingly disparate, list of proteases, blood protein inactivators, fibrinogenases, hemorrhagins, etc., have been isolated from crotalid and viperid venoms. They have the *Present address: Center for Biochemical and Biophysical Sciences and Medicine, Brigham and Women's Hospital, Boston, MA 02115, U.S.A. "To whom reprint requests should be addressed. *Present address: Wheat Board, P O Box 908, Pretoria, Republic of South Africa. 247
248
D. J. STRYDOM et al.
properties in common of possible metalloproteinase character and molecular weights near 24,000 (KRESS and PAROSKI, 1978; ZWILLING and PFLEIDERER, 1967; PFLEIDERER and SUMYK, 1961 ; BJARNASON and TU, 1978 ; WAGNER et al., 1968; SPIEKERMANN et al., 1973; TAKAHASHI and OHSAKA, 1970; OUYANG et al., 1977 ; VAN DER WALT and JOUBERT, 1971 ; NIKAI et al., 1977 ; MORAN and GEREN, 1981 ; LAWRENCE and MORRIS, 1981 ; XU et al., 1981 ; KURECKI and LASKOWSKI, 1978 ; PANDYA and BUDZYNSKI, 1984). The size of these proteins make them readily amenable to sequence studies, which could enable a comparative study of the metalloprotein characteristics of these vertebrate proteases with bacterial metalloproteases such as thermolysin (LATT et al., 1969; TITANI et al., 1972). One of the proteases from the venom of the puff adder, Bitis arietans, named protease A, is easily purified in large quantities (VAN DER WALT and JOUBERT, 1971). This paper describes an investigation into the chemical structure of protease A. MATERIAL AND METHODS Materials Bitis arietans venom was obtained with the cooperation of D. J. Muller, Professional Snake Catcher (Pty) Ltd., 99 Cedar Road, Northcliff, Johannesburg, Republic of South Africa . The venom was collected in containers held at - 80°C and then lyophilized. Trypsin was supplied by Miles Laboratories (Republic of South Africa) as a twice crystallized diphenylcarbamyl chloride-treated, salt-free preparation and pepsin (twice crystallized) was supplied by Worthington Biochemical Corp . (U .S.A .). Diethylaminoethyl cellulose (DEAEcellulose) was a microgranular preparation from Whatman (U .K .) and Sephadex G-50 (fine) was obtained from Pharmacia (Sweden) . Chromatography Sephadex G-50 and DEAE-cellulose columns were prepared as recommended by the manufacturers and the eluates were monitored at 230 nm or 280 nm with a Beckman spectophotometer . Pur(/ication ofprotease A Protease A was prepared from lyophilized Bitis arietans venom according to the procedure of VAN DER WALT and JouBERT (1971), with a few minor alterations. Concentration of protein between chromatography steps was effected by precipitation with ammonium sulfate, followed by dialysis against the appropriate starting buffer . The elution buffer in the final purification on Sephadex G-50 (instead of Sephadex G-75) was0.2 M ammonium acetate, 0.005 M calcium chloride at pH 5.6 . The intial separation and rechromatography steps were done with ammonium acetate instead of sodium acetate buffer, with a slight change in elution pattern. Assay ofprotease A activity Caseinase activity was measured as described by VAN DER WALT and JOUBERT (1971) . Activity against globin was measured by substituting globin for casein in the above assay. The globin was prepared from bovine haemoglobin by themethod of FANELLI et al. (1958) . The resultant globin wasdialyzed against o-phenanthroline (0.001 M) and then repeatedly against water before lyophilization . Assessment of effectors was done by incubating 100 M1 of a solution of protease A in an appropriate buffer with 10 pl of a 10 mM solution of effector for 15 min, and then assaying for residual proteinase activity. Reduction and S-carboxymethylation Reduction of protease A was performed in 1 M Tris, pH 8.6, containing 6 M guanidinium hydrochloride and 0.1 M EDTA, using a 10-fold molar excess of dithiothreitol over cystine-content of the enzyme. Reduced enzyme was S-carboxymethylated with iodoac etic acid, in quantities equimolar to the free sulfhydryl groups in the reaction mixture. Both reduction and alkylation were carried out under anaerobic conditions . The final product was dialyzed against distilled water and lyophilized. S-suftolysis of protease A Protease A was S-sulfitolysed according to the procedure of PECHERE et al. (1958) . Lyophilized protease A (430 mg) was dissolved in 9.2 ml of 0.005 M HCI and diluted with 15 ml 0.001 M HCI. Urea (29.5 g) was added in small portions, with the pH being kept at 3 with 1 N HCI. Sodium sulfite (6 ml, 1.5 M) was added and the pH adjusted to 10 .2 with ammonia, followed by addition of cupric nitrate solution (1 .2 ml, 2 M), the pH being kept at 10.2 . The volume was adjusted to 60 ml and after 1 hr, dialysed with a DOW-beaker dialyzer with hollow
Protease A of Bitis arietans Venom
249
cellulose fibres against 30 liters of 0.01 M ammonium hydrogencarbonate solution . This solution was used directly for a tryptic digest .
Digestion with proteolytic enzymes
S-sulfo protease A (430 mg) in 100 m10.01 M NH,HCO, was digested with two lots of 4.3 mg of trypsin for 4 hr at room temperature. The digestion was stopped by acidification to pH 3 with acetic acid and the digest was lyophilized. Reduced and S-carboxymethylated protease A (420 mg) was dissolved in 2.1 ml formic acid (984`) and diluted with 37 .9 ml water . Pepsin (4 .2 mg) was added and the mixture incubated at 37*C for 2 hr . The mixture was neutralized and lyophilized.
Fractionation ofpeptides
The peptic digest (soluble portion) was fractionated by gel filtration on Sephadex G-50 with 1 % acetic acid as eluant. The fractions were further purified by chromatography on DEAE-cellulose columns using a linear gradient from 0.05 to 0.5 M NH,HCO, . The tryptic digest (soluble portion) of S-sulfocysteinyl protease A was fractionated on DEAE-cellulose columns using a linear gradient from 0.01 to 0.6 M NH,HCO,. Peptide fractions were tested for purity and further purified, using paper chromatography or paper electrophoresis as detailed previously (BOTEs et al., 1971).
Chemical methods
Amino acid analyses were performed with an automatic Beckman amino acid analyser . Samples were hydrolysed with 6 N HCI for 24 hr in sealed tubes and phenol was added to prevent the destruction of tyrosine (SANGER and THOMPSON, 1963). For the determination of tryptophan the samples were hydrolysed with 3 M p-toluene sulphonic acid (Llu and CHANG, 1971). Free sulphydryl groups, in 6 M guanidinium chloride, were assayed in intact protease A samples according to the method of ELLMAN (1959) . Metal analysis was done by atomic absorption on a Varian Techtron AA-5 spectrophotometer .
Carboxy terminal analyses
The C-terminal amino acid of protease A was determined by Amberlite CG-50 catalysed hydrazinolysis as described by BRAUN and SCHROEDER (1967) .
Sequence determination
The N-terminal sequence of reduced and S-carboxymethylated protease A was determined with the Beckman sequencer using the quadrol programme (Beckmann No . 12294 mod) . A large peptide was also sequenced with the sequencer using the peptide program (No. 111374) with N,N-dimethylamino propyne (BRAUNITZER and SCHRANK, 1970) buffer. For small peptides the semi-micro Edman degradation procedure of PEDERSON et al. (1972) was used for manual sequence studies. The phenylthiohydantoin derivatives of amino acids were identified by gas chromatography on an SP-400 column (PIsANo and BRONZERT, 1969) and thin layer chromatography on silica gel (BRENNER et al., 1961 ; CHERBULIEz et al ., 1963). In many cases back hydrolysis of the phenylthiohydantoin to free amino acids with HI (SMrrHiEs et al., 1971) was used as an aid in identification. RESULTS
Inhibition of protease A activity
The effect on caseinase activity at pH 8 of pre-incubation at pH 5.5 of protease A with 0.9 mM concentrations of various metal ions was tested and the results are summarized TABLE 1 . THE EFFECT OF VARIOUS METAL IONS ON CASEINASE ACTIVITY OF PROTEASE A Ion Af. Ba 2" Cd2* Cs* Co'. Cu" Fe'* Hg
Residual activity (%)
Ion
Residual activity (4ti)
96 95 94 95 92 100 97 69
Mg.. Mn'* Ni2* Pb» Sr'* Zn'* Nd3* Water
95 97 100 97 99 98 104 100
250
D . J . STRYDOM et ai.
in Table 1 . Apart from Hg=*, which inhibited the enzyme by 31 %, the effects were negligible . Under the same conditions, o-phenanthroline caused an inhibition of 57% of the activity . Using globin at pH 8 as substrate, protease A activity was tested after 15 min pre-incubation at pH 5.5 or 8 with 10-' M o-phenanthroline. Inhibition was found with o-phenanthroline at both pH values to the extent of 28 and 40% inhibition at pH 5 .5 and 8, respectively, whereas a control experiment with m-phenanthroline did not show any inhibition . Chemical and amino terminal studies
Atomic absorption analysis showed that protease A contains 0.77 moles of Zn2 * per mole (based on a molecular weight of 24,800). Table 2 reports the average results of eleven independent analyses of amino acid composition of protease A. It contains 213 amino acids, including one free sulphydryl group. The carboxyl terminal amino acid for protease A was serine . The N-terminal amino acid sequence of the reduced and Scarboxymethylated protease A is given in Table 3 . Peptic and tryptic peptides ofprotease A
The lyophilized peptic digest was extracted with 5% acetic acid. The acetic acid soluble peptides were dried and extracted with 0.05 M NH,HCO,. These soluble peptides were fractionated by gel filtration on Sephadex G-50 in I% acetic acid (Fig. 1) . Fraction S1 was dried, dissolved in 0.05 M NH4HCO, and further fractionated on a DEAE-cellulose column . A linear gradient of ammonium bicarbonate eluted 18 fractions (P1-P18) (Fig . 2). Fractions P1, P2, P3, P4, P5, P6, P9 and PI I were fractionated further on Sephadex G-25 . The major fractions of P1, P2, P3, P4, P5, P6 and Pll, as well as fraction P10, TABLE 2. AMINO ACID COMPOSITION OF PROTEASE A (GIVEN AS MOLE OF RESIDUE PER MOLE PROTEIN, BASED ON A MOLECULAR WEIGHT OF 24,800)
Amino acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine -Cystine* Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophant Free sulphydryl* Total
Analysis 32 .24 (32) 9 .08 (9) 13 .01 (13) 15 .68 (16) 8 .18 (8) 10 .62 (11) 12 .08 (12) 7 .74 (8) 14 .02 (14) 8 .53 (9) 15 .12 (15) 9 .09 (9) 12 .17 (12) 9 .25 (9) 9 .34 (9) 8 .93 (9) 12.01 (12) 5 .40 (5) 0.76 (1) 213
*Determined as 3-carboxymethyl cysteine . DDteermined as described by Llu and CHANG (1971) . *Determined as described by ELLMAN (1959) .
Protease A of Bibs arierans Venom
251
TABLE 3 . N-TERMINAL SEQUENCE OF REDUCED AND S-CARBOXYMEfHYLATED PROTEASE SEQUENCER (280 lanole was loaded)
Step 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
GC analysis*
Normal
Silylated
Cys/Ser Cys/Ser
Cys/Ser Cys/Ser Asp Pro Asn
Pro Tyr Phe Asp Val Ile/Leu Val Val Val Asn
Tyr Phe Asp Val Ile Val Val Val Asp Asn
Met Val
Met Val
Others Identification Arg Ser Ser Asp Pro Asn Lys Tyr Phe Asn Val Ile Val Val Val Asp Asn Arg Met Val Asn
Step
Arg Ser Ser Asp Pro Asn Lys Tyr Phe Asn Val Ile Val Val Val Asp Asn Arg Met Val Asn
22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41
A USING THE
AUTOMATIC
GC analysis' Normal Silylated Other' Identification Tyr
Tyr Tyr
Thr
Gly Glu Leu Leu Lys Gln Thr
Tyr Tyr Lys Gly Glu Leu Asn Lys Ile Thr
Tyr Tyr Lys Gly Glu Leu Asn Lys Ile Thr
Gly
Val
Val
Val
Val
Tyr Phe
Tyr Phe
Gln
Ile/Leu Ile/Leu
Ile Ile
Gln Tyr Phe Asn Ile Leu
Ile/Leu Ile/Leu
Phe Asn Ile Leu
*Gas chromatography analysis . 'Analysis by thin layer chromatography, back hydrolysis to amino acids, phenanthrenequinone colour reaction for arginine, Pauli-reaction for histidine. *Not identified . The quadrol programme (Beckman No . 122974 mod) was used . The yield of proline at step 5 was 50%. The repetitive yield was 91 .5°10 .
0.4
04l
n
0 .5 ,
0.75 'L ELUATE
VOLUME
I
1
1 .0
(liter)
FIG . 1 . GEL FILTRATION OF THE PEPTIC DIGEST (SOLUBLE PORTION) OF PROTEASE A. Reduced and S-carboxymethylated protease A (420 mg) was digested with pepsin and the soluble portion loaded on a Sephadex G-SO column (3 x 1 .9 x 150 cm) and eluted with 1 % acetic acid . The flow rate was 20 ml/hr, the column temperature was 20°C and the elutee was monitored at 280 nm .
252
D. J . STRYDOM et al.
P3
0 M N 4
P9 0.5 P7
PIO
1P Il
P9 j
i
PI6
P15
P 17
P2 j Pj3 PI4
Pis
1 û ELUATE
VOLUME
(liter)
FIG. 2. CHROMATOGRAPHY OF FRACTION S, ON DEAE-CELLULOSE . Peak S, (Fig . 1) was lyophilized, dissolved in 0.05 M NH,HCO, and loaded on a DEAE-cellulose column (0 .9 x 150 cm) and eluted by a linear gradient of 0.05 -0.5 M NH,HCO, over 2 litres at a flow rate of 50 ml/hr. The column temperature was 20°C and the eluate was monitored at 230 nm .
TABLE 4. AMINOACID COMPOSITION OF THE PEPTIC PEPTIDES OF REDUCED AND S-CARRO:
P1 .1 .1
P1 .1 .2
P1 .1 .3
P2.1
Asp
1.35(1) 2.04(2) 2.06(2)
3.16(3) 0.40 1.01(1) 2.13(2)
3.10(3)
Glu
1.34(1) 1.93(2) 1.51(2)
0.92(1) 2.94(3)
Gly
1.05(1)
1.07(1)
CYs(CM) Thr Ser Pro
Ala Val Met Ile
Leu Tyr
Phe Lys His
Arg Trp
1.00(1)
0.88(1)
1.05(1) 1.00(1) 1.70(2) 1.28(1) 2.82(3) 0.90(1)
0.97(1) 0.38 2.95(3) 1.21(1) 1.13(1) 2.13(2) 2.49(2) 0.81(1) 0.94(1) 2 .80(3)
16 Total Yield (%) 1 .7
2.6
12
3.4
16
P2.3
P3 .2 .2
P4.1 .3 1 .25(2) 3.18(3) 1 .04(1) 1 .90(2) 1 .08(1) 2.05(2)
0.37 0.20 0.13
3.05(3) 0.99(1)
1 .97(2)
1.31(1) 0.2 0.34 0.21
3.02(3)
1.92(2) 1.06(1) 0.94(1)
0.50(1)
0.95(1)
1.00(1) 1 .07(1) 1 .01(1)
0.99(1)
1 .77(2)
1 .94(2) 0.15 1.00(1) 2.91(3) 1.28(1) (1)
57
19
33
6
4.5
12
P5 .4 .1
P5 .4 .2
P5 .4.3
0.92(1)
0.96(1)
0.45(1)
0.94(1)
P4 .2 .1
P6 .3 .1 0.2 0.91(1)
0.94(1) 1 .06(1)
2.05(2) 0.95(1)
1 .11(1)
1.14(1) 0.86(1)
1.1(1) 0.86(1) 1.00(1) 1.20(1) 0.28 0.92(1)
0.95(1) 0.99(1) 10
15
1.9
3
1.4
2
0.9
3
3.0
2
4.3
6
253
Protease A of Bitis arietans Venom
0.10 T8
0 N a
1
Q05
T18 T20T21
r T. 1 T2
TI6
TI5
v
v
n
~
~
i
T19
T24
T22
~
T23
i
w
2
4
ELUATE FIG .
3. CHROMATOGRAPHY
VOLUME
(liter)
THE TRYPTIC DIGEST (SOLUBLE PORTION) OF PROTEASE A ON DEAE-CELLULOSE. S-sulfocysteinyl protease A (430 mg) was digested with trypsin and the soluble portion loaded on a DEAE-cellulose column (1 .9 x 150 cm) and eluted by a linear gradient of 0.01-0.6 M NH 4HCO, over 8 litres at a flow rate of 50 ml/hr . The column temperature was 20°C and the eluate was monitored at 230 nm . OF
hMETHYLATED PROTEASE A (SOLUBLE PORTION) (GIVEN
P6 .3 .2
P6 .3 .3
0.26 1.08(1)
3 .34(3)
0.99(1)
0.43
2.45(3) 0.14
0.4 0.22
0.57(1) 0 .91(1)
1 .80(2) 0.87(1) 0.24
1.01(1) 0.2
6.8
P8 .1
1 .04(1) 1 .10(1)
1 .25(1) 0.22 0.13
0.89(1)
1 .5
8
6.5
5
MOLE
OF
RESIDUE PER MOLE PEPTIDE)
P9 .1 .1
P9.1 .2
P9.1 .3
0.87(1) 1 .10(1)
0.97(1) 1 .07(1) 0.92(1) 0.98(1)
0.84(1) 1 .31(1) 1 .07(1) 1 .06(1)
1 .02(1) 1 .00(1) 1.06(1) 1.94(2) 0.97(1)
1.12(l) 1.04(1) 1.96(2) 1.85(2) 0.75(1) 0.93(1)
0.98(1) 0.99(1) 1 .11(1) 1 .09(1) 0.52(1)
1 .06(1) 0.90(1)
0.88(1) 8
AS
0.86(l) 0.97(1) 0.88(1)
2.1
9
1.03(1) 1.18(1)
2.9
12
0.98(1) 0.90(1)
1 .7
15
P9.1 .4 1 .08(1) 1 .89(2) 2.97(3) 0.92(1) 1.11(1) 1.00(1) 0.97(1)
P9.2 0.85(1)
1 .03(1) 1.97(2) 0.64(1) 1 .00(1)
P10.2 1 .12(1) 1 .96(2) 3.02(3) 1 .03(1) 1.03(1) 0.95(2) 1.03(1)
P11.1 .1
P15
0.98(1)
0.92(1) 2.44(2) 1 .04(1) 1 .04(1) 1 .27(l)
1 .01(1)
0.44
0.72(1) 2.01(2)
1 .87(2) 1.04(1)
0.99(1) 0.93(1)
4.7
11
0.18
14
0.85(1)
6
7.5
12
0.96(l) (1) 3.1
5
5
11
D . J . STRYDOM
25 4
et aL
A
TABLE 5 . AMINO ACID COMPOSITION OF THE TRYPTIC PEPTIDES OF S-SULFOCYSTEINYL PROTEASE PORTION) (GIVEN AS MOLE OF RESIDUE PER MOLE PEPTIDE) T2 .4
Cys(CM) Asp Thr Ser Glu Pro Gly Ala Val Met lie Leu Tyr Phe Lys His Arg Trp
Total Yield (%)
1 .54(2)
T2 .7
T7 .2
1 .26(1)
1 .19(1)
1 .7(1)
2 .07(2)
1 .33(1) 1 .02(1)
0.5 1 .18(1) 0.16
1 .09(1)
0.32
1 .09(1) 0.15
0.31 0.76(1)
1.00(1)
25 .7
4
1 .7(2)
1 .83(2) 0.91(1)
0 .7
4
0.27 0.24 1 .86(2)
7 .2
7
T7 .4
T7 .6
0 .84(1)
1 .09(1)
1 .00(1) 0 .13
4 .0
5
10 .4
4
T8 .1 T9 .1
T10 .1
T11 .9
T17 .1
T21 .2
0 .26
1 .06(1)
0 .62(1) 5 .45(5)
2 .29(3) 4 .86(5)
0 .17 1 .17(1)
0 .17 1 .00(1)
3 .11(3)
1 .38(1) 3 .16(3)
0.13 1 .02(1) 0 .15
0 .34 3 .91(4) 1 .02(1) 0 .64(1) 1 .00(1) 0 .12
0 .32 0 .26 0 .12 0 .14
1 .66(2)
0 .20
1 .16(1) 0 .96(1)
0 .81(1) 0 .14
0.54(2) 1 .12(1) 1 .10(1) 0.97(1) 1 .05(1) 0.97(1) 0.17 0.24
12 12 .1
T8 .6
(SOLUBLE
1 .04(1)
0 .96(1)
8 .9
2
3 .1
9
3 .25(3) 0 .14 0 .93(1) 1 .07(1) 0 .94(1)
0 .12
0 .94(1) 0 .26
1 .85(2) 0 .96(1) 0 .90(1) 1 .00(1)
4 .25(4) 3 .50(4) 3 .46(4) 1 .57(2) 0 .96(1) 0 .80(1) 0 .91(1) 3 .06(3)
(1)
2 .5
3
4 .0
21
TABLE 6 . AMINO ACID SEQUENCES OF THE PEPTIC (P) AND TRYPTIC (T) PEPTIDES OF PROTEASE
2 .2
32
A
Peptide
Sequence results P2 .1 Ala- Glu-Trp-Arg- Glu-Arg-Asp-Leu-Leu-Ser-Arg-Lys-(His,Asx,Lys,Asx,Leu,Ala,Glu) P2 .3 Tyr-Ile-Arg-Pro-(Ile,Leu) P4 .1 .3 Ile-Ser-Thr-His-Asn-Pro-Gln-Cys-Phe-Cys-(Asx,,Ser,Pro,Arg) Val-Met p5 .4 .3 Ile-Ala-Tyr-Val-Gly-Thr P6 .3 .1 P6 .3 .2 Ile-(Ser)-Asn-(Pro,,Met,Tyr) P8 .1 Tyr- Lys-Gly-(GIu,Leu) P9 .1 .1 Met-Cys-Asp-Pro-Lys-His-(Ser, Val,Ala) Val -Gly-Thr-Met-Cys-Asp-Pro-(Lys,His,Ser, Va1,Ala) P9 .1 .2 P9 .1 .3 Ile-Ala-Tyr-Val-Gly-Thr-Met-(Cys)-Asp-(Pro,Lys,His,Ser,Val,Ala) P10 .2 Val-Thr-Gin-Ser-Ala-Ser-Asp-Thr-(Ser,Leu,His,Phe) P11 .1 .1 Val-Gly-Ile-(GIu,Ile) P15 Ile-Glu-Ile-(Trp,Ser,Asx,Arg,Asx,Leu,Cys,Thr) T2 .7 Lys-His-(Asx,Lys) T7 .2 Glu-Arg-Asp-Leu-(Leu,Ser,Arg) T7 .4 Gly- Glu-Leu-(Asn,Lys) T7 .6 Asp-Asn-Pro-Ser The manual semi-micro Edman degradation procedure of PETERSON et al. (1972) was used . 'The sequencer was used with NN-dimethylamino propyne buffer .
were chromatographed on paper, using BPAW as the solvent system, while the major fraction of P9, as well as fractions P7, P8 and P10, were separated by paper electrophoresis at pH 1 .9. The soluble portion of the tryptic digest of S-sulfocysteinyl protease A was fractionated on DEAE-cellulose using a linear gradient of ammonium bicarbonate and yielded 24
Protease A of Bitis arietans Venom
25 5
fractions (Fig . 3). Fractions T2, T7, T8, T9, T10, T11, T17 and T22 were fractionated further by paper chromatography using solvent system BPAW, while pH 1 .9 electrophoresis served to fractionate fraction 21 . Tables 4 and S record the amino acid compositions of some peptic and tryptic peptides of protease A. Sequence studies on some of these peptides are summarized in Table 6. DISCUSSION
Together with earlier data (VAN DER WALT and JOUBERT, 1971), our present results suggest that protease A of Bitis arietans venoms is a metalloprotease with one zinc atom per mole of protein. The pertinent clues are: the inhibition of protease A by the general metal chelator EDTA, as well as by o-phenanthroline, a transition metal chelator ; the failure of m-phenathroline, a non-chelating homologue of o-phenanthroline, to inhibit; the failure of inhibition by serine protease inhibitors (VAN DER WALT and JOUBERT, 1971); the inhibition by Hg (pointing also to a possible sulfhydryl protease activity); the finding of a nearly stoichiometric amount of zinc in the protein molecule . Confirmation of this will have to await definitive studies of the possible metal-binding properties of protease A . The presence of a cysteinyl residue opens the additional possibility that protease A could be a sulphydryl protease, although earlier tests (VAN DER WALT and JOUBERT, 1971) showed that neither phenylmercuric acetate nor N-ethylmaleimide, both sulphydryl protease inhibitors, inhibited protease A. The molecular weight of protease A was found by VAN DER WALT and JOUBERT (1971) to be 21,400 or 24,000, using ultracentrifugation procedures . The amino acid composition is more compatible with the higher molecular weight . Together with this molecular weight, the results we obtained show clearly that protease A consists of a single protein chain of about 213 amino acid residues, terminated on the carboxyl end by serine . The amino-terminal sequence starts with arginine and is given in Table 3. All of the proteolytic digests that were attempted (also including chymotryptic, thermolysin and Staphylococcus aureus V8 protease digests) produced copious quantities of `insoluble core' peptides, which decreased the potential number of peptides that could be isolated. The peptic and tryptic digests have, however, given information on a few isolated areas of protease A, apparently encompassing the majority of methionine residues in protease A, and thereby pave the way for overlapping of cyanogen bromide derived peptides . The existence of a class of metalloproteases of about 24,000 molecular weight in snake venoms is now becoming obvious (Table 7) . Such proteases have now been found in the genera Crotalus, Agkistrodon, Trimeresurus and Bitis. These genera are from crotalids and viperids, both groups being part of the proteroglyphs . A few general studies have suggested the presence of metalloproteases in these families, and their virtual absence in elapid venoms (BERNICK and SIMPSON, 1976 ; KRESS and PAROSKI, 1978). Comparing the information on protease A of Bitis arietans with the other proteases, fibrinogenases and hemorrhagins in Table 7, it is clear that protease A can be considered a representative member of this class of protease . It contains one zinc atom per mole, is inhibited by metal chelating agents, needs calcium for stability and has a molecular weight of about 24,800 . Thus far no information has turned up to suggest homology of protease A with the sequence and structure of any other known protease from any source but snake venom . The amino acid sequence of protease A will therefore be useful in the understanding of the chemistry, biology and evolution of this little known group of proteases.
25 6
D. J. STRYDOM
et al.
TABLE 7. PROPERTIES OF SOME SNAKE VENOM PROTEASES Venom
Protease
Mol. wt
Metal
Inhibitors
Crotalus atrox
a-Protease and two others
23000
Crotalux atrox
4 Hemorrhagins
25000
1 Zn
o-Phenanthroline
Crotales atrox
Protease I
20000
1 Zn
EDTA
Crotalus adamanteus Agkistrodon piscivoris leucostoma
Proteinase 1 Proteinase II
24600 23700
Agkistrodon acutus Agkistrodon acutus
Ac-1 proteinase
24500
Agkistrodon contortrix mokasen Trimerosurus Jlavoviridis Trimeresurus mucrosgwmatus Bitis arietans
Fibrinogenase
22900
Protease H 2
24000
Bitfis arietans
Protease A
Bitfis arietans
Metalloproteinase
Leucostoma peptidase
Hemorrhagins 1, II and 111
25000
EDTA,CN, Hg
1 Zn, 2 Ca
Ca, Mg
MEBs (1968), PFEIDERER and SumYK (1961)
BJARNASON and Tu (1978)
Zn, Ca
1 Zn
KuRECKI and LAsKowsKI (1978)
et
et al. (1977) Xu et ai. (1981) NIKAI
EDTA, cysteine
MoRAN and GEREN (1981)
Cd, Ni, EDTA, cysteine
TAKAHASHI and OHsAKA (1970)
EDTA, cysteine EDTA
1 Zn
PANDYA and BuDzyNSKI (1984)
WAGNER et al . (1968) . SPIEKERMANN al. (1973)
EDTA, o-phenanthroline
EDTA, cysteine
24300
74000
References
EDTA, cysteine
22000
a-Fibrinogenase 21500-23400 Protease A
EDTA, Zn, Ca, cysteine
Activators
o-Phenanthroline, Hg EDTA
Ca
OuYANG (1977)
et al.
VAN DER WALT and JOUBERT (1971) This paper
LAWRENCE and MORRIS (1981)
Acknowledgements -
We thank Mrs A. RUELLE and Dr T. HAYLETT for their assistance with sequencer analyses . Mr N. TALJAARD and Mrs Y. COPPERTHWAITE ably performed the amino acid analyses . We thank Mr T. N. VAN DER WALT of the Department of Analytical chemistry for the atomic absorption analyses . REFERENCES BANDA, M. J. and WERB, Z. (1981) Mouse macrophage elastase . Purification and characterization as a metalloproteinase . Biochem. J. 193, 589. BERNICK, J. J. and SIMPSON, J. W. (1976) Distribution of elastase-like enzyme activity among snake venoms . Comp. Biochem. Physiol. 54B, 51 . BJARNASON, J. B. and Tu, A. T. (1978) Hemorrhagic toxins from Western diamond-back rattlesnake (Crotales atrox) venom: Isolation and characterization of five toxins and the role of zinc in hemorrhagic toxins . Biochemistry 17, 3395 . BOTES, D. P., STRYDom, D. J., ANDERSON, C. G. and CHRISTENSEN, P. A. (1971) Purification and properties of three toxins from Naja nivea (LinnaeusXCape Cobra) venom and the amino acid sequence of toxin d. J. biol. Chem . 246, 3132 . BRAUN, V. and SCHROEDER, W. A. (1967) A reinvestigation of the hydrazinolytic procedure for the determination of C-terminal amino acids. Archs Biochem. Biophys. 118, 241 . BRAUNITZER, G. and SCHRANx, B. (1970) 3-Dimethylamino-l-propin als Puffersubstanz. Hoppe-Seyler's Z. physiol. Chem . 351, 417. BRENNER, M., NDEDERWIEZER, A. and PATAKI, G. (1961) Dünnschicht-chromatographie von aminosäure derivaten aufKieselgel G. N-(2,4-dinitrophenyl)-aminosäuren und 3-phenyl-2-thiohydantoine . Experientia 17, 145 . CAwsToN, T. E. and MURPHY, G. (1981) Mammaliam collagenases . Meth. En4ym. 80, 711 . CHERBuLIEz, E., BAEHLER, B. R., MAnczAr EcK, J., SUSSMAN, A. K. and RABINOWITZ, J. (1963) Etude de structures peptidiques a'l'aide de phenylisothiocyanate . V Sur la formation des phenylthiohydantoines-f°Si et sur leur chromatographie . Helv . chien. Acta 46, 2446.
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257
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