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Structure and effects of A kinin potentiating fraction F (AppF) isolated from Agkistrodon piscivorus piscivorus venom
004&0101(9!9ooo71-2
Toxicon, Vol. 33, No. IO, pp. 13134319, 1995 Copyristn0 1995 Elsevier Scimcc Ltd Printed in Great Britain. All rights resewed 00414101/95 s9.50+0.00
STRUCTURE AND EFFECTS OF A KININ POTENTIATING FRACTION F (AppF) ISOLATED FROM AGKISZ-XODON PISCIVORUS PISCIVORUS VENOM L. A. F. FERREIRA,’ T. Mi5LLRING,Z F. L. A. S. LEBRUN,3 M. RAIDA, R. ZNOTTKA4 and G. G. HABERMEHL** ‘Departamento de Bioquimica e Biofisica, Instituto Butantan, SBo Paulo, Brazil; *Department of Chemistry, School of Veterinary Medicine, Bischofsholer Damm 15, D-30173 Hannover, Germany; ‘Departamento de Fisiologia e Biofisica, Instituto de Ciencias Biomedicas USP, SHo Paulo, Brazil; and 4Lower Saxonian Institute of Peptide Research, Hannover, Germany (Received 23 January 1995; accepted 24 April 1995)
L. A. F. Ferreira, T. Miilhing, F. L. A. S. Lebrun, M. Raida, R. Znottka and G. G. Habermehl. Structure and effects of a kinin potentiating fraction F (AppF) isolated from Agkistrodon piscivorus piscivorus venom. Toxicon 33, 1313-1319, 1995.-The contractile action of bradykinin on isolated smooth muscles is known to be potentiated by special peptides isolated from snake venoms and other animal sources. A fraction F (AppF) has been derived from Agkistrodon piscivorus piscivorus venom. This fraction does not increase the depressor effect of bradykinin on blood pressure, potentiates the action of bradykinin on isolated guinea-pig ileum and prolongs the duration of relaxation in isolated rat duodenum. This fraction was able to inhibit the conversion of angiotensin I to angiotensin II in vitro. The primary structure of this decapeptide is given. INTRODUCTION
Kinins are defined as hypotensive polypeptides which contract extravascular isolated smooth muscle preparations, relax the rat duodenum, decrease the arterial blood pressure of mammals and cause pain. There is evidence that kinins are important in various physiological and pathological conditions in mammals as well as in other animals. Five kinins have been found in mammalian plasma, and one of them, bradykinin, discovered by Rocha e Silva et al. (1949), has been well studied. Ferreira (1965) found in the venom of the snake Bothrops jararaca a factor which potentiates the effects of bradykinin (Bamberg et al., 1969), isolated bradykinin potentiating peptides from a tryptic hydrolysate of human plasma, and studied their activities. Ferreira et al. (1992) found six bradykinin potentiating peptides in the venom of B. jararacussu and studied one of them, denominated peptide P. We have recently isolated a peptide from Agkistrodon piscivorus piscivorus venom, which we named peptide F. It potentiates the effect of bradykinin on the isolated guinea-pig ileum, does not potentiate the effects of bradykinin upon rat arterial blood pressure and *Author to whom correspondence
should be addressed. 1313
1314
L. A. F. FERREIRA et al.
inhibits the angiotensin-converting enzyme from rat plasma. The amino acid sequence has been determined. It fits into a group of other bradykinin potentiating peptides, the N-terminal amino acid being pyroglutamic acid, and the C-terminus consisting of the sequence Ile-Pro-Pro. MATERIALS AND METHODS dried venom was obtained from Ventoxin (Frederick, MD, U.S.A.). Captopril, pyroglutamyl-aminopeptidase (PGAP) and bradykinin were purchased from Sigma Chemical Co. (St Louis, MO, U.S.A.). Peptide P was from the Instituto Butantan (Slo Paulo, Brazil). Angiotensin converting enzyme was purified from rat plasma (Nishimura et al., 1977). Agkistrodon
piscivorus
Isolation of fraction
pisciuorus
F
Dried venom (3 g) was dissolved in 180 ml water, and 540 ml boiling ethanol was added to precipitate proteins. After centrifugation, the solution was lyophilized and dissolved again in 100 ml water; 100 ml 6 M (NH,),SO, solution was added, the precipitate was centrifuged, and after centrifugation, 300 ml isopropanol (98%, HPLC pure) was added for a third precipitation. Lyophilization of the supernatant yielded 632 mg of a colourless material, which was dissolved in 6.0 ml 0.05 M (NH,HCO,) buffer solution and separated by HPLC (glass column TSK Fraktogel HW 40). The single fractions were tested (isolated guinea-pig ileum) for their bradykinin potentiating activity. Fractions C and F (Fig. 1) were active and were rechromatographed by HPLC (Ultropac TSK ODS C, material, 50 pm, column dimension 4.0 x 18.0 cm). Elution was performed with a solvent gradient: Solvent A, 0.005 CH,COONH, in water, pH = 5; solvent B, 80% methanol + 0.005 M CH,COONH,; 4 ml per min flow rate). Potenliating
activity
The potentiating activity of fraction F was determined on the isolated guinea-pig ileum as published previously (Ferreira et al., 1970). One ‘potentiating unit’ is defined as the amount of peptide per ml of incubation solution, able to double the contractions produced by a single dose of bradykinin. Assay on the isolated guinea-pig
ileum
Fraction F was tested on the isolated ileum from female guinea-pigs (Fig. 2). A strip of ileum muscle (l-2 cm) was suspended in a 5 ml muscle chamber with aerated Tyrode solution containing 1 pg/ml of atropine, at 37°C. F and standards were added just before the addition of bradykinin (Fig. 2). Assay on the isolated rat duodenum
Wistar male young rats (200-220 g) were used. A strip of duodenum muscle (l-2 cm) was suspended in a 5 ml muscle chamber with aerated Tyrode solution, pH 7.5, containing 1pg/ml of atropine, at 37°C. The active fraction (400 and 800 ng) was added just before addition of bradykinin (Fig. 3), and the preparation was washed for 60 set after each addition (Fig. 3). Rat arterial blood pressure
Fraction F was tested in normotensive conscious unrestrained rats (200-220 g). The animals were anaesthetized with ether, the femoral vein was canulated for drug injection, and the femoral artery canulated for recording the arterial blood pressure after 24 hr in a Beckman polygraph 411 R-A (0.1 mm/set paper speed and O-200 mm Hg calibrated scale) (Table 1). Angiotensin
converting enzyme
(ACE) inhibition
To measure the inhibition, 50 ng ACE and 2 ng of fraction F were incubated in 1 ml Tyrode solution for 15, 30, 45 and 60 min. Bradykinin (100 ng) was used as substrate. The residual activitv was calculated usine a lon dose-response curve for-spontaneous bradykinin hydrolysis in the absence of ACE under the same condition; as in the assay on the isolated guinea-pig ileum. The fraction was omitted from control samples (Fig. 4).
Fig. 1. HPLC chromatogram of A. p. pisciuorus venom. (a) First preparation and designation of fraction F; (b) last purification for amino acid sequencing and mass spectrum.
Peptidc F
1315
:E ,
R
4
,~,,,1,1,‘,,,,,1,11,,,,,,,,~~,,,~~,~~,,,,,,,,,,,,,,~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
5
IO
I5
20
25
30
35
W 200,
40
45
50
55
60
65
70
75
II0
85
90
2nd nanement “9-2”
Fig. 1.
L. A. F. FERREIRA
1316
ng
9K
9K
BK
BK
FtBK
10
20
40
80
49
52
55
min46
BK
et al.
BK
BK Cop+BK
4001010
20
40200010
56
73
76
5970
60
63
BK
BK BK gpt6K
5
10206005
93
96101
iO4a
Fig. 2. Potentiation of bradykinin by fraction F (AppF) and potentiator standards. Bradykinin activity was assayed by contraction of isolated guinea-pig ileum. Synthetic bradykinin (IMOng) was added to 5ml organ bath at 37°C and 3 min intervals. For measurement of bradykinin potentiating activity, fraction F (F), and standards (Cap: Captopril and Pep: Peptide P), were added 30 set before bradykinin (BK). Similar results to those shown were obtained in three separate experiments.
F+BK ng
min
400
00
F+BK 600
27
30
BK
BK
BK
60 160
60
40
33
36 39
Fig. 3. Activity of fraction F on isolated rat duodenum. Fraction F (400 and 800 ng) was added to isolated rat duodenum just before addition of bradykinin (BK 40-160 ng). The volume of the perfusion bath was 5 ml, and the preparation was washed for 60 set after each addition. Similar results were obtained in three separate experiments. Table
I. Hypotensive
Bradykinin (control) Treatment Hypotensive (mm Hg)
effect of fraction Fraction
F
F upon rat arterial
Fraction F f bradykinin
pressure
(0.5 pg)
(1.0 ng)
(1.0 pg, 0.5 Irg)
Fraction F (5.0 ng)
23.2 k 2.0
8.3 f 1.6
25.7 k 7.6
8.6 f 1.5
Fraction F + bradykinin (5.0 pg, 0.5 ng) 28.5 f 4.3
effect
Hypotensive effect of fraction F upon rat arterial blood pressure of conscious rats. The fraction F (1.0 and 5.0 ug was added just before the injection of bradykinin (BK; 0.5 pg, in 601~s). BK (0.5 pg) was injected alone as control (n = 6; *P -c 0.05 compared with control, Student’s t-test), mean f SE. No significant differences were found.
Peptide F
0
15
30
1317
45
60
Fig. 4. Inhibition of angiotensin converting enzyme (ACE) by fraction F. Inhibition curve obtained by assay on the isolated guinea-pig ileum; fraction F (2 pg) was incubated with 5Opg of ACE for different incubation times (15-60 min), using 1OOpg of bradykinin as substrate (-). Results are shown as means + SE. (n = 3).
Final purifcution
of peptide F
Peptide F was rechromatographed to obtain a material of highest purity for sequencing, at tirst with the aid of a RP ll-Vydac column (4 x 250 mm; 5 pm) with a gradient of 10 mM ammonium hydrogencarbonate (pH 8) as solvent A, and 80% acetonitrile as solvent B. The last purity check was performed by chromatography on a Microbore column packed with Vydac C,, (2 x 100 mm; 5 pm). Solvent A was 0.05% trifluoroacetic acid, and solvent B 80% acetonitrile/O.OS% trifluoroacetic acid. Both solvents were pH 3. No impurity was detectable (Fig. 1).
612.5
556.6
Fig. 5. Mass spectrum of the reaction product after PGAP digestion of peptide F. The small peak at 1224.2 represents the whole pcptide F. and the small peak at 1112.3 the residue after loss of pyroglutamic acid.
1318
L. A. F. FERREIRA et al.
Mass spectrometry
The mass spectroscopic investigations were performed with an API-Biomolecular Mass Analyser, by atmospheric pressure ionization (electrospray). Registration of the molecular fragments was carried out using a quadrupole device (Fig. 5). Amino acid sequence
The sequence was determined computer-controlled program.
with an ABI sequencer according to the Edman degradation,
using a
Enzymatic degradation
Direct sequencing by Edman degradation was not possible with peptide F, owing to the presence of pyroglutamic acid as the N-terminal amino acid, which therefore had to be removed enzymatically. The buffer consisted of 50 mM Na,HPO,, 2 mM EDTA, and 2 mM DTE in 100 ml aqua bidestillata. This solution was brought to nH 8 bv adding dropwise 0.1 M NaOH. Twelve micrograms of peutide F was dissolved in 90 ul of this buffer, and 10 kJ pyroglutamic acid peptidase in 10 ~1 buffer was added: Incubation at 37°C lasted for’8 hr. The reaction then was stopped by adding 0.01 M HCl, and after lyophilization a sample was investigated by mass spectrometry. Purification of the peptide mixture for sequencing was performed by HPLC as described above.
RESULTS AND DISCUSSION
A bradykinin potentiating
fraction F (AppF) was isolated from the venom of Agkby a preparation (fraction F), a log dose-response bradykinin curve for the effect of bradykinin (BK) on an isolated ileum preparation was made first. Fraction F (400 ng) was able to potentiate two-fold the effect of bradykinin on the isolated guinea-pig ileum, and it proved to be more active than captopril and peptide P (Fig. 2). Fraction F did not potentiate the effect of bradykinin (80ng) in relaxing the isolated rat duodenum at concentrations of 400 and 800 ng. Fraction F was able to increase the duration of the relaxing effect of bradykinin on rat duodenum, although the effect was not dose related. Hence, we observed a potentiating by bradykinin of the time of relaxation of duodenum (Fig. 3). After 15 min, 2 pg of fraction F caused 50% inhibition (I,,) of the activity of ACE against bradykinin, as determined by biological activity on the isolated guinea-pig ileum. This result suggests that the potentiating activity of fraction F is not solely due to the inhibition of the endogenous angiotensin-converting enzyme (ACE) present in tissue of the ileum. In addition, injection of fraction F (1 pg and 5 pg) elicited non-dose-related hypotensive effects. No potentiation with bradykinin was observed, even when BK was injected 1, 5 and 15 min after injecting fraction F. Mass spectral analysis (Fig. 5) shows a mass of 1224.2 Da for the whole peptide F, and a second one of 1112.3 Da for the peptide minus pyroglutamic acid. These mass numbers are in accordance with the calculated value of 1223.6 for the intact peptide F, as derived from the sequence: pGlu-Leu-Trp-Pro-Arg-Pro-His-Ile-Pro-Pro. The sequence of this peptide is similar to other bradykinin potentiating peptides from snake venoms. In nearly all of them, we find a sequence Ile-Pro-Pro for the carboxyl terminus of the peptide chain, and it is most probable that this end binds to the active site of kininase II (angiotensin converting enzyme), while pyroglutamic acid at the N-terminal is not degradable by most enzymes and therefore is responsible for prolongation of the binding time. Among these peptides, with pyroglutamic acid being the N-terminus and Ile-Pro-Pro being the Cterminus, peptide F is the first one with a histidine residue adjacent to the Be-Pro-Pro terminus. istrodon piscivorus piscivorus. To determine the potentiation
Peptide F
1319
Agkistrodon piscivorus piscivorus is a snake species which does not possess specifically acting toxins (Clark and Higginbotham, 1971). The symptoms following a bite are a synergistic effect of many enzymes and mediators. Apart from the local reactions, the main systemic effects are directed towards blood pressure and circulatory system. In this respect, the bradykinin potentiating peptides are essential for the understanding of this type of envenomation.
Acknowledgemenrs-Thanks are due to the Internationales Biiro des Forschungsxentrums Jiilich as well as to the Fonds der Chemischen Industrie. This project is also supported by FAPESP and FundaButantan.
REFERENCES Bamberg, U., Elg, P. and Stelwagen, P. (1960) Tryptic and plasmatic peptide fragments increasing the effect of bradykinin on isolated smooth muscle. &and. J. clin. Lab. Inoest. 24 (Suppl. 107), 21-35. Clark, J. M. and Higginbotham, R. D. (1971) Cottonmouth mocassin venom fraction of toxic and allergenic components and interaction with tissue mast cells. Tex. Repf. Eiol. Med. 29, 181. Ferreira, S. I-I. (1965) A bradykinin potentiating factor (BPF) present in the venom of Bothrops jararaca. J. Pharmac. Chemother. 24, 163-169.
Ferreira, S. H., Bartelt, D. C. and Greene, L. J. (1970) Isolation of bradykinin potentiating peptides from Bothrops jararaca venom. Biochemistry 9, 2583-2593. Ferreira, L. A. F., Henriques, 0. B., Lebrun, I., Battista, M. B., Prezoto, B. C., Andreoni, S. S. A., Zelnik, R. and Habermehl, G. (1992) A new bradykinin potentiating peptide (Peptide P) isolated from the venom of Bothrops jararacussu.
Toxicon 30, 33-40.
Nishimura, K., Yoshida, N., Hiwada, K., Ueda, E. and Kokubu, T. (1977) Purification of angiotensin-converting enzyme from human lung. Biochem. biophys. Acta 483, 398408. Rocha e Silva, M., Texeira, W. B. and Rosenfeld, G. (1949) Bradykinin, a hypotensive and smooth muscle stimulating factor released from plasma globulin by snake venoms and by trypsin. Am. J. Physiol. 1!%,261-273.
Toxicon, Vol. 33, No. IO, pp. 13134319, 1995 Copyristn0 1995 Elsevier Scimcc Ltd Printed in Great Britain. All rights resewed 00414101/95 s9.50+0.00
STRUCTURE AND EFFECTS OF A KININ POTENTIATING FRACTION F (AppF) ISOLATED FROM AGKISZ-XODON PISCIVORUS PISCIVORUS VENOM L. A. F. FERREIRA,’ T. Mi5LLRING,Z F. L. A. S. LEBRUN,3 M. RAIDA, R. ZNOTTKA4 and G. G. HABERMEHL** ‘Departamento de Bioquimica e Biofisica, Instituto Butantan, SBo Paulo, Brazil; *Department of Chemistry, School of Veterinary Medicine, Bischofsholer Damm 15, D-30173 Hannover, Germany; ‘Departamento de Fisiologia e Biofisica, Instituto de Ciencias Biomedicas USP, SHo Paulo, Brazil; and 4Lower Saxonian Institute of Peptide Research, Hannover, Germany (Received 23 January 1995; accepted 24 April 1995)
L. A. F. Ferreira, T. Miilhing, F. L. A. S. Lebrun, M. Raida, R. Znottka and G. G. Habermehl. Structure and effects of a kinin potentiating fraction F (AppF) isolated from Agkistrodon piscivorus piscivorus venom. Toxicon 33, 1313-1319, 1995.-The contractile action of bradykinin on isolated smooth muscles is known to be potentiated by special peptides isolated from snake venoms and other animal sources. A fraction F (AppF) has been derived from Agkistrodon piscivorus piscivorus venom. This fraction does not increase the depressor effect of bradykinin on blood pressure, potentiates the action of bradykinin on isolated guinea-pig ileum and prolongs the duration of relaxation in isolated rat duodenum. This fraction was able to inhibit the conversion of angiotensin I to angiotensin II in vitro. The primary structure of this decapeptide is given. INTRODUCTION
Kinins are defined as hypotensive polypeptides which contract extravascular isolated smooth muscle preparations, relax the rat duodenum, decrease the arterial blood pressure of mammals and cause pain. There is evidence that kinins are important in various physiological and pathological conditions in mammals as well as in other animals. Five kinins have been found in mammalian plasma, and one of them, bradykinin, discovered by Rocha e Silva et al. (1949), has been well studied. Ferreira (1965) found in the venom of the snake Bothrops jararaca a factor which potentiates the effects of bradykinin (Bamberg et al., 1969), isolated bradykinin potentiating peptides from a tryptic hydrolysate of human plasma, and studied their activities. Ferreira et al. (1992) found six bradykinin potentiating peptides in the venom of B. jararacussu and studied one of them, denominated peptide P. We have recently isolated a peptide from Agkistrodon piscivorus piscivorus venom, which we named peptide F. It potentiates the effect of bradykinin on the isolated guinea-pig ileum, does not potentiate the effects of bradykinin upon rat arterial blood pressure and *Author to whom correspondence
should be addressed. 1313
1314
L. A. F. FERREIRA et al.
inhibits the angiotensin-converting enzyme from rat plasma. The amino acid sequence has been determined. It fits into a group of other bradykinin potentiating peptides, the N-terminal amino acid being pyroglutamic acid, and the C-terminus consisting of the sequence Ile-Pro-Pro. MATERIALS AND METHODS dried venom was obtained from Ventoxin (Frederick, MD, U.S.A.). Captopril, pyroglutamyl-aminopeptidase (PGAP) and bradykinin were purchased from Sigma Chemical Co. (St Louis, MO, U.S.A.). Peptide P was from the Instituto Butantan (Slo Paulo, Brazil). Angiotensin converting enzyme was purified from rat plasma (Nishimura et al., 1977). Agkistrodon
piscivorus
Isolation of fraction
pisciuorus
F
Dried venom (3 g) was dissolved in 180 ml water, and 540 ml boiling ethanol was added to precipitate proteins. After centrifugation, the solution was lyophilized and dissolved again in 100 ml water; 100 ml 6 M (NH,),SO, solution was added, the precipitate was centrifuged, and after centrifugation, 300 ml isopropanol (98%, HPLC pure) was added for a third precipitation. Lyophilization of the supernatant yielded 632 mg of a colourless material, which was dissolved in 6.0 ml 0.05 M (NH,HCO,) buffer solution and separated by HPLC (glass column TSK Fraktogel HW 40). The single fractions were tested (isolated guinea-pig ileum) for their bradykinin potentiating activity. Fractions C and F (Fig. 1) were active and were rechromatographed by HPLC (Ultropac TSK ODS C, material, 50 pm, column dimension 4.0 x 18.0 cm). Elution was performed with a solvent gradient: Solvent A, 0.005 CH,COONH, in water, pH = 5; solvent B, 80% methanol + 0.005 M CH,COONH,; 4 ml per min flow rate). Potenliating
activity
The potentiating activity of fraction F was determined on the isolated guinea-pig ileum as published previously (Ferreira et al., 1970). One ‘potentiating unit’ is defined as the amount of peptide per ml of incubation solution, able to double the contractions produced by a single dose of bradykinin. Assay on the isolated guinea-pig
ileum
Fraction F was tested on the isolated ileum from female guinea-pigs (Fig. 2). A strip of ileum muscle (l-2 cm) was suspended in a 5 ml muscle chamber with aerated Tyrode solution containing 1 pg/ml of atropine, at 37°C. F and standards were added just before the addition of bradykinin (Fig. 2). Assay on the isolated rat duodenum
Wistar male young rats (200-220 g) were used. A strip of duodenum muscle (l-2 cm) was suspended in a 5 ml muscle chamber with aerated Tyrode solution, pH 7.5, containing 1pg/ml of atropine, at 37°C. The active fraction (400 and 800 ng) was added just before addition of bradykinin (Fig. 3), and the preparation was washed for 60 set after each addition (Fig. 3). Rat arterial blood pressure
Fraction F was tested in normotensive conscious unrestrained rats (200-220 g). The animals were anaesthetized with ether, the femoral vein was canulated for drug injection, and the femoral artery canulated for recording the arterial blood pressure after 24 hr in a Beckman polygraph 411 R-A (0.1 mm/set paper speed and O-200 mm Hg calibrated scale) (Table 1). Angiotensin
converting enzyme
(ACE) inhibition
To measure the inhibition, 50 ng ACE and 2 ng of fraction F were incubated in 1 ml Tyrode solution for 15, 30, 45 and 60 min. Bradykinin (100 ng) was used as substrate. The residual activitv was calculated usine a lon dose-response curve for-spontaneous bradykinin hydrolysis in the absence of ACE under the same condition; as in the assay on the isolated guinea-pig ileum. The fraction was omitted from control samples (Fig. 4).
Fig. 1. HPLC chromatogram of A. p. pisciuorus venom. (a) First preparation and designation of fraction F; (b) last purification for amino acid sequencing and mass spectrum.
Peptidc F
1315
:E ,
R
4
,~,,,1,1,‘,,,,,1,11,,,,,,,,~~,,,~~,~~,,,,,,,,,,,,,,~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
5
IO
I5
20
25
30
35
W 200,
40
45
50
55
60
65
70
75
II0
85
90
2nd nanement “9-2”
Fig. 1.
L. A. F. FERREIRA
1316
ng
9K
9K
BK
BK
FtBK
10
20
40
80
49
52
55
min46
BK
et al.
BK
BK Cop+BK
4001010
20
40200010
56
73
76
5970
60
63
BK
BK BK gpt6K
5
10206005
93
96101
iO4a
Fig. 2. Potentiation of bradykinin by fraction F (AppF) and potentiator standards. Bradykinin activity was assayed by contraction of isolated guinea-pig ileum. Synthetic bradykinin (IMOng) was added to 5ml organ bath at 37°C and 3 min intervals. For measurement of bradykinin potentiating activity, fraction F (F), and standards (Cap: Captopril and Pep: Peptide P), were added 30 set before bradykinin (BK). Similar results to those shown were obtained in three separate experiments.
F+BK ng
min
400
00
F+BK 600
27
30
BK
BK
BK
60 160
60
40
33
36 39
Fig. 3. Activity of fraction F on isolated rat duodenum. Fraction F (400 and 800 ng) was added to isolated rat duodenum just before addition of bradykinin (BK 40-160 ng). The volume of the perfusion bath was 5 ml, and the preparation was washed for 60 set after each addition. Similar results were obtained in three separate experiments. Table
I. Hypotensive
Bradykinin (control) Treatment Hypotensive (mm Hg)
effect of fraction Fraction
F
F upon rat arterial
Fraction F f bradykinin
pressure
(0.5 pg)
(1.0 ng)
(1.0 pg, 0.5 Irg)
Fraction F (5.0 ng)
23.2 k 2.0
8.3 f 1.6
25.7 k 7.6
8.6 f 1.5
Fraction F + bradykinin (5.0 pg, 0.5 ng) 28.5 f 4.3
effect
Hypotensive effect of fraction F upon rat arterial blood pressure of conscious rats. The fraction F (1.0 and 5.0 ug was added just before the injection of bradykinin (BK; 0.5 pg, in 601~s). BK (0.5 pg) was injected alone as control (n = 6; *P -c 0.05 compared with control, Student’s t-test), mean f SE. No significant differences were found.
Peptide F
0
15
30
1317
45
60
Fig. 4. Inhibition of angiotensin converting enzyme (ACE) by fraction F. Inhibition curve obtained by assay on the isolated guinea-pig ileum; fraction F (2 pg) was incubated with 5Opg of ACE for different incubation times (15-60 min), using 1OOpg of bradykinin as substrate (-). Results are shown as means + SE. (n = 3).
Final purifcution
of peptide F
Peptide F was rechromatographed to obtain a material of highest purity for sequencing, at tirst with the aid of a RP ll-Vydac column (4 x 250 mm; 5 pm) with a gradient of 10 mM ammonium hydrogencarbonate (pH 8) as solvent A, and 80% acetonitrile as solvent B. The last purity check was performed by chromatography on a Microbore column packed with Vydac C,, (2 x 100 mm; 5 pm). Solvent A was 0.05% trifluoroacetic acid, and solvent B 80% acetonitrile/O.OS% trifluoroacetic acid. Both solvents were pH 3. No impurity was detectable (Fig. 1).
612.5
556.6
Fig. 5. Mass spectrum of the reaction product after PGAP digestion of peptide F. The small peak at 1224.2 represents the whole pcptide F. and the small peak at 1112.3 the residue after loss of pyroglutamic acid.
1318
L. A. F. FERREIRA et al.
Mass spectrometry
The mass spectroscopic investigations were performed with an API-Biomolecular Mass Analyser, by atmospheric pressure ionization (electrospray). Registration of the molecular fragments was carried out using a quadrupole device (Fig. 5). Amino acid sequence
The sequence was determined computer-controlled program.
with an ABI sequencer according to the Edman degradation,
using a
Enzymatic degradation
Direct sequencing by Edman degradation was not possible with peptide F, owing to the presence of pyroglutamic acid as the N-terminal amino acid, which therefore had to be removed enzymatically. The buffer consisted of 50 mM Na,HPO,, 2 mM EDTA, and 2 mM DTE in 100 ml aqua bidestillata. This solution was brought to nH 8 bv adding dropwise 0.1 M NaOH. Twelve micrograms of peutide F was dissolved in 90 ul of this buffer, and 10 kJ pyroglutamic acid peptidase in 10 ~1 buffer was added: Incubation at 37°C lasted for’8 hr. The reaction then was stopped by adding 0.01 M HCl, and after lyophilization a sample was investigated by mass spectrometry. Purification of the peptide mixture for sequencing was performed by HPLC as described above.
RESULTS AND DISCUSSION
A bradykinin potentiating
fraction F (AppF) was isolated from the venom of Agkby a preparation (fraction F), a log dose-response bradykinin curve for the effect of bradykinin (BK) on an isolated ileum preparation was made first. Fraction F (400 ng) was able to potentiate two-fold the effect of bradykinin on the isolated guinea-pig ileum, and it proved to be more active than captopril and peptide P (Fig. 2). Fraction F did not potentiate the effect of bradykinin (80ng) in relaxing the isolated rat duodenum at concentrations of 400 and 800 ng. Fraction F was able to increase the duration of the relaxing effect of bradykinin on rat duodenum, although the effect was not dose related. Hence, we observed a potentiating by bradykinin of the time of relaxation of duodenum (Fig. 3). After 15 min, 2 pg of fraction F caused 50% inhibition (I,,) of the activity of ACE against bradykinin, as determined by biological activity on the isolated guinea-pig ileum. This result suggests that the potentiating activity of fraction F is not solely due to the inhibition of the endogenous angiotensin-converting enzyme (ACE) present in tissue of the ileum. In addition, injection of fraction F (1 pg and 5 pg) elicited non-dose-related hypotensive effects. No potentiation with bradykinin was observed, even when BK was injected 1, 5 and 15 min after injecting fraction F. Mass spectral analysis (Fig. 5) shows a mass of 1224.2 Da for the whole peptide F, and a second one of 1112.3 Da for the peptide minus pyroglutamic acid. These mass numbers are in accordance with the calculated value of 1223.6 for the intact peptide F, as derived from the sequence: pGlu-Leu-Trp-Pro-Arg-Pro-His-Ile-Pro-Pro. The sequence of this peptide is similar to other bradykinin potentiating peptides from snake venoms. In nearly all of them, we find a sequence Ile-Pro-Pro for the carboxyl terminus of the peptide chain, and it is most probable that this end binds to the active site of kininase II (angiotensin converting enzyme), while pyroglutamic acid at the N-terminal is not degradable by most enzymes and therefore is responsible for prolongation of the binding time. Among these peptides, with pyroglutamic acid being the N-terminus and Ile-Pro-Pro being the Cterminus, peptide F is the first one with a histidine residue adjacent to the Be-Pro-Pro terminus. istrodon piscivorus piscivorus. To determine the potentiation
Peptide F
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Agkistrodon piscivorus piscivorus is a snake species which does not possess specifically acting toxins (Clark and Higginbotham, 1971). The symptoms following a bite are a synergistic effect of many enzymes and mediators. Apart from the local reactions, the main systemic effects are directed towards blood pressure and circulatory system. In this respect, the bradykinin potentiating peptides are essential for the understanding of this type of envenomation.
Acknowledgemenrs-Thanks are due to the Internationales Biiro des Forschungsxentrums Jiilich as well as to the Fonds der Chemischen Industrie. This project is also supported by FAPESP and FundaButantan.
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