G Á B O R K. TÓTH,*1 J Á N O S P A T A R I C Z A . t T A M Á S J A N Á K Y , * M A R I A N N A M Á K . Í M Á R T A Z A R Á N D I , * JULIUS G Y . P A P P t A N D B O T O N D PENKE*
*Department of Medical Chemistry, f Department of Pharmacology, Albert Szent-Györgyi Medical University, H-6720 Szeged, Hungary, and f Central Research Institute of Chemistry, H-1025 Budapest, Hungary ' x
Received 17 January 1995 1
TÓTH. G. K.. J. PATAR1CZA. T. JANÁKY, M. MÁK. M. ZARÁNDI, J. GY. PAPP AND B. PENKE. Synthesis of two peptide scorpion toxins and their use to investigate the aortic tissue regulation. PEPTIDES 16(7) 1167-1172, 1995.—The 37 amino acid residue polypeptides iberiotoxin and charybdotoxin, which contain three disulfide bridges, were chemically synthesized and char-acterized. The physiological effectiveness of these peptides was tested on rabbit aorta in vitro.
Charybdotoxin Iberiotoxin Scorpion toxins Solid-phase synthesis Formation of disulfide bridges Aortic tissue regulation
CHARYBDOTOXIN, a polypeptide containing three disulfide bridges, was isolated from the venom of the scorpion Leiurus quinquestriatus by Miller et al. (25,27). The three-dimensional structure of the isolated 37-mcr polypeptide was investigated by 2D NMR (3-5,32). A polypeptide with 68% homology to charybdotoxin (Fig. 1) was recently isolated and characterized from the venom of the scorpion Buthus tamulus by Galvez et al.
(14,18). This peptide, which has been named iberiotoxin, dis-plays similar biological activities as concerns the Ca~*-activated K* channels (30,33). Recently, Murphy et al. (13) synthesized it and characterized its 3D structure by 2D 'H-NMR spectroscopy (13,20). However, several successful syntheses of these peptides are described in the literature (22,23,31); the methods used are rather expensive (mainly Fmoc chemistry) and the described yields are quite low (approx. 1 %). The synthesis of the appro-priate hexasulfhydryl peptide chains can be carried out with bet-ter yields and simpler methods, but the refolding of the synthetic polypeptides containing multiple disulfide bonds remains one of the most challenging problems to surmount in the production of biologically activc peptides and proteins.
Calcium-activated potassium channels have been shown to play an important role in the regulation of the arterial tone (29).
The presence of a large or "big" conductance type (BKfJ of these channels was demonstrated in the smooth muscle cell membrane of the rabbit aorta with electrophysiological (17) and pharmacological methods (12). Depolarization of the cell
Charybdotoxin:
Glp-Phe-Thr-Asn-Val-5er-Cys-Thr Thr-Ser-Lys-Glu-Cys-Trp-Ser-Val-Cys-Gln-Arg-1
1 »—t H
-Leu-His-Asn-Thr-Ser-Arg-Gly-Lys-Cys-Met-Asn-Lys-Lys-Cys-Arg-Cys-Tyr-Ser-OH Iberiotoxin:
Glp-Phe-Thr-Asp-Val-Asp-Cys-Ser-Val-Ser-Lys-Clu-Cys-Trp-Ser-Val-Cys-Lys-Asp-1
1
L—i H
-Leu-Phe-Gly-Val-Asp-Arg-Gly-Lys-Cys-Met-Gly-Lys-Lys-Cys-Arg-Cys-Tyr-Gln-OH FIG. I. Primary structure of the two scorpion toxins.
membrane (11) and enhancement of intracellular calcium ([Ca2<],) (8) activate BKCa channels. Contraction of rabbit aorta elicited either by depolarization or by receptor stimulation was shown to be enhanced by a partially purified toxin of Leiurus quinquestriatus venom (9) and, subsequently, by nanomolar con-centrations of pure charybdotoxin (10). BK^, is the only known potassium channel in arterial smooth muscle that is blocked by charybdotoxin (29) and, therefore, the increased tone of the rabbit aorta by the toxin can be accepted as a blocking action on BKo, channels (30). In our experiments, isolated aortic tissue of rabbits was used as a tool for comparing the functional effectiveness of charybdotoxin and that of its more selective derivative, iberio-toxin (15). The effect of the iberio-toxins was measured on basal and
1 Requests for reprints should be addressed to Dr Gábor K. Tóth. Department of Medical Chemistry. Albert Szent-Györgyi Medical University, H-6720 Szeged. Dóm tér 8. Hungary.
1167
1168 T O T H ET A L .
0.6
5 0.2
19.47 min
1
V
5 10 15 20 25
TIME (mini
30
a 0 . 4
-10.07 min
1
5 10 15
TIME (mini
20 FIG. 2. HPLC profiles of the purified toxins. HPLC conditions: 4.6 X 250 mm 5 p CI 8 column (Vydac 2I8TP54) at a flow rate of 1.0 ml/min with the solvents A (0.1% aqueous TFA) and B (0.1% TFA in 80% aqueous MeCN) in linear gradient mode (gradient: 16.5-30.0% MeCN in 20 min for iberiotoxin, and 6.0-21.0% MeCN in 30 min for charybdotoxin). (A) Charyb-dotoxin and (B) iberiotoxin.
phenylephrine (PE)-induced isometric tone of aortic ring prepa-rations.
METHOD Synthesis of Peptides
T h e peptides were synthesized by a solid-phase technique, utilizing r-Boc chemistry (26). Side-chain protecting groups were as follows: Arg(Tos), G l u ( O c H e x ) , A s p ( O c H e x ) , His(Z), Tyr(2BrZ), L y s ( 2 C l Z ) , C y s ( M e b ) , Thr(Bzl), and Ser(BzI). Glp, Met, and Trp were unprotected. The peptides were synthesized on 1.0 g B o c G l n P a m resin ( 0 . 5 6 m m o l / g ) and 1.5 g B o c -S e r ( B z l ) - P a m resin (0.37 mmol/g), respectively, and the syn-theses were carried out manually. Couplings were performed with D C C , with the exception of Gin and Arg, which were in-corporated as their HOBt esters. After the coupling of the first Met or Trp, 0 . 5 % o f dithiothreitol w a s added to the c l e a v a g e mixture; this reagent proved to be sufficient to prevent side reactions under the acidolytic cleavage conditions (35). A m i n o acid incorporation were monitored by the ninhydrin test (21).
T h e peptide resins obtained (3.98 g of iberiotoxin and 4 . 4 g of charybdotoxin) were divided into t w o aliquot parts and treated with 6 ml dimethyl sulphide, 2 ml p-cresol, 2 ml p-thiocresol, 2 ml anisole, and 8 0 ml HF at 0°C for 1 h. After evaporation of HF under high vacuum, the mixture of the peptide and the resin w a s w a s h e d with dry diethyl ether, and the whole of the re-mainder was then solubilized without isolation in 2 M urea, 0.1 M glycine, 0.1 M NaCl buffer, pH 8.7, 2 0 0 0 ml volume, and folded (oxidized) by stirring overnight in air. The resulting folded crude peptides containing disulfide bridges were purified on a S H 1 M A D Z U L C 8 A preparative H P L C system. The resin w a s filtered off and the w h o l e 2 0 0 0 - m l solution was pumped onto the c o l u m n and separations were achieved on a 47 x 3 0 0 m m c o l u m n packed with C I S silica gel ( 3 0 0 A pore size, 1 5
-2 0 p m particle size) with solvents A (0.1% aqueous T F A ) and B (0.1% T F A in 80% aqueous M e C N ) using a gradient o f 1 3 -26% M e C N in 32 min for the iberiotoxin and 7 - 2 0 % M e C N in 32 min for the charybdotoxin. The flow rate was 8 0 ml/min and the column eluate was monitored at 2 2 0 nm.
Materials
Phenylephrine hydrochloride (PE) and pentobarbital sodium were purchased from Sigma Chemical Co. (St. Louis, MO). PE was dissolved in double-distilled water for obtaining 1 mmol/1 stock solution and stored at - 2 0 ° C . Dilutions were freshly made every day using K r e b s - H e n s e l e i t solution. Stock solutions of charybdotoxin and iberiotoxin (each 3 pmol/1 concentrations) were prepared in double-distilled water and stored at —20°C. All concentrations indicated in the text and in figures are expressed as final concentrations. The K r e b s - H e n s e l e i t solution consisted of (mmol/1): NaCl 120, KC1 4.2, CaCL 1.5, N a H C O , 20, MgCL
1.2, K H , P 04 1.2, and glucose 11.
Preparation of Isolated Rabbit Aorta
White N e w Zealand rabbits of either sex were anesthetized with 35 mg/kg pentobarbital sodium and then exsanguinated. The thoracic aorta was carefully removed, dissected free of adjoining connective tissue, and immersed in a room-temperature bath of K r e b s - H e n s e l e i t solution. The aorta was cut into 5 - m m rings and suspended in 100ml organ chambers filled with K r e b s H e n -seleit solution (bubbled with 95% 0 - and 5% CO, gas mixture;
pH 7.4 at 37°C).
Measurement of Isometric Tension
Rings were mounted in a recording chamber with a volume of 2 ml for isometric tension recording ( H u g o Sachs Elektronic,
FIG. 3. Fab mass spectra of the synthesized peptides, (a) Charybdotoxin, (b) charybdotoxin after reduction of the disulfide bridges, and (c) iberiotoxin.
a : CT Ident : 2_fl 18_21 Àcq: 7-JUI.-1992 13:43:33 +0 : gal : C 5 U (n/a) -SEQ rAB» Magnet Bpl:470416 TIC: 66914374«
Text :ttfa+gly
4.7E5 4 . 5E5 4.2E5 4 . OES 3.8E5 3.5E5 3. 3E5 3. IES 2. BES 2.6E5 2.4E5 2. IES 1.9E5 1.6E5 1.4E5 1.213 9. 4E4 7.1E4
3950 4000 4 050 4l'oO 4150 42W
42504300 4350 4400 4450 4500 4550 4600 4650
M/1 (Eat) a :CTRÊ0UJTUL Ident:l_31 Xcq: 7-JUL-1992 14:26:45 +1:00 Cal :CSI4(n/a)-SEQ FABt Magnat BpI:8581S2 TIC:1318931328 a Text :++dtt+tfa+gly
4303.9
3950 4000 4050 4100 4150 4200 4250 4300 4350 4400 4450 4500 4550 4600 4650 H /Z (Eat)
1170 T O T H ET A L .
0.08
0.04
5 10
TIME (mini
0.02
- 0 . 0 1
5 10
TIME (mini
FIG. 4. Capillary electropherogram of the purified toxins. Conditions: BioRad Biofocus 3000 apparatus. 50 //m X 50 cm uncoated capillary using 0.1 M phosphate buffer (pH 2.5) and 18 kV voltage. Detection was in 200 nm in single wavelength mode. (A) Charybdotoxin and (B) iberiotoxin.
Type F30, Germany). The recording chamber contained K r e b s -Henseleit solution. A resting tension of 10 m N was applied to the tissues, which were equilibrated for 4 5 min. Mechanical re-sponses o f arterial rings were displayed on a pen recorder ( K U T E S Z Type 175, Hungary).
T w o intact arterial rings from the same aorta were separately mounted on two isometric force transducers: one was used for detecting the constricting effect of PE and the other for measuring the action of charybdotoxin or iberiotoxin on PE-induced in-crease of arterial tone. A cumulative c o n c e n t r a t i o n - e f f e c t curve was constructed on both rings for PE ( 1 5 - 1 6 0 nmol/1), allowing a stable contractile response to develop before progressing to the next concentration o f the contractile agent; 9 0 nmol/1 charybdo-toxin or iberiocharybdo-toxin was applied in 6 2 p\ volume 30 min prior to the addition of the first PE dose. In the control (without charyb-dotoxin or iberiotoxin), 62 p\ of distilled water was incubated for 3 0 min.
Statistical Analysis
Changes in the baseline tension and in PE-induced contrac-tions were expressed in millinewtons. In the text, a minus symbol indicates a decrease of baseline tension compared to the initial resting tension. Values are given as mean ± SEM. Statistical evaluation of the data was performed by o n e - w a y analysis of variance ( A N O V A ) followed by N e w m a n - K e u l s multiple range test. Significance was accepted at the 95% confidence interval.
RESULTS
The peptides thus obtained (35.6 mg iberiotoxin and 15.9 mg charybdotoxin from one aliquot) were judged by using a H e w -lett-Packard H P - 1 0 9 0 liquid chroniatograph to be substantially ( > 9 7 % ) pure. The peptides were chromatographed on a 4 . 6 X 2 5 0 mm 5 p C I S column (Vydac 2 I 8 T P 5 4 ) at a (low rate of" 1.0 ml/min with the solvent system mentioned above in linear gra-dient mode. The retention time for iberiotoxin was 10.07 min (gradient: 1 6 . 5 - 3 0 . 0 % M e C N in 2 0 min) and that for charyb-dotoxin was 19.47 min (gradient: 6 . 0 - 2 1 . 0 % M e C N in 30 min)
(Fig. 2). The electropherogram of the upper peptides were made on a BioRad B i o f o c u s 3 0 0 0 apparatus in 5 0 pm X 5 0 cm un-coated capillary using 0.1 A7 phosphate buffer (pH 2.5) and 18 kV voltage. Detection was in 2 0 0 nm in single wavelength mode (Fig. 4). A m i n o acid analyses were: iberiotoxin: A s p 4.3, Glu 3.1, Ser 2.7, Gly 2.7, Arg 2.0, Tyr 1.0, C y s 5.0, Val 4.0, Phe 1.9, Leu 1.0, Lys 4.6, Met 1.1, Thr 1.1; charybdotoxin: A s p 2.9, Glu 2.7, Ser 3.7, Gly 1.0, Arg 2.6, Tyr 0.8, C y s 4.5, Val 2.0, Phe 1.1,
0 40 80 120 160
nmol/l phenylephrine
FIG. 5. Effect of charybdotoxin and iberiotoxin on phenylephrine-in-duced lone in isolated rabbit aorta. Charybdotoxin ( • , n - 10) and ib-eriotoxin (O, n = 8) increased the tone of aortic ring preparations com-pared to control without toxins ( • , n = 16). *p < 0.05 and **p < 0.01 represent significant differences compared to control; tP < 0.05 and f t / '
< 0.01 refer to significant differences between iberiotoxin and charyb-dotoxin.
SYNTHESIS OF SCORPION TOXINS 1171
Leu 0.9, Lys 3.2, Met 1.0, Thr 2.8, His 0.9, Pro 1.4. Both peptides were coeluted with natural rcfercnce standards and proved to be identical.
The FAB-MS experiments were performed with a VG ZAB-2SEQ type hybrid tandem mass spectrometer, equipped with an LSIMS source (Cs' ion gun used at 30 keV). Results: iheriotoxin:
calculated M + H: 4231.9, found: 4232.6 ± 0.5 [Fig. 3(c)l;
charybdotoxin: calculated M + H: 4296.9, found: 4297.7 ± 0.5 [Fig. 3(a)]; after reduction of the disulfide bridges with dilhioihrcitol:
calculated M + H: 4303.0, found: 4303.9 ± 0.5 |Fig. 3(b)).
Pharmacological Measurements
Baseline tension of isolated aorta was not significantly changed after 30-min incubation of rabbit aortic rings with 90 nmol/l charybdotoxin compared to the control treated with sol-vent (control = - 0 . 1 8 ± 0.14 mN, charybdotoxin = 1.10 + 0.82 mN, n = 10, NS). Similarly, 90 nmol/l iheriotoxin also did not change the resting tension of the rings during a 30-min incubation period (control = 0.0 ± 0.0 mN. iheriotoxin = 0.20 ± 0 . 1 4 mN, n = 8, NS). When PE, as a contracting agent, was added to the organ bath the B K o inhibitors enhanced the amplitude of eon-traction expressed in mN (Fig. 5). Charybdotoxin increased the effect of 160 nmol/l PE only whereas iheriotoxin significantly increased 15, 40, 80, and 160 nmol/l PE-induccd tone. Iherio-toxin was more effective than charybdoIherio-toxin against 40 and 80 nmol/l PE.
DISCUSSION Peptide Synthesis and Purification
The Boc syntheses of the scorpion toxins gave at least com-parable results as the described Fmoe syntheses; however, the cost of a Boc synthesis is far lower than the Fmoe synthesis. The overall yield of the synthetic process mentioned above is higher than the described ones. The key step of the production was find-ing the proper conditions for the formation of the three disulfide bridges by appropriate folding. After the HF cleavage, the solu-bilization and subsequent lyophilization of the crude unfolded peptide was avoided, becausc this procedure resulted in an enor-mous amount of side products. Different pH values were tried for the oxidation. In the first experiments, 0.1 M NHjOH and 2 M urea containing solutions were used, but the pH value was not stable during stirring the reaction mixtures in air to form the disulfide bridges. Therefore, in the latter experiments glycine-NaOH buffers were used. According to our findings the best pH for the folding is 8.7. For the folding, mainly the simple air ox-idation method was used. The formation of disulfide bridges us-ing oxidized/reduced glutathione gave similar results. The pep-tide purification proved to he the most effective, if we made separation only in the last step. The isolation of the hexasulfhy-dryl peptide is unnecessary, and time and work consuming. In some cases an appropriately pure product was resulted in only
one-step preparative RP-HPLC purification—in contrast of the described methods.
Aortic Tissue Regulation
In the present study, the functional effectiveness of charyb-dotoxin and iheriotoxin was tested on the mechanical activity of rabbit aorta in vitro. The two toxins serve as important pharma-cological tools for evaluating the role of calcium-activated po-tassium channels in regulatory pathways of different tissues.
Rabbit aorta was used to test the efficiency of the chemical syn-thesis because only one charybdotoxin-sensitive potassium chan-nel, BKo. is located in the smooth muscle of the arterial tissues (29). The role of B K o channels in the regulation of rabbit aortic tension has recently been demonstrated in detail (2). This study supported the presence of B K o channels in a single smooth mus-cle cell by using patch clamp method and 50 nmol/l charybdo-toxin was able to increase the tone of the blood vessel when 100-200 nmol/l PE was used as contractile agent. We also used PE, a specific ai-adrenergic receptor agonist. Stimulation of the a , receptor results in depolarization (19) and increase of [ C a - ' I (16,34) in rabbit aorta, both of which can open BKf, channels (6,8,11). Charybdotoxin (90 nmol/l), which corresponded to the magnitude of concentrations used by others for blocking B K o channels in rabbit aorta (2,12) and in other arterial preparations (1,7), increased 160 nmol/l PE-induced contraction .but did not affect the baseline tension. In another study, basal tone of rabbit aorta was also not changed by charybdotoxin (28), supporting 1) the lack of a nonspecific contractile action of the toxin, and 2) the absence of functional B K o channels in resting smooth mus-cle. Iheriotoxin also did not influence basal tone and was more effective than charybdotoxin against low concentrations of the contractile agent (40 and 80 nmol/l PE). The latter result is in agreement with the finding that iheriotoxin enhances smooth muscle tone more potently than charybdotoxin (24).
It is important to note that the effectiveness of the toxins in a functional model does not mean selectivity on a particular po-tassium channel. Charybdotoxin also blocks calcium-activated potassium channels other than B K o (IS). Pharmacological ago-nist-antagonist interactions cannot be performed because no spe-cific agonists for B K o channels are available at present. How-ever, the charybdotoxin-sensitive potassium channel is a well-characterized channel in rabbit aorta, and the mechanical responses of this tissue to selected concentrations of these toxins can be considered as antagonism on B K o channels (30). In con-clusion, our synthetic charybdotoxin and iheriotoxin are potent and valuable pharmacological tools for assessing the possible involvement of hyperpolarizing, calcium-activated potassium channels in the physiological and pathologic regulation of tissue responses.
ACKNOWLEDGEMENTS
The authors are indebted to Mr. R. Ferenci, Ms. É. Dôsai-Molnâr, and Ms. M. Fehér for skillful technical assistance and to Bachem Cali-fornia for financial support.
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2.17.
*~>"w l
J. P h a r m . P h a r m a c o l . 1995, 47: 921-925 © 1995 J. P h a r m . P h a r m a c o l Received J a n u a r v 26. 1995
Accepted July 20, 1995