PURIFICATION AND PROPERTIES OF A CARDIOACTIVE TOXIN, CARDIOLEPUTIN, FROM STONEFISH, Synanceja verrucosa

Abstract

Cardioleputin, a new cardioactive toxin, was purified from a stonefish venom using column chromatographies. The purified toxin was found to be an unstable protein that was susceptible to heat and freeze-thawing. This protein showed to have a molecular size of 46,000 daltons, and its amino acid composition was rich in serine and glycine, but low in basic amino acids. The crude venom induced a sudden drop in blood pressure and heart rate of rats right after administration. Both the blood pressure and heart rate returned to their original values as time elapsed, and thereafter continued to show a gradual decrease. In addition, crude venom actively affected the contractile response and suppressed the heart rate of guinea pig atria. The purified toxin caused irreversible inotropical and chronotropical increases in guinea pig atria. The action of the toxin on the atria was completely different from that of lysolecithin. It might be suggested that the toxin acts on the Ca<img SRC="http:/img/fbpe/jvat/v2n2/image2184.gif"> ion channel of the atrial membrane.

cardioactive toxin; cardioleputin; Synanceja verrucosa

Original paper

PURIFICATION AND PROPERTIES OF A CARDIOACTIVE TOXIN, CARDIOLEPUTIN, FROM STONEFISH, Synanceja verrucosa

T. ABE , M. SUMATORA, Y. HASHIMOTO , J. YOSHIHARA , Y. SHIMAMURA , J. FUKAMI

1 Institute of Physical and Chemical Research, Hirosawa 2-1, Wako-shi, Saitama 351, Japan, 2 Laboratory of Chemistry, BATAN, Jl. Cinere Pasar Jumat Kotak Pos 2, Kebayaran Lama, Jakarta Selatan, Indonesia.

ABSTRACT. Cardioleputin, a new cardioactive toxin, was purified from a stonefish venom using column chromatographies. The purified toxin was found to be an unstable protein that was susceptible to heat and freeze-thawing. This protein showed to have a molecular size of 46,000 daltons, and its amino acid composition was rich in serine and glycine, but low in basic amino acids. The crude venom induced a sudden drop in blood pressure and heart rate of rats right after administration. Both the blood pressure and heart rate returned to their original values as time elapsed, and thereafter continued to show a gradual decrease. In addition, crude venom actively affected the contractile response and suppressed the heart rate of guinea pig atria. The purified toxin caused irreversible inotropical and chronotropical increases in guinea pig atria. The action of the toxin on the atria was completely different from that of lysolecithin. It might be suggested that the toxin acts on the Ca ion channel of the atrial membrane.

KEY WORDS: cardioactive toxin, cardioleputin, Synanceja verrucosa

INTRODUCTION

Stonefish, called "Lepu" in Indonesia, are widely distributed in the shallow coral seas of tropical Indo-Pacific oceans. Stonefish are feared by fishermen because of their venom. Victims of stonefish envenoming frequently experience severe pain, edema, cardiac arrhythmia and sometimes death. Stonefish venoms of three species Synanceja verrucosa, S. trachynis and S. horrida were studied in the past. All stonefish species possess pointed spines, each with a pair of venom glands, located in the dorsal, pectoral and anal fines, which secrete toxic compounds. A study of the dorsal spine venom was reported in 1889 by Bottard(4). The hemolytic activity of venom from S. horrida was reported by Duhig and Jones(6). These and other studies revealed that: 1. the venomous activity is preserved in saline, 40% glycerol and by freezing at -20ºC; 2. the LD50 of the venom is 15 mg/kg in rabbits(14); 3. the lethal fraction is a water-insoluble protein that is unstable at pH lower than 5.5 or higher than 9.0 and at temperature over 50ºC; 4. on electrophoresis, the fraction migrates only slightly from the origin(16); 5. hypotension induced at low venom concentrations is likely to be produced by an increase in capillary permeability(17), and 6. the cause of death of lethal doses of venom may be a respiratory arrest by irreversible neuromuscular blockade of the diaphragm(8,11). Regarding S. trachynis venom , venomous properties similar to those of S. horrida venom have been reported(9,20). In addition to these properties, S. trachynis venom shows hyaluronidase activity(3) and blocks the release of transmitter from the presynaptic membranes of murine and frog neuromuscular junctions(10). S. verrucosa venom is nondialyzable and causes hypotension and increase in respiratory rate(15). Thus, information on stonefish venoms from several species show common properties, although their comprehensive characteristics have not yet been clearly identified. Until today, information has slowly been accumulated because of the scarcity of available fish spines and the instability of the venom. This paper describes the study conducted to characterize the isolated toxin from S. verrucosa, as well as its effects on the cardiovascular system.

MATERIAL AND METHODS

MATERIAL: CM-Sephadex C-50, Sephacryl S-200, DEAE-Sephadex A-50 and Sephadex G-75 were purchased from Pharmacia Fine Chemical Co. (Upsala, Sweden). Butyl Toyopearl was provided by Toso Chemical Co. (Tokyo, Japan). Lysolecithin was purchased from Wako Chemical Co. (Tokyo, Japan).

PREPARATION OF STONEFISH CRUDE VENOM: Stonefish (S. verrucosa ) specimens were transported by air on dry ice from Indonesia. Upon arrival, the venomous spines from the dorsal, pectoral and anal fines were removed and kept at - 80ºC until use. Venomous spines (323g) were crushed with a mortar and a pestle under freezing in liquid nitrogen. The frozen powder was homogenized with 646 ml of 50mM acetate buffer, pH 5.2, using a Polytron homogenizer (Kinematica, Lucerne, Switzerland). The homogenate was centrifuged at 15,000xg for 20 min at 2ºC. The supernatant was used as crude stonefish venom.

PURIFICATION BY COLUMN CHROMATOGRAPHY: Sephadex G-75 and Sephacryl S-200 column chromatographies were carried out isocratically. CM-Sephadex C-50, DEAE-Sephadex A-50 and Butyl Toyopearl column chromatographies were performed by the linear gradient method at 4ºC. The fractions were collected by a LKB UltroRac II fraction collector, and the eluates were monitored at 280 nm by a REC-2 recorder.

CONCENTRATION, DESALTING AND PROTEIN ASSAY OF SAMPLES: Cardioactive fractions were concentrated on a Diaflow YM-5 membrane using an Amicon concentration system under nitrogen gas pressure(1). The concentrated samples were dialyzed overnight in appropriate concentrations of a buffer using Visking tubing. For the cardiovascular assay, 0.5 ml of each sample was desalted on a Sephadex G-50 minicolumn (0.5x10cm). Protein concentrations were assayed by the method of Lowry(12).

POLYACRYLAMIDE GEL ELECTROPHORESIS: Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis was conducted in a 7.5% polyacrylamide disc gel at pH 8.7(1).

AMINO ACID ANALYSIS: About 20µg of the lyophilized protein sample was dissolved in 0.1 ml of distilled 5.8 N HCl and deaerated completely by the one-touch degassing cock. The shielded samples were hydrolyzed at 105ºC for 24h. Amino acid and amino sugars were analyzed by an automated Hitachi 835 amino acid analyzer detected by ninhydrin.

ASSAY OF RAT CARDIOVASCULAR SYSTEM: Male Wistar rats weighing 180-200g were anesthetized with Nembutal (pentobarbital) (50mg/kg) and fixed on a dissection table warmed to 37ºC. The right femoral vein was cannulated with a polyethylene tube (SP28, 0.8mm; Natsume, Tokyo, Japan) for sample injection. The left femoral artery was also cannulated for the measurement of the blood pressure. The cannula was connected to a blood pressure transducer P10EZ (Gould, NJ, USA), and the pressure was amplified by a blood pressure amplifier AP-641G (Nihonkoden Co., Tokyo, Japan) at a sensitivity of 50 mmHg/D.V. Electrocardiograms were analyzed by the second indication method(18), using an electrocardiogram amplifier AC-601G (Nihonkoden Co., Tokyo, Japan) at a sensitivity of 0.5 mV/D.V. The heart rate was calculated through the electrocardiograms using a heart rate unit AC-611G (Nihonkoden Co., Tokyo, Japan). Body temperature was also measured by a temperature amplifier unit AW-601H (Nihonkoden Co., Tokyo, Japan).

ASSAY OF GUINEA PIG ATRIA: Female guinea pigs were sacrificed by cervical dislocation. The right atrial strip was carefully isolated from the heart and incubated in 3.5 ml of organ bath filled with Krebs-Hemseleit solution of the following composition (mMol): NaCl 118, KCl 4.7, CaCl2 2.55, MgSO4 1.18, KH2PO4 1.18, NaHCO3 24.88 and Glucose 11.1 at 37ºC through which a mixture of 95% O2 and 5% CO2 was bubbled. Tension was measured by an isometric transducer (Nihonkoden Co., Tokyo, Japan) and a tension amplifier EF-601G (Nihonkoden Co., Tokyo, Japan).

RESULTS

EFFECTS OF CRUDE STONEFISH VENOM ON THE RAT CARDIOVASCULAR SYSTEM: When 0.2 ml of stonefish crude venom diluted 5 times was injected intravenously into a rat, there was an immediate decrease in blood pressure followed by a decrease in the heart rate (Figure 1). Subsequently, both blood pressure and heart rate recovered within a few minutes and continued thereafter to show gradual decrease. The contractile response of guinea pig atria increased quickly upon the addition of stonefish venom and the level remained high (Figure 2). In contrast, the heart rate showed a smooth gradual curve which slightly decreased in the beginning, but increased as time elapsed. These facts show that the crude venom contains cardioactive compounds that affect cardiac muscle contraction, heart rate and blood pressure.

FIGURE 1.
Effects of stonefish S. verrucosa crude venom on rat blood pressure and heart rate. The venom was administered at the arrows.

FIGURE 2.
Effects of stonefish crude venom on guinea pig atria. The venom (50µl) was administered at the arrows, then washed out at the *.

PURIFICATION OF CARDIOLEPUTIN, THE STONEFISH CARDIOACTIVE PROTEIN: A total of 740 ml of crude venom was obtained from 323 g of stonefish spines. The spines were chromatographed on a CM-Sephadex column (5.5x65.5 cm) equilibrated with 50 mM acetate buffer, pH 5.2. The sample was eluted by a linear gradient of 0 to 2 M NaCl (2 l each) in 50 mM acetate buffer, pH 5.2, and collected in 18 ml fractions. The fractions eluted first (total 1.4 l), which had a low affinity for CM Sephadex, caused an increase in cardiac contraction and a decrease in heart rate (Figure 3 A). This fraction was applied to a Sephacryl S-200 column (2.7x65.5) and eluted with 50 mM acetate buffer, pH 5.2. An activity was found at a molecular size of about 50,000 daltons (Figure 3 B). The activity was further purified on a DEAE-Sephadex A-50 column (1.8x36 cm) eluted with a linear gradient of 50 mM tris-HCl, pH 8.1 (150 ml) and the same buffer containing 0.8 M NaCl (150 ml) (Figure 3 C). The resulting blue-colored active fractions were concentrated and applied to a Sephadex G-75 column (2.2x98 cm) (Figure 3 D). The gel filtration was carried out with 50 mM acetate buffer, pH 5.2, and 3.7 ml fractions were collected. Finally, this active fraction was eluted by a linear gradient of 15% ammonium sulfate (100 ml) to 5% glycerol (100 ml) on a Butyl-Toyopearl column (2.2x30 cm). Very high contractile activity was found in fractions number 8 to 12 (Figure 3 E). The active compound, cardioleputin, was separated from the blue protein and showed a single band on polyacrylamide disc gel electrophoresis as shown in Figure 4. The yields of purification procedures are shown in Table 1.

FIGURE 3.
Purification procedures of the stonefish S.verrrucosa venom using column chromatographies. A: CM-Sephadex column chromatography of crude venom. B: Sephacryl S-200 column chromatography of Fr.a. C: DEAE-Sephadex A-50 column chromatography of Fr.b. D: Sephadex G-75 column chromatography of Fr.d. E: Butyl-Toyopearl column chromatography of Fr.e.

FIGURE 4.
Polyacrylamide disc gel electrophoresis of cardioleputin.

TABLE 1.
Purification procedure and yield of cardioleputin.

* Supressive components were contained.

PHYSICAL-CHEMICAL PROPERTIES OF CARDIOLEPUTIN: The molecular size of cardioleputin was estimated to be 46,000 daltons by SDS-polyacrylamide disc gel electrophoresis and 46,300 daltons by amino acid composition. The amino acid composition of cardioleputin was analyzed as shown in Table 2. The protein was found to contain glucosamine, but no cystine was detected. The serine and glycine contents were relatively higher than those of the other amino acids. In addition, the protein was found to be low in components of the basic amino acids. Subsequent studies on the stability of cardioleputin showed that the toxic activity was lost by freeze-thawing, high temperature and dilution.

TABLE 2.
Amino acid composition of cardioleputin

EFFECT OF CARDIOLEPUTIN ON CARDIOVASCULAR SYSTEM: The administration of cardioleputin to guinea pig atria promoted a rapid increase in the contractile response and a moderate increased in the heart rate (Figure 5 A). It is clear that cardioleputin is active on guinea pig atria both inotropically and chronotropically. After washing out the toxin from the atria, neither the contractions nor the heart rate returned completely to normal. From the results, it may be stated that cardioleputin causes irreversible activity in the atria. These effects are different from those of toxins that possess phospholipase A2 activity, since lysolecithin produced by phospholipase A2 caused an increase in the heart rate, but had no effect on the contractile response as shown in Figure 5 B. Furthermore, cardioleputin had no hemolytic activity at the concentration that acted on the atria. This suggests that cardioleputin does not have phospholipase A2 properties.

FIGURE 5.
Effects of cardioleputin (A) and lysolecithin (B) on the contractile response (a) and heart rate (b) in the same guinea pig atria. The toxin (20µl) was administered at the arrows and washed out at the *. Lysolecithin of 10µg was administered at the arrow.

DISCUSSION

Two major toxic effects of the stonefish venom on the cardiovascular and respiratory systems known to date are hypotension and respiratory arrest(2,11,14,15,17,20). Other disorders such as hemolysis, myocardial injury and necrosis, as well as hyaluronidase activity were reported(3,6,9,15). It was found that the lethal activity of stonefish venom is the blockade of respiration leading to the failure of cardiac function(2,5,14). However, although many cardiac disorders caused by the venom are known, isolation of the toxic compounds was not performed. The cardioleputin isolated in our experiments caused an irreversible increase both in contraction and in heart rate of guinea pig atria.

The instability of the stonefish venom has been shown in several studies(9,14,16,20). In this experiment, subsequent purification steps caused lower yields. These may have been caused by the instability of the toxin. Among the toxic effects of stonefish venom identified to date, the components causing hemolysis, cytolysis, vascular permeability and eventual death are known to have a molecular size of about 150,000 daltons(9,13). Cardioleputin was found to have a molecular size of 45,000 daltons, and thus is a molecular species different from the toxic components described above. Other cardioactive venom components that were high-molecular-weight proteins were also found (unpublished data). It can be said that the instability of stonefish venom is caused by these higher-molecular-weight components which are easily denatured. Considering the mechanism of invasion of stonefish venom into the body via a sharp sting injury under water, it is an advantage for the venom to be adapted to the marine environment. The high-molecular-weight proteins of high ionic strength bind irreversibly or cause irreversible reactions under conditions in which washing out in sea water is likely(14).

Well-known cardiotoxins in cobra snake venom affect membranes in a way similar to the action of melittin. They activate membrane phospholipase C, triglyceride hydrolysis and membrane disruption(7). In isolated guinea pig atria, these toxins induce a twitch contractile response and suppress the stimulated contraction(11). These peptide-like toxins are completely different from cardioleputin both in molecular species and in function. However, both toxins show a contractile induction of cardiac muscle. In contrast, stonefish venom shows strong hemolytic and necrotic activities. Thus, it is most likely that stonefish venom contains phospholipase components that also cause hypotension and respiratory arrest. It remains to be determined whether cardioleputin has phospholipase A2 activity. The experiment on the myocardial effects of lysolecithin shown in Figure 5 B suggests that the effect is different phospholipase A2 activity, since cardioleputin induces both chronotropic and inotropic actions, while lysolecithin shows only chronotropic activity. Moreover, cardioleputin produces a faster response in contraction than the chronotropic increase. This effect appears to be similar to ß-adrenergic action rather than phospholipase A2 action. Specifically, purified phospholipase A2 from hornet venom show a similar activity to that of lysolecithin. In other words, it produces a slow increase in inotropic action but a rapid increase in chronotropic response (unpublised data).

These remarkable actions suggest that an increase in Ca ion influx into cells(19) as well as an increase in K ion efflux into pacemaker cells(13) will result in membrane modification by cardioleputin. The toxin would mainly affect the Ca ion channel protein or modify the membrane to be near the channel proteins These properties could explain how the toxin affects the decrease of membrane perturbation. Further detailed analysis will be needed in the future.

ACKNOWLEDGEMENTS

We are indebted to Drs. M. Ridwan and Wandowo for their helpful support in the collection of Lepu. We also thank Drs. T. Tatsuno and Y. Fujimoto for their helpful advice, and Mr. M. Chijimatsu for assistance in amino acid analysis.

REFERENCES

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CORRESPONDENCE TO:

T. ABE - Institute of Physical and Chemical Research, Hirosawa 2-1, Wako-shi, Saitama 351, Japan.

Publication Dates

• Publication in this collection
  08 Jan 1999
• Date of issue
  1996