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The hemolytic activity of suberitine
Comp. Biochem. Physiol. Vol. 73C, No. I, pp. 91 to 93, 1982 Printed in Great Britain
0306-4492/82/050091-03503.00/0 © 1982 Pergamon Press Ltd
THE HEMOLYTIC ACTIVITY OF SUBERITINE L u c I o CARIELLO, ELISABETTA TOSTI and LAURA ZANETTI Laboratory of Biochemistry, Stazione Zoologica Villa Comunale, Naples, Italy (Received 30 October 1981) Abstract- 1. The hemolytic activity of suberitine, a neurotoxic protein isolated from Suherites domuncula, has been examined. 2. The toxin has no phospholipase A activity. 3. The hemolytic activity of suberitine does not exhibit the induction period and is completely inhibited by sucrose. 4. A study of the effect of temperature on the rate of hemolysis shows that suberitine has two values of the critical thermal increment. The optimum pH for the hemolytic activity was 6.0. 5. There was a correlation between the hemolytic and toxic activity.
INTRODUCTION
solution of the appropriate isotonic buffer containing the required concentration of suberitine. At different times, samples of 2 ml of solution were centrifuged and the supernatant was read at 579nm to determine the amount of hemoglobin released (Bernheimer, 1947). Total (1000/,~) of hemolysis was obtained by centrifuging the same amount of erythrocytes and suspending them in the same volume of distilled water. The effect of sucrose on the hemolytic activity was determined by adding 0.15 M sucrose at 5, 10 and 15 rain to the erythrocyte solution prepared as above.
Suberitine, a neurotoxic a n d hemolytic protein, was isolated from the marine sponge, Suberites domuncula. 0.8/~g of this protein (tool. wt 28,000) paralyses a crab of the species Carcinus maenas weighing 10 g in 1 min, a n d also has strong ATPase a n d hemolytic properties (Cariello & Zanetti, 1979). Suberitine consists of four subunits, each with a different chemical composition a n d a different conformation. The neurotoxic protein has a mainly //-structure, with ~t-helix present only in small a m o u n t s (Cariello et al., 1980). Since the specific p h o t o o x i d a t i o n of the cysteinyl residue completely inhibits the paralytic action, cystein is probably involved in the active site of the protein (Cariello et al., 1981). How suberitine acts upon cell m e m b r a n e s is not properly understood. Results obtained using the giant axon of the crayfish, Astacus leptodacty/us, show that the toxin blocks the action potential by depolarizing the m e m b r a n e potential from - 7 3 mV to - 6 3 inV. The effects are irreversible (Cariello et al., 1980). There we report the results of a study of the hemolytic property of suberitine that did not show phospholipase A activity.
Phospholipasic activity Phospholipase A activity was measured by following the decrease in acyl ester bonds that occurs during the conversion of lecithin into lysolecithin. Ester content was determined by a hydroxamic acid method according to Magee & Thompson (1960). Samples containing 1 mg/ml of phosphate buffer of lecithin (BDH) were shaken with 0.1 ml diethyl ether for 10 rain at room temperature. Suberitine (3.57 x 10 -6 M) dissolved in 5 mM CaC12 was added and at fixed time intervals (15 and 30 min) the reaction was stopped by adding 2 ml of EtOH and 1 ml of hydroxylamine dissolved in NaOH 3.5 N (final concentration of hydroxylamine 16.7mg/ml). The mixture was acidified by adding 0.6 ml of 3.3 N HC1 and then shaken, after which 0.5 ml of FeCI 3 reagent was introduced to develop the color. The samples were read against a reagent blank without lecithin at 525 nm. The standard curve was determined according to Stern & Shapiro (1953).
MATERIALS AND METHODS
Neurotoxic actit,ity The bioassay was performed according to Cariello & Zanetti (1979) using the crab, Carcinus maenas.
Preparation ~1' suheritine and erythrocyte,s Suberitine was purified by gel filtration on Sephadex columns according to Cariello & Zanetti (1979) and stored in 0.15M sodium acetate pH 7.6 at -20°C. Concentrations of the stock toxin solution were determined according to Lowry's method (Lowry et al., 1951). Appropriate concentrations of suberitine were prepared before each set of experiments by diluting the stock solution in different buffers: isotonic mono and di-phosphate 66 mM between pH 5.8 and 7.2 and isotonic Tris-HC1, 0.1 M, pH 8.0. Human erythrocytes were centrifuged from citrate blood, washed three times with isotonic buffer and resuspended in the same buffer with a volume to obtain a final concentration of 30 x 106 cells/ml.
RESULTS AND DISCUSSION Our results demonstrate that suberitine has no phospholipase A activity. Figure l shows the course of hemolysis induced by various concentrations of suberitine. It can be seen that the peak of hemolysis has already occurred with 7.14 x 1 0 - ~ M suberitine after 20 min. The shape of the hemolysis curve is more or less sigmoid depending on the concentration of suberitine. This plot is very similar to that of other lysins, in particular streptolisin O (Bernheimer, 1974). As may be seen from Fig. 1, the hemolytic action of suberitine does not occur in two phases; indeed the induction period is not
Hemolytic tests Erythrocyte suspensions were prepared for individual experiments by diluting (1:1 v/v) the stock solution with a
91
92
Luclo CARIELLO et al.
100
]
9O ~ 7o~ E
60~ '
~60
10
20
30 rain
40
50
60
Fig. 1. Percentage hemolysis caused by suberitine on the red blood cells in the time at pH 6.0 at different concentrations. G - - 10.71 × l O - 6 M ; ~ 7.14 × l O - 6 M ; *-3.57 × l O - 6 M ; A 1.78 × 10-6M;E2--8.92 × 10-TM.
m
-~ 4-
E
.c
.
2-
5
6
pH
8
7
9
Fig. 3. Rate of hemolysis of red blood cells as a function of pH. Suberitine concentration 10.71 x 10 -6 M.
• t
0.80-
"~ 0.60o E '~ 0,40
°.2°i 32
0 0
2 4 6 8 Concentration (M X 1 0 6)
Fig. 2. Rate of hemolysis as a function of suberitine concentration (7.14 x 10 6 M 0.89 x 10 6 M).
present. The addition of sucrose completely inhibited the hemolysis. The relation between the rate of hemolysis and suberitine concentrations was obtained from the curves in Fig. 1. As shown in Fig. 2, rate of hemolysis was directly proportional to suberitine concentration. A series of isotonic buffers at various pH values between 5.8 and 8.1 was used to study the effect of the pH on the hemolytic activity. Figure 3 shows the rate of hemolysis induced b)f 10.71 x 10 ~' M suberitine at different pH values. The plot was obtained by drawing the slopes at zero time of the curves of hemolysis at different pH values. The activity is completely lost above pH 7 and is highest at pH 6.0. Also the neurotoxic activity increases tenfold at pH 6.0 (0.08 7 paralyzed a crab weighing 10g in 1 min). This result shows that the two activities were closely related. However the paralysing action is not caused by the hemolytic activity. The effect of temperature on the rate of hemolysis
33
34 1/T x 104
35
36
Fig. 4. Arrhenius plot showing the temperature effect on the rate of hemolysis. Suberitine concentration 3.57 x 10 6M.
was determined by measuring the slopes of curves obtained from 3-36cC, Figure 4 shows the Arrhenius plot. Suberitine had a much higher ~ value between 3 and 10'C (23,300) than between 22 and 3&C. The shift from the higher to lower p value occurred at a b o u t 15'C. It is possible that the change in # value at a critical temperature is associated with a change in the molecular configuration, probably due to a p~ "tial deactivation of suberitine that shows a relevant content of/~-structure.
REFERENCES
BERNHEIMER A. W. (1947) Comparative kinetics of hemolysis induced by bacterial and other hemolysins. J..qen. Physiol. 30, 337 353. CARIELLO L. ~,~ ZANETTI L. (1979) Suberitine, the toxic protein from the marine sponge, Suberites domuncu/a. Comp.
Biochem. Physiol. 64C, 15 19.
The hemolytic activity of suberitine CAR|ELLO L., SALVATO B. & JORI G. (1980) Partial characterization of suberitine, the neurotoxic protein purified from Suherite.~ domuncula. Comp. Biochem. Physiol. 67B, 337 344. CARIELLO L., SALVATO B. & JORI G. (1981) The role of the cysteinyl and one of the tryptophyl residues in the neurotoxic action of suberitine. Experientia 37~ 801 803. LOWRY O. H., ROSEBROUGH N. J., FARR A. L. & RANDALL
93
R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265 275. MAGEE W. L. & THOMPSON R. H. S. (1960) The estimation of phospholipase A activity in aqueous systems. Biochem. J. 77, 526-534. SI"ER~q 1. & SHAPmO B. (1953) A rapid and simple method for determination of esterified fatty acids and for total fatty acids in blood. J. clitl. Pathol. 6, 158 160.
0306-4492/82/050091-03503.00/0 © 1982 Pergamon Press Ltd
THE HEMOLYTIC ACTIVITY OF SUBERITINE L u c I o CARIELLO, ELISABETTA TOSTI and LAURA ZANETTI Laboratory of Biochemistry, Stazione Zoologica Villa Comunale, Naples, Italy (Received 30 October 1981) Abstract- 1. The hemolytic activity of suberitine, a neurotoxic protein isolated from Suherites domuncula, has been examined. 2. The toxin has no phospholipase A activity. 3. The hemolytic activity of suberitine does not exhibit the induction period and is completely inhibited by sucrose. 4. A study of the effect of temperature on the rate of hemolysis shows that suberitine has two values of the critical thermal increment. The optimum pH for the hemolytic activity was 6.0. 5. There was a correlation between the hemolytic and toxic activity.
INTRODUCTION
solution of the appropriate isotonic buffer containing the required concentration of suberitine. At different times, samples of 2 ml of solution were centrifuged and the supernatant was read at 579nm to determine the amount of hemoglobin released (Bernheimer, 1947). Total (1000/,~) of hemolysis was obtained by centrifuging the same amount of erythrocytes and suspending them in the same volume of distilled water. The effect of sucrose on the hemolytic activity was determined by adding 0.15 M sucrose at 5, 10 and 15 rain to the erythrocyte solution prepared as above.
Suberitine, a neurotoxic a n d hemolytic protein, was isolated from the marine sponge, Suberites domuncula. 0.8/~g of this protein (tool. wt 28,000) paralyses a crab of the species Carcinus maenas weighing 10 g in 1 min, a n d also has strong ATPase a n d hemolytic properties (Cariello & Zanetti, 1979). Suberitine consists of four subunits, each with a different chemical composition a n d a different conformation. The neurotoxic protein has a mainly //-structure, with ~t-helix present only in small a m o u n t s (Cariello et al., 1980). Since the specific p h o t o o x i d a t i o n of the cysteinyl residue completely inhibits the paralytic action, cystein is probably involved in the active site of the protein (Cariello et al., 1981). How suberitine acts upon cell m e m b r a n e s is not properly understood. Results obtained using the giant axon of the crayfish, Astacus leptodacty/us, show that the toxin blocks the action potential by depolarizing the m e m b r a n e potential from - 7 3 mV to - 6 3 inV. The effects are irreversible (Cariello et al., 1980). There we report the results of a study of the hemolytic property of suberitine that did not show phospholipase A activity.
Phospholipasic activity Phospholipase A activity was measured by following the decrease in acyl ester bonds that occurs during the conversion of lecithin into lysolecithin. Ester content was determined by a hydroxamic acid method according to Magee & Thompson (1960). Samples containing 1 mg/ml of phosphate buffer of lecithin (BDH) were shaken with 0.1 ml diethyl ether for 10 rain at room temperature. Suberitine (3.57 x 10 -6 M) dissolved in 5 mM CaC12 was added and at fixed time intervals (15 and 30 min) the reaction was stopped by adding 2 ml of EtOH and 1 ml of hydroxylamine dissolved in NaOH 3.5 N (final concentration of hydroxylamine 16.7mg/ml). The mixture was acidified by adding 0.6 ml of 3.3 N HC1 and then shaken, after which 0.5 ml of FeCI 3 reagent was introduced to develop the color. The samples were read against a reagent blank without lecithin at 525 nm. The standard curve was determined according to Stern & Shapiro (1953).
MATERIALS AND METHODS
Neurotoxic actit,ity The bioassay was performed according to Cariello & Zanetti (1979) using the crab, Carcinus maenas.
Preparation ~1' suheritine and erythrocyte,s Suberitine was purified by gel filtration on Sephadex columns according to Cariello & Zanetti (1979) and stored in 0.15M sodium acetate pH 7.6 at -20°C. Concentrations of the stock toxin solution were determined according to Lowry's method (Lowry et al., 1951). Appropriate concentrations of suberitine were prepared before each set of experiments by diluting the stock solution in different buffers: isotonic mono and di-phosphate 66 mM between pH 5.8 and 7.2 and isotonic Tris-HC1, 0.1 M, pH 8.0. Human erythrocytes were centrifuged from citrate blood, washed three times with isotonic buffer and resuspended in the same buffer with a volume to obtain a final concentration of 30 x 106 cells/ml.
RESULTS AND DISCUSSION Our results demonstrate that suberitine has no phospholipase A activity. Figure l shows the course of hemolysis induced by various concentrations of suberitine. It can be seen that the peak of hemolysis has already occurred with 7.14 x 1 0 - ~ M suberitine after 20 min. The shape of the hemolysis curve is more or less sigmoid depending on the concentration of suberitine. This plot is very similar to that of other lysins, in particular streptolisin O (Bernheimer, 1974). As may be seen from Fig. 1, the hemolytic action of suberitine does not occur in two phases; indeed the induction period is not
Hemolytic tests Erythrocyte suspensions were prepared for individual experiments by diluting (1:1 v/v) the stock solution with a
91
92
Luclo CARIELLO et al.
100
]
9O ~ 7o~ E
60~ '
~60
10
20
30 rain
40
50
60
Fig. 1. Percentage hemolysis caused by suberitine on the red blood cells in the time at pH 6.0 at different concentrations. G - - 10.71 × l O - 6 M ; ~ 7.14 × l O - 6 M ; *-3.57 × l O - 6 M ; A 1.78 × 10-6M;E2--8.92 × 10-TM.
m
-~ 4-
E
.c
.
2-
5
6
pH
8
7
9
Fig. 3. Rate of hemolysis of red blood cells as a function of pH. Suberitine concentration 10.71 x 10 -6 M.
• t
0.80-
"~ 0.60o E '~ 0,40
°.2°i 32
0 0
2 4 6 8 Concentration (M X 1 0 6)
Fig. 2. Rate of hemolysis as a function of suberitine concentration (7.14 x 10 6 M 0.89 x 10 6 M).
present. The addition of sucrose completely inhibited the hemolysis. The relation between the rate of hemolysis and suberitine concentrations was obtained from the curves in Fig. 1. As shown in Fig. 2, rate of hemolysis was directly proportional to suberitine concentration. A series of isotonic buffers at various pH values between 5.8 and 8.1 was used to study the effect of the pH on the hemolytic activity. Figure 3 shows the rate of hemolysis induced b)f 10.71 x 10 ~' M suberitine at different pH values. The plot was obtained by drawing the slopes at zero time of the curves of hemolysis at different pH values. The activity is completely lost above pH 7 and is highest at pH 6.0. Also the neurotoxic activity increases tenfold at pH 6.0 (0.08 7 paralyzed a crab weighing 10g in 1 min). This result shows that the two activities were closely related. However the paralysing action is not caused by the hemolytic activity. The effect of temperature on the rate of hemolysis
33
34 1/T x 104
35
36
Fig. 4. Arrhenius plot showing the temperature effect on the rate of hemolysis. Suberitine concentration 3.57 x 10 6M.
was determined by measuring the slopes of curves obtained from 3-36cC, Figure 4 shows the Arrhenius plot. Suberitine had a much higher ~ value between 3 and 10'C (23,300) than between 22 and 3&C. The shift from the higher to lower p value occurred at a b o u t 15'C. It is possible that the change in # value at a critical temperature is associated with a change in the molecular configuration, probably due to a p~ "tial deactivation of suberitine that shows a relevant content of/~-structure.
REFERENCES
BERNHEIMER A. W. (1947) Comparative kinetics of hemolysis induced by bacterial and other hemolysins. J..qen. Physiol. 30, 337 353. CARIELLO L. ~,~ ZANETTI L. (1979) Suberitine, the toxic protein from the marine sponge, Suberites domuncu/a. Comp.
Biochem. Physiol. 64C, 15 19.
The hemolytic activity of suberitine CAR|ELLO L., SALVATO B. & JORI G. (1980) Partial characterization of suberitine, the neurotoxic protein purified from Suherite.~ domuncula. Comp. Biochem. Physiol. 67B, 337 344. CARIELLO L., SALVATO B. & JORI G. (1981) The role of the cysteinyl and one of the tryptophyl residues in the neurotoxic action of suberitine. Experientia 37~ 801 803. LOWRY O. H., ROSEBROUGH N. J., FARR A. L. & RANDALL
93
R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265 275. MAGEE W. L. & THOMPSON R. H. S. (1960) The estimation of phospholipase A activity in aqueous systems. Biochem. J. 77, 526-534. SI"ER~q 1. & SHAPmO B. (1953) A rapid and simple method for determination of esterified fatty acids and for total fatty acids in blood. J. clitl. Pathol. 6, 158 160.