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A toxic protein from eel serum
Toxicon, 1964, Vol. 2, pp . 79-80. Peraamon Press Ltd., Printed in Great Britain
SHORT COMMUNICATIONS A TOXIC PROTEIN FROM EEL SERUM* E. RocCA and F. GHiRErrl Department of Physiology, Statione Zoologica, Naples, Italy
is known that the serum from fishes belonging to the family Murenidae is toxic to laboratory animals [1,2]. Serum collected from the eel Anguilla vulgaris was used in the present study. Lethality was determined in crabs and mice. Fractionation of the serum proteins on DEAE cellulose column showed that the lethal activity was associated with a single fraction (Fig. 1) . Conventional methods were used for the purification of the toxic fraction from the eel serum. The precipitate collected between 25 and 35 per cent (v/v) saturated ammonium sulfate at pH 7 and 4° , was dissolved in water and dialyzed in the cold against0 -15 M NaCl . The solution was then diluted with an equal volume of saline. The pH was adjusted to 5 -5 with 2 M acetate buffer, and 0-6 volumes 25 per cent ethanol at -5° was added slowly with continuous stirring. The precipitate, dissolved in 0 -05 M phosphate buffer at pH 8 -0, was dialyzed against 0-01 M phosphate at pH 7-6, or against 0-02 MTris-phosphate buffer at pH 7-5. This fraction, which appears as a single band on starch gel electrophoresis, was further purified by chromatography on hydroxylapatite or DEAE cellulose column (Table 1). From the hydroxylapatite, prepared according to TisEmus [3], four fractions were obtained by stepwise elution with phosphate buffer. The whole activity was retained in IT
TABLE. 1 . PURIFICATION OF THE TOXIN FROM EEL SERUM.
Voltune ml
Protein mg
70-0
Precipitate with ammonium sulfate
22-5
74-5
0.15
497
7-2
18-7
0-065
288
15 .0
8-4
0-030
280
Active fractions from hydroxylapatite column
3-3
Total activity
Total serum Precipitate with ethanol
4,350
Activity
1,320
Activity is expressed in arbitrary units and defined as the amount of protein in mg which kills all crabs (100 g body weight) in an average time of 5 min. ' This work was supported under a contract (li)A-91-591-EUC-2815) of the European Research Office, in the U.S . Army . 79 J
80
E . ROCCA and F. GHIRETTI 3" 0r-
FIG. 1 . FRACTIONATION OF EEL SERUM ON DEAE CELLULOSE COLUMN (2 .5 X 30 C.m.). 25 ML DIALYZED SERUM CONTAINING 1'5 G PROTEIN WERE USED. ELUTION WITH TRIS-PHOSPHATE BUFFER AT LINEAR GRADIENT OF PH . (1)
(2) (3) (4) (5)
Washing with 0'02 M Tris-phosphate pH 8'3 Gradient to 0-05M 6'8 11 0-10M 6'2 0'20 M 5'6 0'20 M NaH,PO,11
10 ml fractions collected at 12 min interval . Temp. 0° . Abscissa : Number of fractions . Ordinate : Absorption at 280 ma . Lethal activity in crabs (0'5 ml/15 g body weight) indicted in arbitrary units (shaded area) .
the fraction eluted with 0-1 M buffer at pH 6-7, which contained about 50 per cent of the total protein. From the DEAE cellulose column, several fractions were obtained by linear gradient elution from 0-02 M at pH 7-5 to 0'2 M at pH 6-5 Tris-phosphate buffer . The fractions which appeared between pH 7'3 and 7'0 were found to contain 30-40 per cent of the total proteins, and the entire lethal activity . Additional proof of the proteic nature of the toxin was obtained by hydrolysis of the active fraction. Treatment with trypsin or papain was invariably followed by total inactivation . [1] [2] [3]
REFERENCES Mosso, A., Arch. ItaL BloL, 10, 141, 1888 . GHIREITI, F. and ROCCA, E., Some experiments on ichthyotoxin. In Venomous and Poisonous Animals and Noxious Plants of the Pacific Region, Eds. KEEGAN, H . L . and MACFARLANE, W . V., Pergamon Press, Oxford, 211-216, 1963 . TISELIUs, A., HIERTEN and LEVIN, O., Arch. biochem . Blophys. 65, 132, 1956.
SHORT COMMUNICATIONS A TOXIC PROTEIN FROM EEL SERUM* E. RocCA and F. GHiRErrl Department of Physiology, Statione Zoologica, Naples, Italy
is known that the serum from fishes belonging to the family Murenidae is toxic to laboratory animals [1,2]. Serum collected from the eel Anguilla vulgaris was used in the present study. Lethality was determined in crabs and mice. Fractionation of the serum proteins on DEAE cellulose column showed that the lethal activity was associated with a single fraction (Fig. 1) . Conventional methods were used for the purification of the toxic fraction from the eel serum. The precipitate collected between 25 and 35 per cent (v/v) saturated ammonium sulfate at pH 7 and 4° , was dissolved in water and dialyzed in the cold against0 -15 M NaCl . The solution was then diluted with an equal volume of saline. The pH was adjusted to 5 -5 with 2 M acetate buffer, and 0-6 volumes 25 per cent ethanol at -5° was added slowly with continuous stirring. The precipitate, dissolved in 0 -05 M phosphate buffer at pH 8 -0, was dialyzed against 0-01 M phosphate at pH 7-6, or against 0-02 MTris-phosphate buffer at pH 7-5. This fraction, which appears as a single band on starch gel electrophoresis, was further purified by chromatography on hydroxylapatite or DEAE cellulose column (Table 1). From the hydroxylapatite, prepared according to TisEmus [3], four fractions were obtained by stepwise elution with phosphate buffer. The whole activity was retained in IT
TABLE. 1 . PURIFICATION OF THE TOXIN FROM EEL SERUM.
Voltune ml
Protein mg
70-0
Precipitate with ammonium sulfate
22-5
74-5
0.15
497
7-2
18-7
0-065
288
15 .0
8-4
0-030
280
Active fractions from hydroxylapatite column
3-3
Total activity
Total serum Precipitate with ethanol
4,350
Activity
1,320
Activity is expressed in arbitrary units and defined as the amount of protein in mg which kills all crabs (100 g body weight) in an average time of 5 min. ' This work was supported under a contract (li)A-91-591-EUC-2815) of the European Research Office, in the U.S . Army . 79 J
80
E . ROCCA and F. GHIRETTI 3" 0r-
FIG. 1 . FRACTIONATION OF EEL SERUM ON DEAE CELLULOSE COLUMN (2 .5 X 30 C.m.). 25 ML DIALYZED SERUM CONTAINING 1'5 G PROTEIN WERE USED. ELUTION WITH TRIS-PHOSPHATE BUFFER AT LINEAR GRADIENT OF PH . (1)
(2) (3) (4) (5)
Washing with 0'02 M Tris-phosphate pH 8'3 Gradient to 0-05M 6'8 11 0-10M 6'2 0'20 M 5'6 0'20 M NaH,PO,11
10 ml fractions collected at 12 min interval . Temp. 0° . Abscissa : Number of fractions . Ordinate : Absorption at 280 ma . Lethal activity in crabs (0'5 ml/15 g body weight) indicted in arbitrary units (shaded area) .
the fraction eluted with 0-1 M buffer at pH 6-7, which contained about 50 per cent of the total protein. From the DEAE cellulose column, several fractions were obtained by linear gradient elution from 0-02 M at pH 7-5 to 0'2 M at pH 6-5 Tris-phosphate buffer . The fractions which appeared between pH 7'3 and 7'0 were found to contain 30-40 per cent of the total proteins, and the entire lethal activity . Additional proof of the proteic nature of the toxin was obtained by hydrolysis of the active fraction. Treatment with trypsin or papain was invariably followed by total inactivation . [1] [2] [3]
REFERENCES Mosso, A., Arch. ItaL BloL, 10, 141, 1888 . GHIREITI, F. and ROCCA, E., Some experiments on ichthyotoxin. In Venomous and Poisonous Animals and Noxious Plants of the Pacific Region, Eds. KEEGAN, H . L . and MACFARLANE, W . V., Pergamon Press, Oxford, 211-216, 1963 . TISELIUs, A., HIERTEN and LEVIN, O., Arch. biochem . Blophys. 65, 132, 1956.