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Purification and characterization of a lethal factor in venom from the crown-of-thorns starfish (Acanthaster planci)
0041-0101188 53 .00+ .00 ~~' 1988 Pergnmon Press pk
T°.c&'°n Vol . 26. No . I I, pp . 1077-1083, 1988 . Printed in Grent Britain .
PURIFICATION AND CHARACTERIZATION OF A LETHAL FACTOR IN VENOM FROM THE CROWN-OF-THORNS STARFISH (ACANTHASTER PLANCK KAZUO SHIOMI, SHINSUKE YAMAMOTO, HIDEAKi YAMANAKA
and
TAKEAKI KIKUCHI
Department of Food Science and Technology, Tokyo University of Fisheries, Tokyo 108, Japan (Accepled .(or puhlicution 8 June 1988) H . YAMANAKA and T. KIKUCHI . Purification and characterization of 8 lethal factor in venom from the crown-of-thorns starfish (Acanthaster planci) . Toxicon 26, 10771083, 1988 . - A lethal factor in venom of the crown-of-thorns starfish (Acanthaster planci) was obtained in an electrophoretically pure state by chromatography on CM-cellulose and Sephadex G100 . The purified lethal factor is a basic (pI 10.6) glycoprotein (carbohydrate content 3 .5%) . The mol. wt was estimated to be 20,000 by gel filtration or 25,000 by SDS-disc electrophoresis, suggesting that the lethal factor has no subunit structure . Despite its basicity, the lethal factor was richer in acidic amino acids than in basic amino acids. The lethal factor had an Lnm of 0 .43 mg/kg (i .p . injection into mice) . Hemolytic, edema-forming and capillary permeability-increasing activities, though very weak, were also exhibited by the lethal factor, while hemorrhagic and phospholipase A activities were not present. K . SHIOMI, $ . YAMAMOTO,
INTRODUCTION
Starfish (Acanthaster planci), an echinoderm found throughout the tropical Indo-Pacific Ocean, has been incriminated not only because they damage coral reefs but also because they have numerous venomous spines on the body surface. Contact with the venomous spines may result in an extremely painful wound, redness, swelling, protracted vomiting, numbness and paralysis (HALSTEAD, 1965). TAIRA et al. (1975) reported that crude venom extracted from the spines exhibits lethal and hemolytic activities . Based on stability and chromatographic behavior, proteinaceous principles were assumed to be responsible for both activities . Recently phospholipase A, edema-forming, capillary permeability-increasing, hemorrhagic and myonecrotic activities were additionally detected in crude venom (SHiOMI et al., 1985a) . Although such a variety of biological activities are displayed by the A . planci venom, no toxic factors have been obtained in a pure state and therefore not fully characterized . The present study was undertaken to purify a lethal factor in the A . planci venom and clarify its biological and physico-chemical properties .
THE CROWN-OF-THORNS
MATERIALS AND METHODS Starfish Specimens of A . planci were caught at Kabira, Ishigaki Island, Okinawa Prefecture, in October 1986, shipped to our laboratory in the frozen state and stored at -20 °C until used . 1077
1078
K. SHIOMI et al .
Pur~cation procedure Spines (68 g) were collected from a specimen (580 g) and homogenized with two volumes of buffered saline (0 .01 M phosphate buffer containing O.15 M NaCI, pH 7 .0). The homogenate was centrifuged at 15,000 Xd for 15 min. The supernatant was dialyzed against 0.01 M phosphate buffer (pH 7 .0) using a Zeineh dialyzer (model LD-l; Funakoshi, Tokyo, Japan) and applied to a column (2 .5 x 40 cm) of CM-cellulose (Brown, Berlin, U.S.A .) previously equilibrated with the same buffer (first CM~ellulose column chromatography). Materials absorbed by the column were eluted by 0.6 M NaCI in the buffer and dialyzed against the buffer . The dialysate was again applied to a column (1 .8 x 45 cm) of CM~ellulose (second CM-cellulose column chromatography). The column was washed with the buffer and then eluted by a linear gradient of NaCI ranging from 0 to 0.6 M in the buffer (800 ml in total volume). Fractions of 8 ml were collected at a flow rate of 53 ml/hr. Lethal fractions were combined, dialyzed against distilled water and lyophilized. The dried product was dissolved in 6 ml of buffered saline and layered onto a column (2.5 x 90 cm) of Sephadex G-100 (Pharmacia, Uppsala, Sweden). Elution was achieved with buffered saline and fractions of 6 ml were collected at a flow rate of 10 ml/hr. Lethal fractions were combined, dialyzed against distilled water and lyophilized. Bioassay Lethality was examined by i.p . injection of 1 ml of test solution into ddY male mice (Sankyo Labo Service, Tokyo, Japan) weighing about 20 g. Deaths were scared 96 hr after injection. For the purified lethal factor, groups of five mice were injected with five different doses ranging from 0.33 to 0.67 mg/kg and the t,n,° was calculated according to the method of Lrt~Ln and Wtt coxox (1949) . Hemolytic activity against sheep erythrocytes was estimated as reported previously (Srnorn et al., I985b). One hemolytic unit (HU) was defined as the amount of protein required to cause 50% hemolytis. Edema-forming activity was assayed using mice by the method of Y~utuw~ et al. (1976); edema ratio was expressed as the percentage in weight of the envenomated foot relative to the saline-injected foot . Capillary permeability-increasing activity and hemorrhagic activity were estimated using male Wistar rats (Sankyo Labo Service, Tokyo, Japan) weighing 180-200 g, as described in our previous paper (Srno~a et al ., 1985a) . Following intradermal injection of venom solution (0 .1 ml) in the depilated back skin, 1 % Evans blue solution (0.1 ml) was injected i.v . to trace permeability changes. The rats were killed 2 hr later and the cross diameters of each blue weal were measured from the visceral side . For the determination of hemorrhagic activity, the injection of Evans blue solution was omitted and the formation of hemorrhagic spot was observed from the visceral side as well. Phospholipase A activity was examined by measuring the clearing of an egg yolk suspension, according to the method of M~ttttverrt (1965) . Analytical disc electrophoresis Analytical disc electrophoresis was performed on a 7% polyacrylamide gel with ß-alanine-acetic acid buffer (pH 4.5) according to the method of RetsFEt.u et al. (1962) . ARer running at 5 mA per gel, the gel was stained for proteins with Amido Black lOB or for carbohydrates by the method of Zwct~uxtus et al. (1969) . Electrojocusing
Electrofocusing on a 7 .5% polyacrylamide gel was carried out at 200 V for 3 hr . Bio-Lyte 3/10 (Bio-Rad Laboratories, Richmond, U.S .A .) was used as carrier ampholite at a concentration of 2% . The gel was stained with Coomasie brilliant blue G-250. in order to estimate the pH gradient formed, colored pI marker proteins (Oriental Yeast, Tokyo, Japan) were electrofceused together with the purified lethal factor. Molecular weight determination
The mol. wt of the purified lethal factor was determined either by gel filtration or by sodium dodecyl sulfate (SDS) disc electrophoresis. Gel filtration was performed by high-performance liquid chromatography (HPLC) on a column (0.75 X 30 cm) of TSK-GEL G3000SW (Toyo Soda, Tokyo, Japan) with 0.01 M phosphate buffer (pH 7.0) containing 0.3 M NaCI . The flow rate was maintained at 0.7 ml/min and the elutee monitored by absorbance at 280 nm . Bovine serum albumin (mol . wt 67,000), ovalbumin (43,000), ribonuclease A (13,700) and cytcehrome c (12,500) were used as standard mol. wt proteins . SDS-disc electrophoresis was carried out as described by WEeert and Oseoxrt (1969) using a 10% polyacrylamide gel and 0.l M phosphate buffer (pH 7.2) containing 0.1 % SDS. Prior to electrophoresis, samples were dissolved in 1 % SDS and 5% 2-mercaptcethanol and boiled for I S min. The gel was stained with Coomasie brilliant blue R-250. The reference proteins used were bovine serum albumin, ovalbumin, chymotrypsinogen A (mol . wt 25,000) and ribonuclease A. Protein and carbohydrate determinations Protein was estimated by the method of Lowtty et al. (1951) using bovine serum albumin as a standard and carbohydrate by the phenol-sulfuric acid method (Dueo~s et al., 1956) using o-glucose as a standard .
A Lethal Factor in Starfish Venom
1079
0 .6
0 .3
0 .4
a z
w 0
d u c R a
0 .2
0 N
c 0 .N .+ a w a+
C O u c 0 u
a
Fraction
nu~bsr
FIG. I . CM-CELLUL06E COLUMN CHROMATOGRAPHY OF A. ptanci YeNGM. Lethal fractions obtained in the first CM~ellulose column chromatography were again applied to a CM-cellulose column (1 .8 x 45 cm) equilibrated with 0.01 M phosphate buffer (pH 7.0). A linear gradient of 0-0.6 M NaCI in the buffer was started at fraction 41 . Fractions of 8 ml were collected at a flow rate of 53 ml/hr. The lethal factor was eluted in fractions indicated by a bar.
Amino acid analysis The purified lethal factor was hydrolyzed with 6N HCl in an evacuated tube at 110'C for 24 hr and then applied to a Hitachi 835 amino acid analyzer. The amino acid composition was calculated from three determinations. Tryptophan was estimated spectrophotometrically by the method of GoouwIN and MORTON (1946).
RESULTS AND DISCUSSION
Purification ofa lethal factor Although as much as 70% of proteins in the crude venom were passed through a column of CM~ellulose, they showed no lethality (first CM-cellulose column chromatography). Isocratic elution by 0.6 M NaCI in 0.01 M phosphate buffer (pH 7.0) afforded one protein peak with lethality, which accounted for only 4% of proteins in the crude venom. In the second CM-cellulose column chromatography using a linear gradient elution of 0-0.6 M NaCI in the buffer, three protein peaks were observed (Fig. 1). Lethality was detected only in the symmetrical peak which was eluted last, indicating that the lethal factor is fairly basic. The lethal fraction obtained was then chromatographed on Sephadex G-100. As illustrated in Fig. 2, the lethal factor appeared in a symmetrical protein peak . Thus, 4.0 mg of the purified lethal factor was obtained. As mentioned above, CM-cellulose column chromatography was very effective for the purification of the lethal factor . In this step large quantities of contaminated proteins were separated from the lethal factor . Furthermore, analytical disc electrophoresis revealed that the homogeneous lethal factor existed in the second half of the lethal peak (second CM-
(080
K. SHIOMI et at.
0 .3 E C 0 a0 N a+ w
0 .2
m u C w 0 H
a 40
50
60 Fraction
70
80
90
nu~ber
FIG . Z. GEL FILTRATION ON SEPHADEX G-IOO OF THE LETHAL FRACI70N OBTAINED IN CM-CELLUL06E COLUMN CHROMATOGRAPHY. Lethal fractions obtained in the second CM~ellulose column chromatography (Fig. 1) were subjected to gel filtration on a Sephadex G-100 column (2 .5 x 90 cm) with 0.01 M phosphate buffer containing 0.15 M NaCI (pH 7.0). Fractions of 6 ml were collected at a flow rate of 10 ml/hr. The lethal factor was eluted in fractions indicated by a bar .
cellulose column chromatography). Therefore, apart from the yield, the pure lethal factor can be obtained by CM-cellulose column chromatography alone, without the following gel filtration on Sephadex G-100. Physicochemical properties of the purified lethal factor
The homogeneity of the purified lethal factor was supported by the following results; it gave a single band in analytical disc electrophoresis (Fig . 3A), SDS-disc electrophoresis (Fig. 3B) or electrofocusing and also gave a single symmetrical protein peak in HPLC. In analytical disc electrophoresis the purified lethal factor was visualyzed by both protein and carbohydrate staining, suggesting that it is a glycoprotein . its carbohydrate content was estimated to be 3.5% .The mol. wt was determined to be 20,000 by gel filtration (Fig . 4A) or 25,000 by SDS-disc electrophoresis (Fig. 4B). Since similar values were obtained by both methods, the lethal factor appeared to exist in a monomeric form having no subunit structure. As expected from the behavior on CM-cellulose (Fig. 1), the lethal factor was shown to be basic; its isoelectric point was estimated to be 10.6 by electrofocusing. The amino acid composition is listed in Table 1 . In spite of its basic nature, the lethal factor was richer in acidic amino acids than in basic amino acids, suggesting that most of the acidic amino acid residues exist as an amide form . Biological activities of the purified lethal factor
The i.p. LDP of the purified lethal factor was determined to be 0.43 mg/kg (95% confidence interval, 0.37-0.50 mg/kg) . As with the crude venom (Sx1oM1 et al., 1985a), lethal doses of the purified factor induced flaccidness, numbness and occasional convulsion in mice and killed them within 96 hr. The purified lethal factor showed a hemolytic activity of 18.2 HU/mg against sheep erythrocytes . Edema-forming and capillary permeability-
108 1
A Lethal Factor in Starfish Venom
FIG . 3 . POLYACRYLAMIUE GEL ULSC ELECTROPHORESIS OF THE PURIFIED LETHAL FACTOR.
Origin is at top. (A) Analytical electrophoresis. A 7% polyacrylamide gel and ß-alanine-acetic acid buffer (pH 4.5) were used . The protein band was visualized with Amido Black IOB. (H) SDS electrophoresis. A 10% polyacrylamide gel and 0.1 M phosphate buffer (pH 7.2) wntaining 0.1 SDS were used . Prior to electrophoresis, the sample was treated with 1% SDS and 5% 2mercaptcethanol at 100'C for 15 min. The protein band was visualized with Coomassie brilliant blue R-250.
0
Retention tine
(min)
Relative mobility
FIG . 4 . MOLECULAR WEIGHT DETERMINATION OF THE PURIFIED LETHAL FACTOR BY G3000SW (A) ANll SDS-DISC ELECTROPHORESIS (B) .
HPLC ON TSK-0EL
The reference proteins used are: l, bovine serum albumin; 2, ovalbumin; 3, chymotrypsinogen A; 4, ribonuclease A; and 5, cytochrome c.
1082
K. SHIOMI et al. TABLE I .
AMINO ACID COMPOSITION OF THE PURIFIED LETHAL FACTOR
Amino acid Asx Thr Ser Glx
Pro
Gly
Ala Val Cys (half) Met Ile Leu Tyr Phe Trp Lys His
Arg
Mole %* 12.5 5.6 8.5 6.9 5.7 9.3 6.0
?.6
0.8 1 .3 3.1 8.1 4.8 3.0 2.2t 9.6 2.8 2.6
* Average values calculated from three deteIiIIinations . t Determined spectrophotometrically. increasing activities were also displayed by the lethal factor; i.m. injection into mouse foot of65 ~g ofthe lethal factor caused an edema of 123% (average of three determinations) and intradermal injection into rat back skin of 130 ieg a blue weal of 5 mm (average of three determinations) in diameter . On the other hand, the lethal factor was devoid of hemorrhagic activity at a dose of 130 hg. In addition, it seemed to be fret from phospholipase A activity since no clearing of an egg yolk suspension was observed even at a dose of 32 hg. All of the above biological activities examined with the purified lethal factor are manifested by the crude venom (SHIOMI et al., 1985a) . The purified lethal factor, however, lacked hemorrhagic and phospholipase A activities . It is also significant that the hemolytic, edema-forming and capillary permeability-increasing activities of the lethal factor are very weak as compared to those of the crude venom (SHIOMI et al., 1985a). For example, as low as 9 ftg of the crude venom could induced a 130% edema while more than 65 ~g of the lethal factor was needed to cause the same magnitude ofedema. From these results, it is apparent that besides the lethal factor under study, toxic factors bearing the biological activities other than lethal activity are contained in the crude venom. This is in accordance with the assumption of TAIItA et al. (1975) that lethal and hemolytic activities are elicited by different proteinaceous principles . Thus, the lethal factor does not seem to be a major principle responsible for the local effects such as swelling and redness induced by A planci stings . For a better understanding on the local effects, it is necessary to purify and characterize other toxic factors . Acknowledgements - We are grateful to Professor S. KONOSU and Dr A. SHINAGAWA, Faculty of Agriculture, the University of Tokyo, for measuring the amino acid composition .
A Lethal Factor in Starfish Venom
l0ß3
REFERENCES Dueo[s, M., G[[.t,ES, K . A ., HAt~L~rox, J. K., Raee[ts, P. A. and S~+m~t, F. (1956) Calorimetric method for determination of sugars and related substances . Analyt. Chem. 28, 350 . Gooow[N, T. W. and MoR'['ox, R. A. (1946) The spectrophotometric determination of tyrosine and tryptophan in proteins . Biochem. J. 40, 628. HA[srE~o, B. W. (1965) Starfishes, brittle stars, sea urchins, etc. In : Poisonous and Venomous Marine Animals of the World, Vol. I, Invertebrates, p. 537. Washington, DC: U.S . Government Printing Office . L[rc[tF[et.n, J. T. Jr . and W[[.coxox, F. (1949) A simplified method of evaluating dose~ffect experiments. J. Pharmac. exp. Ther . 96, 99 . LOWRY, O. H., ROSEHROUGH, N. J., F~xx, A. L. and RANDALL, R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265. M~wNETn, G. V. (1965) The action of phospholipase A on lipoproteins . Biochim, biophys. Acta 98, 554. RrJSe~cn, R. A., LEW[s, U. J. and WILL[AAfS, D. E. (1962) Disc electrophoresis of basic proteins and peptides on polyacrylamide gels. Nature 195, 281 . SH[oiw, K., hro[t, K., YAMANAKA, H. and K[RUCIi[, T . (1985a) Biological activity of crude venom from the crownof-thorns starfish Acanthaster planci. Nippon Suisan Gakkaishi 51, 1151 . SMOMI, K., TANAKA, E., YAMANAKA, H. and K[[cUCEn, T. (1985b) Isolation and characterisation of a lethal hemolysin in the sea anemone Parasicyonis actinostoloides. Toxicon 23, 865. Taxa, E., TANAHARA, N. and Fux~rsu, M. (1975) Studies on the toxin in the spines of starfish Acanthaster planci . I. Isolation and some properties of the toxin found in spines . Ryukyu Daigaku Nogakubu Gakujutsu Hokoku 22, 203. WEeE[t, K. and Osaoßx, M. (1969) The reliability of molecular weight determination by dodecyl sulfate polyacrylamide gel electrophoresis. J. biol . Chem . 244, 4406 . YAMAKAWA, M., Noz~[a, M. and HOtc~sra, Z. (1976) Fractionation of Sakishima-habu (Trimeresurus elegans) venom and lethal, hemorrhagic, and edema-forming activities of the fractions . In : Animal, Plant, and Microbial Toxins, Vol. 1, p. 97 (OHS~tu, A., HAYASH[, K. and SAWAI, Y., Eds). New York: Plenum Press. ZAC[tax[US, R. M., Zet.[;, T. E., MORR[SON, T. H. and Wooo[.oc[c, J. J. (1969) Glycoprotein staining following electrophoresis on acrylamide gels . Analyt . Biochem. 30, 148.
T°.c&'°n Vol . 26. No . I I, pp . 1077-1083, 1988 . Printed in Grent Britain .
PURIFICATION AND CHARACTERIZATION OF A LETHAL FACTOR IN VENOM FROM THE CROWN-OF-THORNS STARFISH (ACANTHASTER PLANCK KAZUO SHIOMI, SHINSUKE YAMAMOTO, HIDEAKi YAMANAKA
and
TAKEAKI KIKUCHI
Department of Food Science and Technology, Tokyo University of Fisheries, Tokyo 108, Japan (Accepled .(or puhlicution 8 June 1988) H . YAMANAKA and T. KIKUCHI . Purification and characterization of 8 lethal factor in venom from the crown-of-thorns starfish (Acanthaster planci) . Toxicon 26, 10771083, 1988 . - A lethal factor in venom of the crown-of-thorns starfish (Acanthaster planci) was obtained in an electrophoretically pure state by chromatography on CM-cellulose and Sephadex G100 . The purified lethal factor is a basic (pI 10.6) glycoprotein (carbohydrate content 3 .5%) . The mol. wt was estimated to be 20,000 by gel filtration or 25,000 by SDS-disc electrophoresis, suggesting that the lethal factor has no subunit structure . Despite its basicity, the lethal factor was richer in acidic amino acids than in basic amino acids. The lethal factor had an Lnm of 0 .43 mg/kg (i .p . injection into mice) . Hemolytic, edema-forming and capillary permeability-increasing activities, though very weak, were also exhibited by the lethal factor, while hemorrhagic and phospholipase A activities were not present. K . SHIOMI, $ . YAMAMOTO,
INTRODUCTION
Starfish (Acanthaster planci), an echinoderm found throughout the tropical Indo-Pacific Ocean, has been incriminated not only because they damage coral reefs but also because they have numerous venomous spines on the body surface. Contact with the venomous spines may result in an extremely painful wound, redness, swelling, protracted vomiting, numbness and paralysis (HALSTEAD, 1965). TAIRA et al. (1975) reported that crude venom extracted from the spines exhibits lethal and hemolytic activities . Based on stability and chromatographic behavior, proteinaceous principles were assumed to be responsible for both activities . Recently phospholipase A, edema-forming, capillary permeability-increasing, hemorrhagic and myonecrotic activities were additionally detected in crude venom (SHiOMI et al., 1985a) . Although such a variety of biological activities are displayed by the A . planci venom, no toxic factors have been obtained in a pure state and therefore not fully characterized . The present study was undertaken to purify a lethal factor in the A . planci venom and clarify its biological and physico-chemical properties .
THE CROWN-OF-THORNS
MATERIALS AND METHODS Starfish Specimens of A . planci were caught at Kabira, Ishigaki Island, Okinawa Prefecture, in October 1986, shipped to our laboratory in the frozen state and stored at -20 °C until used . 1077
1078
K. SHIOMI et al .
Pur~cation procedure Spines (68 g) were collected from a specimen (580 g) and homogenized with two volumes of buffered saline (0 .01 M phosphate buffer containing O.15 M NaCI, pH 7 .0). The homogenate was centrifuged at 15,000 Xd for 15 min. The supernatant was dialyzed against 0.01 M phosphate buffer (pH 7 .0) using a Zeineh dialyzer (model LD-l; Funakoshi, Tokyo, Japan) and applied to a column (2 .5 x 40 cm) of CM-cellulose (Brown, Berlin, U.S.A .) previously equilibrated with the same buffer (first CM~ellulose column chromatography). Materials absorbed by the column were eluted by 0.6 M NaCI in the buffer and dialyzed against the buffer . The dialysate was again applied to a column (1 .8 x 45 cm) of CM~ellulose (second CM-cellulose column chromatography). The column was washed with the buffer and then eluted by a linear gradient of NaCI ranging from 0 to 0.6 M in the buffer (800 ml in total volume). Fractions of 8 ml were collected at a flow rate of 53 ml/hr. Lethal fractions were combined, dialyzed against distilled water and lyophilized. The dried product was dissolved in 6 ml of buffered saline and layered onto a column (2.5 x 90 cm) of Sephadex G-100 (Pharmacia, Uppsala, Sweden). Elution was achieved with buffered saline and fractions of 6 ml were collected at a flow rate of 10 ml/hr. Lethal fractions were combined, dialyzed against distilled water and lyophilized. Bioassay Lethality was examined by i.p . injection of 1 ml of test solution into ddY male mice (Sankyo Labo Service, Tokyo, Japan) weighing about 20 g. Deaths were scared 96 hr after injection. For the purified lethal factor, groups of five mice were injected with five different doses ranging from 0.33 to 0.67 mg/kg and the t,n,° was calculated according to the method of Lrt~Ln and Wtt coxox (1949) . Hemolytic activity against sheep erythrocytes was estimated as reported previously (Srnorn et al., I985b). One hemolytic unit (HU) was defined as the amount of protein required to cause 50% hemolytis. Edema-forming activity was assayed using mice by the method of Y~utuw~ et al. (1976); edema ratio was expressed as the percentage in weight of the envenomated foot relative to the saline-injected foot . Capillary permeability-increasing activity and hemorrhagic activity were estimated using male Wistar rats (Sankyo Labo Service, Tokyo, Japan) weighing 180-200 g, as described in our previous paper (Srno~a et al ., 1985a) . Following intradermal injection of venom solution (0 .1 ml) in the depilated back skin, 1 % Evans blue solution (0.1 ml) was injected i.v . to trace permeability changes. The rats were killed 2 hr later and the cross diameters of each blue weal were measured from the visceral side . For the determination of hemorrhagic activity, the injection of Evans blue solution was omitted and the formation of hemorrhagic spot was observed from the visceral side as well. Phospholipase A activity was examined by measuring the clearing of an egg yolk suspension, according to the method of M~ttttverrt (1965) . Analytical disc electrophoresis Analytical disc electrophoresis was performed on a 7% polyacrylamide gel with ß-alanine-acetic acid buffer (pH 4.5) according to the method of RetsFEt.u et al. (1962) . ARer running at 5 mA per gel, the gel was stained for proteins with Amido Black lOB or for carbohydrates by the method of Zwct~uxtus et al. (1969) . Electrojocusing
Electrofocusing on a 7 .5% polyacrylamide gel was carried out at 200 V for 3 hr . Bio-Lyte 3/10 (Bio-Rad Laboratories, Richmond, U.S .A .) was used as carrier ampholite at a concentration of 2% . The gel was stained with Coomasie brilliant blue G-250. in order to estimate the pH gradient formed, colored pI marker proteins (Oriental Yeast, Tokyo, Japan) were electrofceused together with the purified lethal factor. Molecular weight determination
The mol. wt of the purified lethal factor was determined either by gel filtration or by sodium dodecyl sulfate (SDS) disc electrophoresis. Gel filtration was performed by high-performance liquid chromatography (HPLC) on a column (0.75 X 30 cm) of TSK-GEL G3000SW (Toyo Soda, Tokyo, Japan) with 0.01 M phosphate buffer (pH 7.0) containing 0.3 M NaCI . The flow rate was maintained at 0.7 ml/min and the elutee monitored by absorbance at 280 nm . Bovine serum albumin (mol . wt 67,000), ovalbumin (43,000), ribonuclease A (13,700) and cytcehrome c (12,500) were used as standard mol. wt proteins . SDS-disc electrophoresis was carried out as described by WEeert and Oseoxrt (1969) using a 10% polyacrylamide gel and 0.l M phosphate buffer (pH 7.2) containing 0.1 % SDS. Prior to electrophoresis, samples were dissolved in 1 % SDS and 5% 2-mercaptcethanol and boiled for I S min. The gel was stained with Coomasie brilliant blue R-250. The reference proteins used were bovine serum albumin, ovalbumin, chymotrypsinogen A (mol . wt 25,000) and ribonuclease A. Protein and carbohydrate determinations Protein was estimated by the method of Lowtty et al. (1951) using bovine serum albumin as a standard and carbohydrate by the phenol-sulfuric acid method (Dueo~s et al., 1956) using o-glucose as a standard .
A Lethal Factor in Starfish Venom
1079
0 .6
0 .3
0 .4
a z
w 0
d u c R a
0 .2
0 N
c 0 .N .+ a w a+
C O u c 0 u
a
Fraction
nu~bsr
FIG. I . CM-CELLUL06E COLUMN CHROMATOGRAPHY OF A. ptanci YeNGM. Lethal fractions obtained in the first CM~ellulose column chromatography were again applied to a CM-cellulose column (1 .8 x 45 cm) equilibrated with 0.01 M phosphate buffer (pH 7.0). A linear gradient of 0-0.6 M NaCI in the buffer was started at fraction 41 . Fractions of 8 ml were collected at a flow rate of 53 ml/hr. The lethal factor was eluted in fractions indicated by a bar.
Amino acid analysis The purified lethal factor was hydrolyzed with 6N HCl in an evacuated tube at 110'C for 24 hr and then applied to a Hitachi 835 amino acid analyzer. The amino acid composition was calculated from three determinations. Tryptophan was estimated spectrophotometrically by the method of GoouwIN and MORTON (1946).
RESULTS AND DISCUSSION
Purification ofa lethal factor Although as much as 70% of proteins in the crude venom were passed through a column of CM~ellulose, they showed no lethality (first CM-cellulose column chromatography). Isocratic elution by 0.6 M NaCI in 0.01 M phosphate buffer (pH 7.0) afforded one protein peak with lethality, which accounted for only 4% of proteins in the crude venom. In the second CM-cellulose column chromatography using a linear gradient elution of 0-0.6 M NaCI in the buffer, three protein peaks were observed (Fig. 1). Lethality was detected only in the symmetrical peak which was eluted last, indicating that the lethal factor is fairly basic. The lethal fraction obtained was then chromatographed on Sephadex G-100. As illustrated in Fig. 2, the lethal factor appeared in a symmetrical protein peak . Thus, 4.0 mg of the purified lethal factor was obtained. As mentioned above, CM-cellulose column chromatography was very effective for the purification of the lethal factor . In this step large quantities of contaminated proteins were separated from the lethal factor . Furthermore, analytical disc electrophoresis revealed that the homogeneous lethal factor existed in the second half of the lethal peak (second CM-
(080
K. SHIOMI et at.
0 .3 E C 0 a0 N a+ w
0 .2
m u C w 0 H
a 40
50
60 Fraction
70
80
90
nu~ber
FIG . Z. GEL FILTRATION ON SEPHADEX G-IOO OF THE LETHAL FRACI70N OBTAINED IN CM-CELLUL06E COLUMN CHROMATOGRAPHY. Lethal fractions obtained in the second CM~ellulose column chromatography (Fig. 1) were subjected to gel filtration on a Sephadex G-100 column (2 .5 x 90 cm) with 0.01 M phosphate buffer containing 0.15 M NaCI (pH 7.0). Fractions of 6 ml were collected at a flow rate of 10 ml/hr. The lethal factor was eluted in fractions indicated by a bar .
cellulose column chromatography). Therefore, apart from the yield, the pure lethal factor can be obtained by CM-cellulose column chromatography alone, without the following gel filtration on Sephadex G-100. Physicochemical properties of the purified lethal factor
The homogeneity of the purified lethal factor was supported by the following results; it gave a single band in analytical disc electrophoresis (Fig . 3A), SDS-disc electrophoresis (Fig. 3B) or electrofocusing and also gave a single symmetrical protein peak in HPLC. In analytical disc electrophoresis the purified lethal factor was visualyzed by both protein and carbohydrate staining, suggesting that it is a glycoprotein . its carbohydrate content was estimated to be 3.5% .The mol. wt was determined to be 20,000 by gel filtration (Fig . 4A) or 25,000 by SDS-disc electrophoresis (Fig. 4B). Since similar values were obtained by both methods, the lethal factor appeared to exist in a monomeric form having no subunit structure. As expected from the behavior on CM-cellulose (Fig. 1), the lethal factor was shown to be basic; its isoelectric point was estimated to be 10.6 by electrofocusing. The amino acid composition is listed in Table 1 . In spite of its basic nature, the lethal factor was richer in acidic amino acids than in basic amino acids, suggesting that most of the acidic amino acid residues exist as an amide form . Biological activities of the purified lethal factor
The i.p. LDP of the purified lethal factor was determined to be 0.43 mg/kg (95% confidence interval, 0.37-0.50 mg/kg) . As with the crude venom (Sx1oM1 et al., 1985a), lethal doses of the purified factor induced flaccidness, numbness and occasional convulsion in mice and killed them within 96 hr. The purified lethal factor showed a hemolytic activity of 18.2 HU/mg against sheep erythrocytes . Edema-forming and capillary permeability-
108 1
A Lethal Factor in Starfish Venom
FIG . 3 . POLYACRYLAMIUE GEL ULSC ELECTROPHORESIS OF THE PURIFIED LETHAL FACTOR.
Origin is at top. (A) Analytical electrophoresis. A 7% polyacrylamide gel and ß-alanine-acetic acid buffer (pH 4.5) were used . The protein band was visualized with Amido Black IOB. (H) SDS electrophoresis. A 10% polyacrylamide gel and 0.1 M phosphate buffer (pH 7.2) wntaining 0.1 SDS were used . Prior to electrophoresis, the sample was treated with 1% SDS and 5% 2mercaptcethanol at 100'C for 15 min. The protein band was visualized with Coomassie brilliant blue R-250.
0
Retention tine
(min)
Relative mobility
FIG . 4 . MOLECULAR WEIGHT DETERMINATION OF THE PURIFIED LETHAL FACTOR BY G3000SW (A) ANll SDS-DISC ELECTROPHORESIS (B) .
HPLC ON TSK-0EL
The reference proteins used are: l, bovine serum albumin; 2, ovalbumin; 3, chymotrypsinogen A; 4, ribonuclease A; and 5, cytochrome c.
1082
K. SHIOMI et al. TABLE I .
AMINO ACID COMPOSITION OF THE PURIFIED LETHAL FACTOR
Amino acid Asx Thr Ser Glx
Pro
Gly
Ala Val Cys (half) Met Ile Leu Tyr Phe Trp Lys His
Arg
Mole %* 12.5 5.6 8.5 6.9 5.7 9.3 6.0
?.6
0.8 1 .3 3.1 8.1 4.8 3.0 2.2t 9.6 2.8 2.6
* Average values calculated from three deteIiIIinations . t Determined spectrophotometrically. increasing activities were also displayed by the lethal factor; i.m. injection into mouse foot of65 ~g ofthe lethal factor caused an edema of 123% (average of three determinations) and intradermal injection into rat back skin of 130 ieg a blue weal of 5 mm (average of three determinations) in diameter . On the other hand, the lethal factor was devoid of hemorrhagic activity at a dose of 130 hg. In addition, it seemed to be fret from phospholipase A activity since no clearing of an egg yolk suspension was observed even at a dose of 32 hg. All of the above biological activities examined with the purified lethal factor are manifested by the crude venom (SHIOMI et al., 1985a) . The purified lethal factor, however, lacked hemorrhagic and phospholipase A activities . It is also significant that the hemolytic, edema-forming and capillary permeability-increasing activities of the lethal factor are very weak as compared to those of the crude venom (SHIOMI et al., 1985a). For example, as low as 9 ftg of the crude venom could induced a 130% edema while more than 65 ~g of the lethal factor was needed to cause the same magnitude ofedema. From these results, it is apparent that besides the lethal factor under study, toxic factors bearing the biological activities other than lethal activity are contained in the crude venom. This is in accordance with the assumption of TAIItA et al. (1975) that lethal and hemolytic activities are elicited by different proteinaceous principles . Thus, the lethal factor does not seem to be a major principle responsible for the local effects such as swelling and redness induced by A planci stings . For a better understanding on the local effects, it is necessary to purify and characterize other toxic factors . Acknowledgements - We are grateful to Professor S. KONOSU and Dr A. SHINAGAWA, Faculty of Agriculture, the University of Tokyo, for measuring the amino acid composition .
A Lethal Factor in Starfish Venom
l0ß3
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