Tealia Nematocyst Toxin Purification
Abstract—A toxin from the nematocysts of the anemone
Tealia crassicornis has been partially purified. The
toxin is nondialyzable, of molecular weight greater
than 3500, and is heat labile, i.e. inactivated after
heating for 30 minutes at 100°0. The LD is estima-
ted to be the toxin from 2:3 X 10° nematocysts per kg
of the crab, Pachygrapsus crassipes. This correspon-
ded to 3-68mg of protein per kg of crab.
INTRODUCTION
Nematocyst toxin studies were revolutionized when Phillips and
Abbott (1957) developed "a method of obtaining purified suspen-
sions of nematocysts from sea anemones (Actiniaria) in order
to obtain a toxic preparation which could be considered nema-
tocyst in origin..." Blanquet (1968) isolated mainly acontial
microbasic mastigophore nematocysts from the sea anemone Aip-
tasia pallida and found that the toxin caused leg autonomy and
death in fiddler crabs; Hessinger and Lenhoff (1973a,b 1974a,b)
found that it also affected ionic conductance in crayfish giant
nerve fibers. Shapiro (1968a,b) used whole tentacle extracts
from the sea anemone Condylactis gigantea and found that the
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Tealia Nematocyst Toxin Purification
extract caused rigid paralysis and death in crayfish, and af-
fected the firing of all classes of lobster and crayfish axons,
probably due to the toxin interacting with the nerve membrane
ionic conductance.
Tealia crassicornis (Muller, 1776), a brightly colored red
and green sea anemone of Central California was used in this
study. There have been no investigations of toxins from this
animal. The predominant nematocyst types of T. crassicornis
are basitrichs and microbasic p-mastigophores, along with spi-
rocysts. The toxic extract from isolated, discharged nemato-
cysts was characterized and assayed for toxicity in the crab,
Pachygrapsus crassipes (Randall, 1839) by injection.
MATERIALS AND METHODS
T. crassicornis was collected subtidally from kelp beds
off Hopkins Marine Station in Pacific Grove, California. The
method reported by Phillips (1956) with several modifications
was employed inthe isolation of undischarged nematocysts from
the whole anemone. Three types of isolating media were used,
1MSodium Citrate in distilled water, 1MSucrose in O-45/Umilli-
pore filtered sea water, and a combination of the two where
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Tealia Nematocyst Toxin Purification
the primary isolation was with citrate and the final purifica-
tion with sucrose. Figure 1 presents a flow chart describing
the isolation and purification procedures.
Not all nematocysts were discharged upon addition of dis-
tilled water and a hemocytometer was used to estimate the num-
ber of nematocysts, both discharged and undischarged, in the
extract prior to final centrifugation.
A protein determination (Lowry, 1951) was performed before
and following final centrifugation using a Bovine Serum Albumin
Standard. The amount of protein released per nematocyst was
estimated.
Toxin assays were performed on the crab, P. crassipes,
which is abundant in the rocky intertidal of the Hopkins Marine
Station. Crabs ranging in size from 11•0 - 47-3grams were in-
jected and maintained in separate containers at 16-18°0 and
fresh water was supplied 3-4 times daily. The injection was
into the venous sinus accessible in the upper joint of the left
leg of the crab. The volume injected ranged from O-055 - 1-5ml.
No effect other than death was noted. Toxin in distilled water
and toxin in Crustacean Ringer's solution (Pantin, 1934) were
compared. Control injections of distilled water and Crustacean
Ringer's were included in the toxin assays.
Toxin in Crustacean Ringer's was obtained by dissolving the
toxin in this solution after concentrating distilled water pre-
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Tealia Nematocyst Toxin Purification
parations by evaporation under vacuum at 30°0.
Toxin preparations were freed of isolation medium contam-
inants by dialysis using Spectrapor Membrane Tubing (Spectrum
Medical Industries, Inc.) against Crustacean Ringer's at 400.
Thermolability of the toxin was determined by heating at
100°0 for 30 minutes in a boiling water bath.
Molecular size of the toxin was estimated using gel fil-
tration on BioGel P-100 (Bio Rad Laboratories) at 50-150 mesh.
The effect of the toxin on the circumesophageal connective
nerve of the lobster, Homarus americanus, was tested. The nerve
was placed over a series of chloride coated silver wires with a
plastic container in the middle for the Crustacean Ringer's.
Stimulation was given upstream and propagated action potentials
recorded downstream from the Crustacean Ringer's. The Crustacean
Ringer's was then replaced by the toxin and action potentials a-
gain recorded. The recordings themselves were made with P8A.C.
pre-amplifiers (Grass Instrument). The outputs were viewed on a
Tektronix 502 oscilliscope and permanent recordings before and
after the change to toxic extract were made on photographic film.
(Kao and Fuhrman, 1967). The stimulus was set at 8volts, duration
0•82msecs., frequency 8°6pulses per sec., and 20volts per om sen¬
sitivity.
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Tealia Nematocyst Toxin Purification
RESULTS
Nematocyst Isolation
Using the sodium citrate as an isolation medium, 7•5 X io
nematocysts were isolated from 500ml. of anemone. Of the total
number, 36% were dischargeable upon treatment with distilled
water; in the sodium citrate with final washing in sucrose,
1•94 X 10' nematocysts were isolated, 32% were dischargeable;
and with the use of sucrose throughout isolation, 7°8 X 10°ne-
matocysts were obtained with 40% dischargeable.
Toxic Extract from Purified Nematocysts
The toxic preparation obtained from nematocysts isolated
using sodium citrate alone contained 1e5mg of protein per ml re¬
leased from 9.0 X 102 nematocysts per ml. or 1-67 X 10-2Ug of
protein per nematocyst. Toxin prepared from nematocysts iso-
lated using sucrose in the final washing contained 2•8mg of pro-
tein per ml. released from 2•0 X 10° nematocysts per ml. or
1•4 X 10 JUg of protein per nematocyst. The amount of protein
released per nematocyst for preparations obtained using sucrose
alone was not determined. These preparations were assayed for
toxicity at one or two dose levels. The results are presented
in Table 1. All toxin preparations caused death of the assay
animal. The lack of such response at the lower dose level of
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Tealia Nematocyst Toxin Purification
namatocysts prepared with sucrose above may indicate a slight
difference in relative toxicity. The time to death of crabs
injected with the toxin in Crustacean Ringer's appears to be
somewhat shorter than for distilled water preparations. Dial-
ysis against Crustacean Ringer's did not result in a loss of
toxicity indicating that the molecular size of the toxin is
greater than 3500. Heat treatment destroyed toxicity.
The LD of the toxin from citrate prepared nematocysts
and dialyzed against Crustacean Ringer's was estimated by tes-
ting groups of three crabs at the following dilutions: full
strength, 32/100, 1/10, 32/1000, and 1/100. The LD appears
to be close to an amount of toxin present in 2'3 X 10° nemato¬
cysts or 3-68mg of protein per kg of crab.
Gel filtration resulted in the separation of the protein
into two fractions, one with a molecular size excluded and,
therefore, larger than 100,000, the other, a smaller sized
molecular component greater than the marker, cytochrome C, of
molecular weight 12,270, utilized in the fractionation. Neither
of these preparations could be shown to possess the toxicity
alone, although the high molecular weight component seemed to
produce an anesthetic effect on all injected crabs. As is shown
in Figure 2, the high molecular weight fraction appears to block
axonal conductance when tested on the circumesophageal connective
nerve of the lobster.
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Tealia Nematocyst Toxin Purification
DISCUSSION
Nematocysts from the sea anemone T. crassicornis have
been isolated, their discharge collected and tested for tox-
icity. An injection of 1°25 X 10' nematocysts per kg of crab,
P. crassipes, or about 20mg. of protein per kg crab generally
produced death. The approximate LDgo of 2:3 X 10° nematocysts
per kg P. crassipes, or 3°68mg protein per crab, was about
1/3000 as toxic as that observed by Shapiro (1967) for Condyl-
actis gigantea using the crayfish as an assay animal. Due to
the pore size used in dialysis, the molecular weight must be
greater than 3500 because upon injection after dialysis, death
was still evident. That the toxin is proteinaceous seems likely
when molecular size is considered along with thermolability. Fur-
ther investigation is required to elucidate the effect of the tox-
in on axonal conduction.
Acknowledgements—A thank-you is not enough for the untiring
patience, guidance, and advice shown to me by Dr. F.A. Fuhrman and
Dr. J.H. Phillips, Jr. Not only did they make this investigation
possible, but they gave me something much more meaningful -- a
better understanding of the way in which all true "pursuers of
knowledge" must reason and act.
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Tealia Nematocyst Toxin Purification
LITERATURE CITED
Blanquet, R., 1968. Properties and Composition of the Nemato-
Toxin of the Sea Anemone, Aiptasia Pallida. Comp. Bio-
chem. Physiol., 25:893-902.
Kao, C.Y. and Fuhrman, F.A., 1967. Differentiation of the Ac-
tions of Tetrodotoxin and Saxitoxin. Toxicon, 5:25.
Lowry, O.H., Rosenbrough, N.J., Farr, A.L., and Randall, R.J,
1951. Protein Measurement with the Folin phenol reagent.
J. Biol. Chem., 193:265-272.
Phillips, J.H., 1956. Isolation of Active Nematocysts of Metri¬
dium Senile and Their Chemical Compositions. Nature,
London, 178:93
Phillips, J.H., and Abbott, D.P., 1957. Isolation and Assay of
the Nematocyst Toxin of Metridium Senile Fimbriatum.
Bio. Bull., 113, No.2:296-301.
Shapiro, B.I., 1968. Purification of a Toxin from Tentacles of
the Anemone Condylactis Gigantea. Toxicon, 5:253-259.
Shapiro, B.I. and Lilleheil, G., 1969. The Action of Anemone
Toxin on Crustacean Neurons, Comp. Biochem. Physiol.,
28:1225-1241.
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Tealia Nematocyst Toxin Purification
FIGURE CAPTIONS
Figure 1
Procedure for the isolation of toxin from purified nematocysts.
Figure 2
Effect of the toxin on axonal conductance of the circumesopha-
geal connective nerve of a lobster, Homarus americanus.
Axonal conductance before the toxin was added.
(a)
(b) Axonal conductance 2 minutes following the replacement of
Crustacean Ringer's by the toxin.
Axonal conductance 4 minutes following the replacement of
(c)
Crustacean Ringer's by the toxin.
I VOLUME IM SODIUM CITRATE
IN DISTILLED WATER
I VOLUME T. CRASSICORNIS (500 ml.)
OR
I VOLUME IM SUCROSE IN 0-45 U
MILLIPORE FILTERED SEA WATER
MACERATE IN WARING BLENDER (APPROXIMATELY 15 MINUTES)
FILIER THROUGH CHEESECLOTH WITH VACUUM
DISCAD EESOUE
FILTRATE (NEMATOCYSTS, TISSUE,
CITRATE OR SUCROSE
CENTRIFUGE (15 MINUTES, 5°c, 750G)
DISCARD SUPERNATANT
WASH, RESUSPEND, AND RECENTRIFUGE
IN IM SODIUM CITRATE OR IM SUCROSE
UNTIL SUPERNATANT CLEAR
DRAIN THOROUGHLY, RINSE RESIDUAL
ISOLATION MEDIUM FROM INSIDE SURFACE
OF CENTRIFUGE TUBE WITHOUT DISTURBING PELLET
DISCHARGE NEMATOCYSTS BY ADDING A VOLUME OF DISTILLED WAIER
APPROXIMATELY EQUINALENT TO PELLET VOLUME, LET STAND 12-18hrs.
CENTRIFUGE (2000 G, 15 MINUTES)
DISCAR0 PELET
ERNAL
FIGURE


1
Simulus
stimulus
G.
h.
ARA
Figure 2
e
Tealia Nematocyst Toxin Purification
TABLE CAPTIONS
Table 1
Results of nematocyst toxicity using the crab, Pachygrapsus
crassipes.
e
ISOLATION
MEDIUM
sucrose
sucrose
citrate
citrate/
sucrose
ontrol/
distilled
water
ISOLATION
MEDIUM
citrate
citrate/
sucrose
control/
Crustacean
Ringer s
TOXIN IN DISTILLED WATER
DOSE PER KG. CRAE
INJECTION
nematocyst no.
mg. protein
(volume:ml)
.O5mi/iog-
1.O X 10
O.lml
O.5ml/iog-
1.05 X 10
1.O5ml
.O5m1/iog-
O.27mg
1.7 X 10
.187m1
.O5m1/iog-
O.65mg
5.O X 10
O.24m1
O.5mi/iog-
O. Omg
O.O
1.08m1
TOXIN IN CRUSTACEAN RINGER'S
DOSE PER KG. CRAB
INJECTION
mg. protein
nematocyst no.
(volume:ml)
O.l1mg
6.8 X 10
O.O5ml/iog-
.O8m1
1.2 X 10
.O5ml/iog-
O.16mg
O.O6ml
O.O
O.5mi/iog-
O. Omg
O.88m1
CRAB
WEIGHT
19.9g
21.0g
37.Ag
47.38
21.6g
CRAB
WEIGHT
15.0g
11.og
17.6g
TIME TO
LEATH
36hrs.
3éhrs.
36hrs.
TIME TO
DEATH
24hrs.
24hrs.
TABLE
ro
ISOLATION
MEDIUM
citrate
citrate
citrate/
sucrose
control/
Crustacean
Ringer's
ISOLATION
MEDIUM
citrate
INJECTION
(volume:ml)
.O5m1/iog-
.081m1
O. 5ml/iog-
.67m1
.O5ml/iog-
.81m1
O.5ml/log-
.12m1
INJECTION
(volume:ml)
.O5m1/iog-
.O7m1
DIALYZED TOXIN
LOSE PER KG. CRAB
mg. protein
nematocyst no.
7.3 X 10
O.12mg
O.96mg
6.0 X 102
O.31mg
2.4 X 100
O. Omg
O.O
TOXIN AFTER HEATING
DOSE PER KG. CRAB
mg. protein
nematocyst no.
6.3X 10
O. 1mg
CRAB
WEIGHT
16.2g
13.4g
24.Ag
16.2g
CRAB
WEIGHT
13.2g
TIME TO
DEATH
12hrs.
12hrs.
12hrs.
TIME TO
DEATH
TABLE 1 (CONT