Comp. Biochem. Physiol. Vol. 112B, No. 1, pp.

105-114, 1995

Pergamon

Elsevier Science Ltd

Printed in Great Britain
0305-0491/95 $9.50 + 0.00

0305-0491(95)00086-0

Small-molecule antioxidants in marine organisms"

antioxidant activity of mycosporine-glycine
Walter C. Dunlap* and Yorihiro. YamamotoCl:
*Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1 Komaba.
Meguru-ku, Tokyo 153, Japan; and ~'Department of Chemistry and Biotechnology, Faculty of
Engineering, University of Tokyo, Hongo, Tokyo 113, Japan
The antioxidant activities of aqueous extracts of tissues from four different marine species containing
mycosporine-like amino acids were examined by their peroxyl radical-trapping abilities using the
phosphatidylcholine peroxidation inhibition assay. The coral trout (Plectropomus leopardus) lens
extract had low oxidation inhibition activity; Porphyra tenera and Lissoclinum patella extracts had
moderately strong inhibition activities while the aqueous extract of Palythoa tubereulosa showed the
greatest oxidation inhibition activity of the extracts tested; the last two extracts contained
mycosporine-glycine. In these sample extracts, mycosporine-glycine was reactive to peroxyl radicals
whereas the iminomycosporine-like amino acids, shinorine, porphyra-334, palythine, asterina-330 and
palythinol, were oxidatively robust. Purified mycosporine-glycine inhibited peroxyl radical-initiated
autoxidation of phosphaditylcholine in a concentration-dependent manner. These results suggest that
myeosporine-glycine may function as a biological antioxidant in marine organisms.
Key words: Antioxidants; Coral trout; Lissoclinum patella; Mycosporine-like amino acids;
Mycosporine-glycine; Oxidative stress; Palythoa tuberculosa; Porphyra tenera.
Comp. Biochem. Physiol. l12B, 105-114, 1995.

Introduction
There is growing awareness of the separate and
interacting detrimental effects of environmental
ultraviolet (UV) radiation and that of reactive
oxygen species on aerobic organisms (Tyrrell,
1991; Shick, 1993). Environmental UV radiation is a recognized biological hazard affecting
survival, physiology and growth of many organisms, including marine invertebrates and algae
living in shallow-water environments (Jokiel,
1980; Jokiel and York, 1982; Hader and Worrest, 1991). Multiple harmful effects of UV
include damage to DNA and proteins, inactivation of enzymes, oxidation of lipids, disruption of membranes and inhibition of algal

photosynthesis and growth (reviewed by Harm,

1980; Worrest, 1982; Renger et al., 1986; Kyle,
1987), which may involve active oxygen species
(DiGiulio et al., 1989; Tyrrell, 1991; Dykens
et al., 1992; Shick, 1993).
The ultraviolet-B (290-320 nm) region overlaps the long wavelength absorption of DNA
and is largely responsible for the direct damaging effects of sunlight. Many other cellular
components can also absorb UVB and UVA
(320-380 nm) radiation causing oxidative damage by photodynamically generating reactive
oxygen intermediates (Jagger, 1983; Cunningham et al., 1985). These intermediates include
singlet oxygen (IO2) and the oxygen radicals,
Correspondence to: W. C. Dunlap, Australian Institute of superoxide anion (O2-'), hydroperoxyl radical
Marine Science, PMB No. 3, Townsville, M.C., Qld. (HO2'; the protonated form of 02-') and
4810, Australia.
hydroxyl radical (HO.), which result from unReceived 5 October 1994;revised6 February 1995;accepted coupling at electron transfer sites or via autoxi16 February 1995.
z~Present address: Research Center for Advanced Science dation reactions (reviewed in Cadenas, 1989).
and Technology,Universityof Tokyo, 4-6-1 Komaba, Liberation of hydrogen peroxide in cellular
Meguro-ku, Tokyo 153, Japan.
processes can also lead to the formation of the
105

106

W. C. Dunlap and Y. Yamamoto

highly reactive hydroxyl radical in a Fenton-like

reaction involving superoxide and cellular Fe 3
(Halliwell and Gutteridge, 1984; Imlay, 1988).
Free radicals and other active derivatives of
oxygen are also the inevitable by-products of
biological redox reactions. Aerobic organisms
have evolved a variety of defense mechanisms
against the toxic effects of active oxygen species.
Essentially, these antioxidation defenses are of
three general classes including water-soluble reductants (glutathione, ascorbate, urate) in the
cytosol, fat-soluble antioxidants (e-tocopherol,
fl-carotene) residing in cellular membranes, and
the antioxidant enzymes, glutathione peroxidase, ascorbate peroxidase, catalase, and superoxide dismutase. Cellular enzyme defenses are
often inducible under conditions of UVA-mediated oxidative stress (Lesser et al., 1990; Shick
et al., 1991; Tyrrell, 1991) and have been identified in the prevention of hyperoxic toxicity in
marine algal-invertebrate endosymbiotic associations (Dykens and Shick, 1982; Matta and
Trench, 1991; Dykens et al., 1992; Shick, 1993).
Whereas the protective role of antioxidant enzymes in marine organisms is well recognized,
the structure and biofunctions of small-molecule antioxidants (reductants) in the photophysiology of marine organisms are yet poorly
understood.
A physiochemical defense against the damaging effects of environmentally relevant UV radiation involves absorbing potentially harmful
wavelengths with UV-absorbing compounds
and dissipating the energy without generating
photochemical reactions or photosensitizing
the formation of reactive intermediates. Many
marine organisms contain UV-absorbing
mycosporine-like amino acids (MAAs) which
are characterized by a cyclohexenone or cyclohexenimine chromophore conjugated with the
nitrogen substituent of an amino acid, having
absorption maxima ranging from 310 to 360 nm
(Nakamura et al., 1982). The chemical structures and wavelength of maximum absorbance
('~max) for common MAAs are provided in
Fig. 1. MAAs have been identified in taxonomically diverse organisms: a marine heterotrophic
bacterium (Arai et al., 1992), cyanobacteria
(Garcia-Pichel and Castenholz, 1993), algae (Ito
and Hirata, 1977; Takano et al., 1979; Tsujino
et al., 1980; Carretto et al., 1990; Karentz et al.,
1991), marine invertebrates (Nakamura et al.,
1981; Grant et al., 1985; Chioccara et al.,
1986a,b; Karentz et al., 1991; Shick et al., 1992;
Carroll and Shick, 1993), within algal-invertebrate symbioses (Takano et al., 1978a,b; Hirata
et al., 1979; Dunlap and Chalker, 1986; Lesser
and Stochaj, 1990; Shick et al., 1991; Stochaj
et al., 1994), and in fish organs (Chioccara et al.,
1980; Dunlap et al., 1989). A protective function

CO2H
o

OO H ~

OCH3

NH

OCH3
NH

HO OH ~

LCO2H

LCO2H

Mycosporine-Gly

Shinorine

~ n a x = 310nm

;~max = 334nrn

CO2H
"~L" N

NH

~OCH3

HOOH v

NH

OCH3

HOOH v

NH

LCO2H

LCO2H

Porphyra-334

Palythine

Zrnax= 334nm

Zmax = 320nm
CH3

C".
HOOH ~

NH

HOOH v

NH

LCO2H

LCO2H

Asterina-330

Palythinol

~nax = 330nm

~nax = 332nm

Fig. 1. Molecularstructures and wavelengthof maximum

absorbance ()~max)for mycosporine-likeamino acids commonly found in marine organisms.

for these compounds is inferred by their efficient

UVA- and UVB-absorbing properties, together
with the often observed correlation between
MAA concentrations and natural or experimental levels of UV exposure.
Jokiel (1980) demonstrated that UV tolerance
affords many coral reef organisms the ability to
survive in shallow waters. Shallow-water corals
appear to be acclimatized to such potentially
stressful conditions (Siebeck, 1981, 1988;
Masuda et al., 1993) and usually contain higher
concentrations of UV-absorbing compounds
(MAAs) than do conspecifics living at greater
depths (Maragos, 1972; Dunlap et al., 1986;
Scelfo, 1986; Shick et al., 1995). UVA and UVB
radiation penetrates clear tropical seawater to
ecologically important depths (Fleischmann,
1989), and this depth-dependent relationship
is hypothesized to be an adaptation to
shield organisms from prevailing levels of solar
UV radiation. Further evidence has been
demonstrated by inducing changes in MAA
tissue concentrations by transplanting or

107

Antioxidants in marine organisms

experimentally altering the exposure of algae

(Carreto et aL, 1990) or corals and other anthozoans to UV radiation (Jokiel and York, 1982;
Scelfo, 1986; Shick et al., 1991; Kinzie, 1993;
Gleason, 1993).
The occurrence of high concentrations of
MAAs in marine organisms exposed to high
levels of solar radiation is consistent with their
providing protection as a UV-absorbing sunscreen. In this study we examine the hypothesis
that certain MAAs may also serve in a complementary fashion as antioxidants for protection
against photooxidative stress induced by
oxyradicals. Examination of the oxidation
properties of MAAs in aqueous extracts of
marine organisms provides in vitro evidence
that mycosporine-glycine, but not iminomycosporines, has moderate antioxidation activity
as a free radical scavenger and may function as
a biological antioxidant in marine organisms.

Materials and Methods

Preparation of sample extracts
Specimens of the zoanthid Palythoa tuberculosa (Cnidaria) and the ascidian Lissoclinum
patella (Chordata) were collected from 2-4 m
depth by scuba diving at Davies Reef
(14738'E: 1851'S), on the Great Barrier Reef,
Australia, in June 1990. Specimens were stored
frozen at -20C until extracted. Coral trout
(Plectropomus leopardus) were caught at Davies
Reef by handline and the ocular lenses were
immediately excised and stored frozen. The
freeze-dried red alga Porphyra tenera (Rhodophyta) was a gift from the Yamamoto Nori
Research Institute, Ota-ku, Tokyo. The freezedried red alga Mastocarpus stellatus collected
from Schoodic Pt., Maine, U.S.A. was provided
by Professor J. M. Shick (U. Maine).
Freeze-dried P. tenera (2.05 g) was extracted
four times with 25 ml volumes of 80/'0 aqueous
methanol. The combined extract was filtered
through Whatman glass fiber GF/C filters. The
organic solvent was removed under reduced
pressure on a rotary evaporator at 40C. The
aqueous extract (c. 20 ml) was refiltered (GF/C)
and passed through tandem C-18 Sep-Pak
(Millipore) cartridges to remove pigments and
suspended lipid. The clear extract was diluted
with distilled water until a 1:4 dilution gave an
absorption maximum of about 1.8 absorbance
units/cm at 334 nm. MAA concentrations were
quantified and lyophilized aliquots (2.0 ml) were
stored at -20C until use.
Sample extracts were similarly prepared from
frozen tissue samples of L. patella, P. tuberculosa and the occular lens of P. leopardus. The
lyophilized extracts were freshly reconstituted in

distilled water (2.0 ml) for comparison of antioxidant activities in the phosphatidylcholine
peroxidation inhibition assay (PC-assay).
Analysis of mycosporine-like amino acids
MAAs were separated and quantified by
reverse-phase, isocratic high-performance liquid
chromatography (HPLC) as described previously (Dunlap and Chalker, 1986; Dunlap
et al., 1989; Shick et aL, 1992). Separation was
achieved on a Brownlee RP-8 column (Spheri-5,
4.6 mm i.d. 250 mm) protected with an RP-8
guard column (Speri-5, 4.6 mm i.d. x 30 mm)
and an aqueous mobile phase of 25% (or 55%)
methanol and 0.1% acetic acid (v:v) at a flow
rate of 0.8 ml/min. Quantitation of MAAs in
prepared lyophylized standards was determined
by chromatographic analysis with dual wavelength absorbance detection at 313nm and
340 nm (Waters model 440 detector) from isolated MAA fractions: mycosporine-glycine (mycosporine-Gly), palythine and palythinol from
P. tuberculosa (Ito and Hirata, 1977; Takano
et al., 1978a,b; Hirata et aL, 1979), asterina-330
from P. leopardus (Dunlap et al., 1989) porphyra-334 from P. tenera (Takano et al., 1979)
and shinorine from M. stellatus (Carroll and
Shick, 1993), using molar extinction coefficients
given by those authors and Tsujino et al. (1980).
Concentrations of MAAs in tissue extracts used
for the PC-assays, and MAA oxidation reactions initiated with AAPH (see below), were
determined by HPLC analysis with peak detection at 320 nm (Shimadzu variable wavelength
detector) which was calibrated using prepared
MAA standards.
Phosphatidylcholine
assay (PC-assays)

peroxidation

inhibition

PC-assays were conducted according to published methods (Kohen et al., 1988) using
2,2'-azobis(2-amidinopropane) dihydrochloride
(AAPH, Wako Pure Chemicals Industries,
Osaka) and soybean phosphatidylcholine (PC)
as radical inititor and lipid substrate, respectively. A 100mM stock solution of AAPH
(27.1 mg/ml) was prepared in 0.1 M NaC1,
stored frozen between use and replaced weekly.
Soybean phosphatidylcholine (PC) was purified
by the method of Singleton et al. (1965) and a
stock solution was prepared in methanol at a
concentration of 40 mg/ml (50 raM). For each
assay, 0.4 ml of the PC solution was evaporated
in a round-bottom flask under reduced pressure
at room temperature. An aqueous solution of
0.1M NaC1 and 100pM EDTA (4.0ml) was
added to the dry residue and the contents were
agitated vigorously on a vortex mixer to form a
multilamellar liposome emulsion.
The control rate of PC oxidation was

108

W. C. Dunlap and Y. Yamamoto

determined by combining 15 #1 of the AAPH

solution with 1.5 ml of liposome emulsion; the
initial concentrations of PC and AAPH in the
PC-assay were 5.0 and 1.0 mM, respectively.
The reaction dispersion was maintained in atmospheric equilibrium at 37C in a thermostated, shaking waterbath. The formation of
phosphatidylcholine hydroperoxide (PC-OOH)
was determined initially and measured at
consecutive 10-min intervals (see below). The
control rate of PC peroxidation (1.39#M
PC-OOH/min) was calculated by linear regression, and inhibition rates were determined
by adding 5-60 #1 aliquots of sample extracts to
the PC peroxidation reaction.
PC-OOH concentrations in the PC-assays
were determined chromatographically by the
method described by Kohen et al. (1988). Assay
aliquots (30/~1) were injected onto a Supelco
silica LC-Si column (4.6mm i.d. x 250mm)
with a mobile phase consisting of 40mM
NaH2PO4 in methanol (10:90) at a flow rate of
1.0 ml/min. PC-OOH, which had a peak retention time of 6.3 min, was detected by UV absorption at 234 nm. A calibration standard for
quantitation of PC-OOH was prepared and
purified by methods previously described
(Yamamoto et al., 1984, 1987).

2.0-

Porphyra tenera

Coral trout lens

Plectropomus

leopardus

334nm

332nr

1.0-

~O.Oi

o
t-t~

~ 2.0[ Lissoclinum
patella

~alythoa tuberculosa

312nm

~,~nrn

1.0

Oxidation of MAAs in tissue extracts

AAPH-initiated oxidation reactions of
MAAs in sample tissue extracts were conducted
without phosphatidylcholine as a competing
substrate. AAPH (60 #1; 100 mM) and 60 #1 of
tissue extracts concentrated 10-fold (lyophylized
tissue extracts (2ml) reconstituted in 200#1
H20) were combined in 6.0 ml of distilled water.
UV-visible spectra and chromatographic analysis were determined initially, and at each hour
for 8-10hr. The chromatographic conditions
for MAA analyses are as described above.

Purification of mycosporine-Gly for PC-assay

Mycosporine-Gly was fractionated from the
Palythoa extract on a YMC (Japan) semipreparative amino column (YMC-pack NH2,
SH-643-5, S-5, 120A; 20 mm i.d. x 250 mm)
using a mobile phase consisting of 40mM
NHaOAc and 17.5 mM HOAc in 80% methanol
at a flow rate of 8.0 ml/min; mycosporine-Gly
eluted with a retention time of 44.4min.
Methanol was evaporated from the mycosporine-Gly fraction under reduced pressure
and the aqueous ammonium acetate residue was
removed by lyophylization and sublimation
at room temperature. The mycosporine-Gly
residue was redissolved in a small volume of
water to provide a concentrate that was
quantified by photometric analyses using published molar absorptivity data (Ito and Hirata,

0.0
200

300

4~

200

300

400

Wavelength (nm)

Fig. 2. UV-visible light absorption spectra (200-450 nm of

aqueous extracts of sample tissue (dilution 1 : 4).

1977). The antioxidant activity of mycosporlneGly was determined with the addition of appropriate quantities of sample to give 15 and 30 # M
concentrations of mycosporine-Gly in the PCassay. Ascorbic and uric acids (analytical grade)
were purchased from Wako Pure Chemical
Industries Ltd. (Osaka) for comparison of PC
peroxidation inhibition rates.

Results and Discussion

Aqueous tissue extracts were prepared from
the coral trout (Plectropomus leopardus) ocular
lens, the red alga Porphyra tenera (Japanese
"nori"), the colonial ascidian Lissoclinum
patella, and the colonial zoanthidian Palythoa
tuberculosa. The UV-visible light absorption
spectra for these extracts are given in Fig. 2

A n t i o x i d a n t s in m a r i n e o r g a n i s m s

showing strong absorption in the UV-A/B region between 310-340 nm which is characteristic of mycosporine-like amino acids; MAA
concentrations in the sample tissue extracts are
provided in Table 1.
The extract of coral trout lens showed a peak
absorption maximum at 332 nm which is consistent with asterina-330 being the predominant
MAA component in this tissue (Dunlap et al.,
1989). The extract of P. tenera had a peak
absorption maximum at 334nm due to
the major MAA component, porphyra-334
(Takano et al., 1979). The extract from the
zooanthid P. tuberculosa showed a peak absorbance maximum at 312 nm due to the predominance of mycosporine-Gly ('~max = 310 nm)
while containing smaller quantities of palythine
(}~max = 320 nm), palythinol (~'max = 332 nm), and
a trace of asterina-330 ()~max = 330 nm), which is
consistent with previous analyses (Dunlap and
Chalker, 1986).
The extract of L. patella shows a peak absorption maximum at 332 nm which is near to the
334 nm absorption maximum reported for the
"cell sap" of its prokaryotic symbiont,
Prochloron sp. (Lewin, 1994). Chromatographic
analysis of the MAA composition of this tunicate gives shinorine ()]'max = 334nm) and mycosporine-Gly
(max = 310 nm) in a 2.4:1 molar
ratio (Table 1). The influence of shinorine on the
peak absorbance maximum at 332 nm is due to
its higher concentration and greater absorptivity
(E = 44,600; Tsujino et al., 1980) than mycosporine-Gly (e =28,100; Ito and Hirata,
1977). Our results differ from those reported by
Lesser and Stochaj (1990) who quantified mycosporine-Gly as the major MAA component in
L. patella and its Prochloron symbiont.
The PC peroxidation inhibition assay is based
on the peroxidation reaction of phosphatidylcholine (containing linoleate phospholipid)
which proceeds by a free-radical chain mechanism where PC. and PCOO. are chain carrying
radicals (Barclay and Ingold, 1981; Yamamoto
et al., 1982). Oxidation is initiated by the thermal unimolecular decomposition of an azo compound (A-N=N-A) under aerobic conditions to
produce peroxyl radicals at a constant rate
(Barclay et al., 1984; Yamamoto et al., 1984).

109

Lipid autoxidation occurs by the general mechanism:

initiation:
A - - N ~ N - - A -~--*2A. + N2
A" + 02
AOO. + PC

------*AOO"
----*PC. + AOOH

propagation:
PC. + 02
--~PCOO.
PCOO. + PC
.~PCOOH+ PC..
The initiator AAPH used in this study has a
decomposition half-life in water of approximately 1 week at 37C producing A. and AOO.
radicals at a near constant rate in the PC-assay.
Since the initiation of peroxyl radicals is constant, the oxidation rate of PC and formation of
PCOOH is also constant, as is observed in
Fig. 3. Antioxidants that competitively scavenge
AOO. or PCOO. radicals will retard the rate of
autoxidation in the PC-assay, and the extent of
inhibition is an indication of antioxidant activity. We have used the water-soluble initiator
AAPH, and the water-soluble antioxidants in
our sample extracts are more likely to scavenge
AOO. radicals initiated in the aqueous phase
than PCOO. radicals propagated in the lipid
phase. More detailed discussions on the chemical mechanisms of lipid peroxidation and inhibition are given by Porter (1986) and Niki
(1987).
Comparison of the PC-assay results (Fig. 3
and Table 2) shows that the coral trout lens
extract (containing mostly asterina-330) has little inhibitory effect on the control rate of PC
peroxidation. The Porphyra extract (containing
mostly porphyra-334) and the Lissoclinum extract
(containing
mycosporine-Gly and
shinorine) were moderately active. The aqueous
extract of Palythoa tuberculosa was the most
active of the samples tested by the PC-assay and
also had the most complex mixture of MAAs
(mycosporine-Gly, palythine, palythinol and a
trace of asterina-330). For this reason, the
Palythoa extract was initially chosen to examine
the reactivity of MAAs to peroxyl radicals
produced from AAPH.

T a b l e 1. C o n c e n t r a t i o n o f m y c o s p o r i n e - l i n e a m i n o a c i d s ( M A A s ) in a q u e o u s
tissue e x t r a c t s o f s a m p l e o r g a n i s m s

Mycosporine-Gly
Shinorine
Porphyra-334
Palythine
Asterina-330
Palythinol

Coral trout
lens

Porphyra
tenera

Lissoclinum
patella

Palythoa
tuberculosa

---102 # M
818 # M
--

-90/tM
862 p M
----

351 # M
845 # M
-----

1061 p M
--343 /~M
38 p M
163 # M

110

W. C. Dunlap and Y. Yamamoto

100-

Coral Trout Lens

extract

80-

Control

Control

e. tenera

extract

60p.L

15~L

60-

40-

601aL
20-

:=L
v

0
9
o0_

0-(

100 -

L patella

extract

80-

60

Control

extract

40-

60p,L

0<

10 20 30 40 50 60

i
10

i
20

i
30

Control

151.[L

20-

P. tuberculosa

5p.L
151aL
301iL
60~L
i
40

I
50

i
60

Time (min)
Fig. 3. Phosphatidylcholine peroxidation inhibition assays with test aliquots of sample tissue extracts.
Prolonged oxidation of the Palythoa extract
with A A P H effected a systematic change to the
M A A absorption profile in the UV-A/B region,
causing a bathochromic shift with a concomitant reduction in peak absorbance (Fig. 4). This
change would be consistent with the decomposition of the short-wavelength absorbing component, mycosporine-Gly, which was readily
confirmed by H P L C analysis (Fig. 5). The kinetics for the AAPH-initiated oxidation of
M A A s in all four sample extracts is given in
Fig. 6. Only mycosporine-Gly (Lissoclinum and
Palythoa extracts) had decomposed whereas the
imino-carbonyl M A A s (shinorine, porphyra334, palythine, asterina-334 and palythinol)
were oxidatively stable.
The mechanism and products of mycosporine-Gly oxidation were not determined in
this study. However, in accordance with free
radical thermodynamics (Buettner, 1993), these

results suggest that imino-MAAs have a higher

one-electron oxidation potential than the reduction potential of the alkylperoxyl radicals
produced by A A P H in the initiation and propagation of lipid oxidation, whereas mycosporineGly has a lower oxidation potential and is
capable of reacting with alkylperoxyl radicals to
inhibit radical-chain lipid peroxidation.
The ability of purified mycosporine-Gly to
inhibit lipid peroxidation was confirmed by the
PC-assay (Fig. 7). Mycosporine-Gly showed a
concentration-dependent ability to inhibit phosphatidylcholine peroxidation and was moderately active at 30/JM. The molar antioxidative
efficiency of mycosporine-Gly, however, is considerably less than the water-soluble reductants,
ascorbic and uric acids.
Mycosporine-Gly was absent from both the
coral trout lens and Porphyra extracts (Table l).
Accordingly, the aqueous lens extract showed

Table 2. Rates of PC-OOH formation in the phosphatidylcholine peroxidation inhibition assays treated with aliquots of sample extracts
Rate of PC-OOH formation (pM/min)
Extract volume
(control) 5 #1
15 ,ul
30 /~1
60 #1
Coral trout lens extract 1.39
--1.12
P. tenera extract
1.39
-1.10
0.41
L. patella extract
1.39
1.01
-0.36
P. tuberculosa extract
1.39
0.99
0.57
0.30
0.11

Antioxidants in marine organisms

1.2-

1.0-

trout Ions extract

Coral

t/)
It.

111
P. tenera oxtra~

1.o~=oooooeooeoeoooo
~

(1)

t~

t ;~nax= 312.5nm
/ AbSmax= 0.523

~,,,,.

t-

0,60.6-

~ m a x -- 31510nm

/_K

0.4-

. M . , / / " . . . . ~ kmax = 320.Onto

"~'.S."'
.... N
Absmax= 0.277

o
.Q
<

OOOOooo0oOOOoO

0.2-

"%.._
~ 0.0

0.0

20O

3(30

400

>m

/.. pate//= extract

Fig. 4. Spectrophotometric analyses (200~,25 nm) of Palythoa tuberculosa extract oxidized with A A P H at initial
conditions (--), t = 4 hr (----) and t = 8 hr (....).

rr

0.8-

very little antioxidation activity (Fig. 3) in which

tissue proteins were denatured in the initial
extraction procedure with 80% methanol. The
extract of Porphyra tenera (Japanese "nori" or
"purple laver") showed moderate antioxidation
activity, presumably due to the presence of
other endogenous antioxidants. The antioxidarive properties of algal extracts are well recoga) t = 0 h

Palythine
Asterina-330
i ~Palythinol
(/3
:2)
or-

~aaglon@a=oaol~0|0~a=
[]

[]

[IQ

[]
a

0.6-

[]
[]

[]

o.o

O~
.~
0.016
O
/)
..Q
<C

P. tuberculosa extract

1,011nOooOOOeOoooOOO O

Mycosporine-Gly

o 1.2-

Wavelength (nm)

0.016-

0.4 -

t:] i::]

a[]

Pal,/thine

Ab"terin~O

0.2 - no~ino~

at~:j

Mycosporine-Gly nag

0.0

i
2

i
4

i
6

o Palythbol

[]0

a ,M~x~e-Gly o?t~an~

i
8

i
10

10

Time (h)

Fig. 6. Kinetic data for the AAPH-initiated oxidation o f

M A A s in sample tissue extracts.

nized (Fujimoto et al., 1985) and the active

reductants may include ascorbate, phenols and
polyphenolic compounds (Le Tutour, 1990; and
references therein).
Although mycosporine-Gly may occur in
small quantities in non-symbiotic algae (Carreto
et al., 1990; Karentz et al., 1991) it is a significant component (Table 1) in the marine invertebrates, L. patella and P. tuberculosa, both of
which have algal symbionts. Lissoclinum patella
is a colonial ascidian having the extracellular
oxygenic-prokaryotic symbiont, Prochoron sp.,
and Palythoa tuberculosa is a colonial zoanthid
harbouring the photoautotrophic dinflagellate
endosymbionts, Symbiodinium sp. (zooxanthellae). Mycoporine-Gly is also present in the
zooxanthellate corals, Acropora formosa

b) t = 8 h

100

80

Control

-~ 60
-iO

40

MG(lOpM)

20
/
o

1'o

1'5

2'0

Time (rain)
Fig. 5. Chromatographic analysis of M A A s in Palythoa
tuberculosa extract (detection: 320 nm) oxidized with A A P H
at (a) initial conditions (t = 0 hr) and (b) t = 8 hr.

MG (301~M}
UA (IO~.M)
~ (IO~M)

10 2'0 30 40 50 60
Time (min)

Fig. 7. Phosphatidylcholine peroxidation inhibition assay

for purified mycosporine-Gly (MG), uric acid (UA) and
ascorbic acid (AA).

112

W. C. Dunlap and Y. Yamamoto

(Dunlap and Chalker, 1986), Acropora microphthalma (Shick et al., 1995) and Porites astroides
(Gleason, 1993). While mycosporine-Gly is
commonly found in invertebrate-algal symbiosis, it is apparently not an obligate feature.
Mycosporine-Gly was reported in the aliciid sea
anemone Phyllodiscus semoni, but not in the
stoloniferan octocoral Clavularia sp., both of
which have endosymbiotic zooxanthellae (Shick
et al., 1991). In the case of the zooanthellate sea
anemone Anthopleura elegantissima, the function of mycosporine-Gly may be replaced by
the oxo-carbonyl MAA, mycosporine-taurine
(Stochaj et al., 1994), wherein taurine is substituted for glycine providing the conjugated
nitrogen substituent of the oxo-carbonyl mycosporine chromophore.
The greater capacity of the Palythoa extract
to inhibit PC peroxidation compared with the
Lissoclinum extract (Fig. 3; Table 2) may be due
in part to the higher concentration of mycosporine-Gly in the Palythoa extract (Table 1).
However, comparison of the oxidation kinetics
(Fig. 6) shows a significant delay (2 hr) in the
onset of mycosporine-Gly oxidation in the Palythoa extract. The initial delay in the AAPH-initiated oxidation of mycosporine-Gly indicates
that a more active (yet unidentified) antioxidant
is present at low concentration in the Palythoa
extract.
Although we are aware that the results of
in vitro studies, cannot be applied directly to
complex in vivo systems, the results of our study
suggest that mycosporine-Gly has moderate antioxidant activity and may provide some protection against photooxidative stress induced by
oxygen radicals in photoautotrophic symbiosis.
The presence of mycosporine-Gly (or mycosporine-taurine) in the hyperoxic tissues of
invertebrate-algal symbioses is consistent with
an antioxidative function, whereas the oxidative
robustness of the imino-MAAs, generally found
in a wide variety of marine organisms, is in
keeping with their primary function as a stable
sunscreen to provide long-term UV protection.
Acknowledgements--W.D. is grateful to Professor Isao
Karube for appointment to the Toyo Suisan endowed
Visiting Chair of Marine Biotechnology at the Research
Center for Advanced Science and Technology, University of
Tokyo. We thank Ms Erika Komuro for skilled technical
assistance, Professor Malcolm Shick and Dr Bruce Chalker
for critically reviewing our manuscript, Mr Sieve Clarke for
preparing the scientific illustrations, and the master and
crew of the R.V. Harry Messel for logistics support. This is
contribution No. 731 from the Australian Institute of
Marine Science (Marine Photobiology Project).

References
Arai T., Nishijima M., Adachi K. and Sano H. (1992)
Isolation and structure of a UV absorbing substance from

the marine bacterium Micrococcus sp. AK-334. MBI

Report 1992, pp. 88-94. Marine Biotechnology Institute,
2-35-10 Hongo, Bunkyo-ku, Tokyo 113, Japan.
Barclay L. R. C. and Ingold K. U. (1981) Autoxidation of
biological molecules. 2. Autoxidation of a model membrane. Comparison of the autoxidation of egg lecithin
phosphatidylcholine in water and chlorobenzene. J. Am.
Chem. Soc. 103, 6478-6485.
Barclay L. R. C., Locke S. J., MacNeil L. M., Vankessel J.,
Burton G. W. and Ingold K. U. (1984) Autoxidation of
micelles and model membranes. Quantitative kinetic
measurements can be made by using either water-soluble
or lipid-soluble initiators with water-soluble or lipidsoluble chain-breaking antioxidants. J. Am. Chem. Soc.
106, 2479-2481.
Buettner G. (1993) The pecking order of free radicals
and antioxidants: lipid peroxidation. ~-tocopherol, and
ascorbate. Archs Biochem. Biophys. 300, 535-543.
Cadenas E. (1989) Biochemistry of oxygen toxicity. Ann.
Rev. Biochem. 58, 79-110.
Carreto J. I., Carignan M. O., Daleo D. and Demarco S. G.
(1990) Occurrence of mycosporine-like amino acids in
the red-tide dinoflagellate Alexandrium excavatum: UVphotoprotective compounds. J. Plankton Res. 12,
909 922.
Carroll A. K. and Shick J. M. (1993) Dietary accumulation
of UV-absorbing compounds in the green sea urchin. Am.
Zool. 33, 63A.
Chioccara F., Della Gala A., De Rosa M., Novellino E. and
Prota G. (1980) Mycosporine amino acids and related
compounds from the eggs of fishes. Bull. Soc. Chim. Belg.
89, 1101-1106.
Chioccara F., Misuraca G., Novellino E. and Porta G.
(1986a) Occurrence of two new mycosporine-like amino
acids mytilins A and B in the edible mussle. Mytilus
galloprovincialis. Tetrahedron Lett. 34, 3181-3182.
Chioccara F., Zeuli L. and Novellino L. E. (1986b) Occurrence of mycosporine related compounds in sea urchin
eggs. Comp. Biochem. Physiol. 85B, 459 461.
Cunningham M. L., Johnson J. S., Giovanazzi S. M. and
Peak M. J. (1985) Photosensitized production of superoxide anion by monochromatic (290-405 nm) ultraviolet
irradiation of NADH and NADPH coenzymes. Photochem. Photobiol. 42, 125-128.
DiGiulio R. T., Washburn P. C., Wenning R. J., Winston
G. W. and Jewell C. S. (1989) Biochemical responses in
aquatic animals: a review of determinants of oxidative
stress. Envir. Toxicol. Chem. 8, 1103-1123.
Dunlap W. C. and Chalker B. E. (1986) Identification and
quantitation of near-UV absorbing compounds (S-320) in
a hermatypic scleractinian. Coral Reefs 5, 155 159.
Dunlap W. C., Chalker B. E. and Oliver J. K. (1986)
Bathymetric adaptations of reef-building corals at Davis
Reef, Great Barrier Reef, Australia. III. UV-B absorbing
compounds. J. exp. Mar. Biol. Ecol. 104, 239 248.
Dunlap W. C., Williams D. McB., Chalker B. E. and
Banaszak A. T. (1989) Biochemical photoadaptations in
vision: UV-absorbing pigments in fish eye tissues. Comp.
Biochem. Physiol. 93B, 601-607.
Dykens J. A. and Shick J. M. (1982) Oxygen production
by endosymbiotic algae control superoxide dismutase
activity in their animal host. Nature 297, 579-580.
Dykens J. A., Shick J. M., Benoit C., Buettner G. R. and
Winston G. W. (1992) Oxygen radical production in
the sea anemone Anthopleura elegantissima and its
endosymbiotic algae. J. exp. Biol. 168, 219-241.
Fleischmann E. M. (1989) The measurement and penetration of ultraviolet radiation into tropical water. Limnol. Oceanogr. 34, 1623-1629.
Fujimoto K., Ohmura H. and Kaneda T. (1985) Screening
for antioxigenic compounds in marine algae and bromophenols as effective principles in a red alga Polysiphonia ulceolate. Bull. Japan. Soc. Sci. Fish. 51, 1139-1143.

Antioxidants in marine organisms

Garcia-Pichel F. and Castenholz R. W. (1993) Occurrence
of UV-absorbing, mycosporine-like compounds among
cyanobacterial isolates and an estimate of their screening
capacity. Appl. Environ. Microbiol. 59, 163-169.
Gleason D. F. (1993) Differential effects of ultraviolet
radiation on green and brown morphs of the Caribbean
coral Porites astreoides. Limnol. Oceanogr. 38, 1452-1463.
Grant P. T., Middleton C., Plack P. A., Thomson R. H.
(1985) The isolation of four aminocyclohexenimines
(mycosporines) and a structurally related derivative of
cyclohexane-1:3-dione (gadusol) from the brine shrimp.
Artemia. Comp. Biochem. Physiol. 80B, 755-758.
Hader D.-P. and Worrest R. C. (1991) Effects of enhanced
solar radiation on aquatic ecosystems. Photobiol. Photochem. 53, 717-725.
Halliwell B. and Gutteridge J. M. C. (1984) Role of iron in
oxygen radical reactions. In Methods o f Enzymology,
Vol. 105, Oxygen radicals in Biological Systems (Edited by
Packer L.), pp. 47-56. Academic Press, Sydney.
Harm W. (1980) Biological Effects o f Ultraviolet Radiation.
Cambridge University Press, Cambridge.
Hirata Y., Uemura D., Ueda K. and Takano S. (1979)
Several compounds from Palythoa tuberculosa (Coelenterata). Pure appl. Chem. 51, 1875-1883.
Imlay J. A. (1988) Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro.
Science 240, 640-642.
Ito S. and Hirata Y. (1977) Isolation and structure of
a mycosporine from the zoanthid Palytoa tuberculosa.
Tetrahedron Lett. 28, 2429-2430.
Jagger J. (1983) Physiological effects of near-ultraviolet
radiation on bacteria. Photochem. Photobiol. Rev. 7, 1-75.
Jokiel P. L. (1980) Solar radiation and coral reef epifauna.
Science 207, 1069-1071.
Jokiel P. L. and York Jr R. H. (1982) Solar ultraviolet
photobiology of the coral reef coral Pocillopora damicornis and symbiotic zooxanthellae. Bull. Mar. Sci. 32,
301-315.
Karentz D., McEuen F. S., Land M. C. and Dunlap W. C.
(1991) Survey of mycosporine-like amino acid compounds in Antarctic marine organisms: potential protection from ultraviolet exposure. Mar. Biol. 108, 157-166.
Kinzie III R. A. (1993) Effects of ambient levels of solar
ultraviolet radiation on zooxanthellae and photosynthesis
of the reef coral Montipora verrucosa. Mar. Biol. 116,
319-327.
Kohen R., Yamamoto Y., Cundy K. C. and Ames B. N.
(1988) Antioxidant activity of carnosine, homocarnosine,
and anserine present in muscle and brain. Proc. natn.
Acad. Sci. U.S.A. 85, 3175-3179.
Kyle D. J. (1987) The biochemical basis for photoinhibition
of photosystem II. In Photoinhibition (Edited by Kyle
D. J., Osmund C. B. and Arntzen C. J.), pp. 197-226.
Elsevier, Amsterdam.
Lesser M. P. and Stochaj W. R. (1990) Photoadaptation and
protection against active forms of oxygen in the symbiotic
procaryote Prochloron sp. and its ascidian host. AppL
Environ. Microbiol. 56, 1530-1535.
Lesser M. P., Stochaj W. R., Tapley D. W. and Shick J. M.
(1990) Bleaching in coral reef anthozoans: effects of
irradiance, ultraviolet radiation, and temperature on the
activities of protective enzymes against active oxygen.
Coral Reefs 8, 225~32.
Le Tutour B. (1990) Antioxidative activities of algal extracts, synergistic effect with vitamin E. Phytochemistry
29, 3759-1765.
Lewin R. A. (1994) The cell sap of Prochloron (Prochlorophyta). Phycologia 33, 71-76.
Maragos J. E. (1972) A study of Hawaiian reef corals.
Doctoral dissertation, University of Hawaii, Honolulu,
Hawaii.
Masuda K., Goto M., Maruyama T. and Miyachi S.
(I 993) Adaptation of solitary corals and their zooxanthel-

113

lae to low light and UV radiation. Mar. Biol. 117,

685-691.
Matta J. L. and Trench R. K. (1991) The enzymatic
response of the symbiotic dinoflagellate Symbiodinium
microadriaticum (Freudenthal) to growth in vitro under
varied oxygen tensions. Symbiosis 11, 31 45.
Nakamura H., Kobayashi J. and Hirata Y. (1981) Isolation
and structure of a 330nm u.v.-absorbing substance
Asterina-330 from the starfish Asterina pectinifera. Chem.
Lett. 1413-1414.
Nakamura H., Kobayashi J. and Hirata Y. (1982)
Separation of mycosporine-like amino acids in marine
organisms using reverse-phase high-performance liquid
chromatography. J. Chromat. 250, 113-118.
Niki E. (1987) Antioxidants in relation to lipid peroxidation.
Chem. Phys. Lipids 44, 227-253.
Porter N. (1986) Mechanisms for the autoxidation of
polyunsaturated lipids. Acc. Chem. Res. 19, 262~68.
Renger G., Voss M., Grabe P. and Shulze S. (1986) Effect
of UV irradience on different partial reactions of the
primary process of photosynthesis. In Stratospheric
Ozone Reduction, Solar Ultraviolet Radiation and Plant
Life (Edited by Worrest R. C. and Caldwell M. M.),
pp. 171-184. Springer, Berlin.
Scelfo G. M. (1986) Relationship between solar radiation
and pigments of the coral Montipora verrucosa and its
zooxanthellae. In Coral Reef Population Biology (Edited
by Jokiel P. L., Richmond R. H. and Rogers R. A.),
pp. 440-451. Hawaiian Institute of Marine Biology Technical Report No. 37, Honolulu.
Shick J. M. (1993) Solar UV and oxidative stress in algalanimal symbiosis. In Frontiers of Photobiology (Edited by
Shima A., Ichihashi M., Fujiwara Y. and Takebe H.),
pp. 561-564. Elsevier Science, Amsterdam.
Shick J. M., Dunlap W. C., Chalker B. E., Banaszak A. T.
and Rosenzweig T. K. (1992) Survey of ultraviolet radiation absorbing mycosporine-like amino acids in organs
of coral reef holothuroids. Mar. Ecol. Prog. Ser. 90,
139-148.
Shick J. M., Lesser M. P., Dunlap W. C., Stochaj W. R.,
Chalker B. E. and Wu Won J. (1995) Depth-dependent
responses to solar ultraviolet radiation and oxidative
stress in the zooxanthellate coral Acropora microphthalma. Mar. Biol. 122, 41-51.
Shick J. M., Lesser M. P. and Stochaj W. R. (1991)
Ultraviolet radiation and photooxidative stress in
zooxanthellate Anthozoa: the sea anemone Phyllodiscus
semoni and the octocoral Clavularia sp. Symbiosis 10,
145-173.
Siebeck O. (1981) Photoreactivation and depth-dependent
UV tolerance in reef corals in the Great Barrier
Reef/Australia. Naturwissenschaften 68, 426-428.
Siebeck O. (1988) Experimental investigations of UV tolerance in hermatypic corals (Scleractinia). Mar. Ecol. Prog.
Ser. 43, 95 103.
Singleton W. S., Gray M. S., Brown M. L. and White J. L.
(1965) Chromatographically homogeneous lecithin from
egg phospholipids. J. Am. Oil Chem. Soc. 42, 53-56.
Stochaj W. R., Dunlap W. C. and Shick J. M. (1994) Two
new UV-absorbing mycosporine-like amino acids from
the sea anemone Anthopleura elegantissima and the effects
of spectral irradiance on chemical composition and content. Mar. Biol. 118, 149-156.
Takano S., Nakanishi A., Uemura D. and Hirata Y. (1979)
Isolation and structure of a 334-nm UV-absorbing substance, Porphyra-334 from the red alga Porphyra tenera
Kjellman. Chem. Lett. 419-420.
Takano S., Uemura D. and Hirata Y. (1978a) Isolation
and structure of a new amino acid, palythine, from
the zoanthid Palythoa tuberculosa. Tetrahedron Lett.
2299-2300.
Takano S., Uemura D. and Hirata Y. (1978b) Isolation
and structure of two new amino acids, palythinol and

114

W. C. Dunlap and Y. Yamamoto

palythene, from the zoanthid, Palythoa tuberculosa.

Tetrahedron Lett. 4909-4912.
Tsujino I., Yabe K. and Sekikawa I. (1980) Isolation and
structure of a new amino acid, shinorine, from the red
alga Chondrus yendoi Yamada et Mikami. Bot. Mar. 23,
65 68.
Tyrrell R. M. (1991) UVA (320-380nm) radiation as an
oxidative stress. In Oxidative Stress: Oxidants and Antioxidants (Edited by Sies H.), pp. 57-83. Academic Press,
Sydney.
Worrest R. C. (1982) Review of the literature concerning the
impact of UV-B radiation upon marine organisms, In The
Role of Solar Ultraviolet Radiation in Marine Ecosystems

(Edited by Calkins J.), pp. 429 457. Plenum Press, New

York.
Yamamoto Y., Brodsky M. H., Baker J. C. and Ames B. N.
(1987) Detection and characterization of lipid hydroperoxides at picomole levels by high performance liquid
chromatography. Analyt. Biochem. 160, 7-13.
Yamamoto Y., Haga S. and Kamiya Y. (1984) Oxidation
of lipids V. Oxidation of methyl linoleate in aqueous
dispersion. Bull. Chem. Soc. Jap. 57, 1260-1264.
Yamamoto Y., Niki E. and Kamiya Y. (1982) Oxidative of
lipids. I. Quantitative determination of the oxidation of
methyl lineolate and methyl linolenate. Bull. Chem. Soc.
Japan 55, 1548-1550.