ABSTRA
1. Green canthaxanthin-carotenoproteins were iso¬
lated and partially purified from three species
7
of marine idoteid isopods - lootea monter
sis
(Maloney), Idotea resecata (Stimpson), and Idotea
kirchanskii (Miller and Lee).
The proteins have similar absorption maxima at
2.
280 nm. and 683 nm., similar electrophoretic
properties, and a molecular weight of greater
than 480,000.
3. Although shown not to be lipoproteins, the pro-
teins show both lipophilic and hydrophilic
properties.
4. A comparison of canthaxanthin-carotenoproteins
and astaxanthin-carotenoproteins is made.
(2)
INTRODUCTION
Carotenoproteins, complexes in which a carotenoid
and a protein are in stoichiometric combination,
were first discovered in the invertebrates in 1883
(Merejkowsky, 1883) and since have been shown to have
a very wide distribution (Cheesman et al., 1967).
o
Most study of the invertebrate carotenoproteli
ns has
centered on three proteins - crustacyanin, the blue
protein of the lobster carapace (Wald, Nathanson, Jencks,
& Tarr, 1948; Jencks & Buten, 1964; Cheesman, Zagalsky
& Ceccaldi, 1966), ovoverdin, the green pigment of
lobster eggs (Kuhn & Sorensen, 1938; Stern & Salomon,
1937, 1938), and ovorubin, the red protein of the
eggs of the gastropod Pomacea
inalicul
ata (Cheesman,
1958; Norden, 1962). Of these, crustacyanin has been
analyzed the most thoroughly, probably due to its
availability and ease of pur
ification. All of these
carotenoproteins have astaxanthin as their prosthetic
group and for quite some time it was believed that
astaxanthin was the only carotenoid found as a caro¬
tenoid prosthetic group in invertebrate caroteno¬
proteins.
In 1966, astaxanthin esters were found to be
the prosthetic group in the egg carotenoproteins in
(3)
Eunagurus (Cheesman & Prebble, 1966) and in a sub-
sequent publication in the same year canthaxanthin
was found in the green carotenoprotein of the marine
isopod Idotea montereyonsis (Maloney) (Lee, 1966a).
Canthaxanthin was also shown to be involved in the
ea resecata (Stimpson) (lee a
carotenoprotein of Ido
Gilchrist, 1972) and in a variety of Anostraca (Gil¬
christ, 1968). Other than very preliminary work
little is known about these canthaxanthin-caroteno¬
proteins.
Recently, studies conducted on the egg astaxanthin-
carotenoproteins of a variety of aquatic invertebrates
indicated these proteins to exhibit remarkable simi-
larities (Zagalsky, 1971). This relatedness combined
with wide distribution may imply that these proteins
have similar functions, perhaps developmental in
nature. The function of the canthaxanthin-caroteno¬
proteins is unknown, but at least in the isopods,
they may be involved in color change (Lee, 1966a,
1972).
There were therefore two basic interests in
initiating this study of canthaxanthin-carotenoproteins:
1) To compare them with the astaxanthin-caroteno-
proteins; and 2) To determine if there exists inter-
(4)
specific similarities between the canthaxanthin-
rotenoproteins of differont isopod species.
For this purpose, Idotea montereyensis (Maloney),
ta (Stimpson), and Idotea kirchanskii
Idotea rese
(Miller & Lee) - three closely related species of
ma
ine isopods - were used. The present studies
were undertaken to compare various properties of the
reen carotenoproteins isolated from three species
of marine idoteid isopods, including absorption
spectra, electrophoretic patterns, composition,
.
minimum molecular weight, and stability to light and
heat.
AN
THOD
ATERIALS
Samples of the green variety of I. montereyensi
skii were collected from beds of the
and I. kird
eelgrass, Phy
scouleri, both at Tomales
adi
Bay, Marin County, California and at Seventeen Mile
Drive, Monterey County, California. Samples of the
brown coloured ve
riety of I. resecata were collected
D7
ra off the Hopkins
from the kelp!
oysis
Marine Station, Pacific Grove, California.
c
(5)
ate
The following materials were utilized: Sephadex
G-200 (Tharmacia, Inc.); DEAE-Cellex 50 (Bio-Rad
Laboratories); and Cyanogum-41 (E-C Apparatus Corp.).
All other chemicals were of reagent grade.
Phosphate buffers were prepared by mixing solutions

I KHyPO, and NagHPO, to the appropriate pH.

teins

eno
xtraction andp
car
iono
From 100-200 animals, all of which had been
maintained in the laboratory in cold aerated sea
water, were starved at least seven hours to remove
+
gut contents. They were subsequently frozen unoll
extracted. Green protein complexes were extracted
in 0.01 M phosphate buffer (KH,PO, + NagHPO,), pH 7.0
ing the method of Lee (1966a); the crude extract
was centrifuged at 12,000 RPM at 0°0. for one hour
to remove nonaqueous residues (Gilchrist, 1968).
Solid ammonium sulfate was added to the solution to
igit to 30 percent saturation and the resulting
bri
precipitate spun down and discarded. The green
itated
protein, which remained in solution, was prec
ith 70 percent ammonium sulfate, centrifuged and
redissolved in 0.001 M phosphate buffer, pH 7.0.
0
(6)
The crude protein extract was placed in concen¬
trated solution on columns of Sephadex G-200 and
eluted with 0.1 M phosphate buffer, pH 7.0. Columns
were approximately 2.5 x 60 cm. and were thoroughly
equilibrated in the cold with 0.1 M phosphate buffer,
pH 7.0 before the protein solutions were added.
The partially purified green protein was resatur¬
ated with 70 percent ammonium sulfate, centrifuged,
and redissolved in a small amount of 0.001 M phosphate
buffer, pH 7.0. The green solution was placed on
DEAE-Cellex 50 Anion Exchange columns, prepared by
the method of Lee (1966a), and was eluted with a
igth of the perfusing
stepwise increase in ionic sti
phosphate buffer.
urification of the Sephadex G-200 treated
protein complexes was also attempted by means of
treatment with diethyl ether and dialysis against
istilled water. Diethyl ether treatment involved
lightly shaking the protein solution with two volumes
of diethyl ether, centrifuging the mixture for five
minutes at 0°0., and removing the upper ether layer
with suction; the lower aqueous layer was saturated
with 70 percent ammonium sulfate, centrifuged, and the
resulting precipitate redissolved in 0.001 M phosphate
(7)
buffer, pH 7.0 and recentrifuged to remove denatured
tein and lipid.
Dialysis of the Sephadex G-200 purified protein
was performed in distilled water with stirring and
frequent changes of water for two days at 5°0. under
conditions of minimal illumination. The resulting
precipitated protein was centrifuged, redissolved in
0.2 M phosphate buffer, pH 7.0, and recentrifuged.
Absorption
The absorption spectra of the carotenoproteins
of the three species of isopods were obtained on a
Beckman DBG Recording Spectrophotometer at successive
fication. The ratio of absorbance
sages of pui
at the major peaks in the ultraviolet range (280 nm.)
and the visible portion of the spectra (683 nm.) were
recorded and calculated as E00/683.

ectrophoresis
D
Electrophoresis was performed using an E-O
Vertical Gel electrophoresis unit. Five percent
Cyanogum-41 gels were utilized with Tris-NagEDTA-
ric Acid buf.
er at pH 8.4. All material was run
between 200 and 300 v., in order to maintain a current
(8)
of 60-120 amps., for three to five hours. Proteins
were stained for with Amido Black 10B and a lipo-
crimson prestain (Gurr Inc.) was used to determine
the presence of lipoproteins. All other procedures
were carried out as noted in E-C Technical Bulletin
No. 140 (E-C Apparatus Corp.).
Apoprotein
Protein complexes which had been purified by
passage through columns of Sephadex G-200 and re¬
precipitated with 70 percent ammonium sulfate were
treated with 6 M urea for one, fifteen, and 30 minute
intervals. Spectra were taken of the resulting
solutions and electrophoresis of protein samples
treated with 6 M urea for 30 minutes was performed,
sing 6 M urea-Cyanogum-41 gels and Tr
s-NaEDTA¬
Boric Acid-6 Murea buffer, pH 8.4.
In a subsequent experiment, Sephadex G-200
ified carotenoproteins were treated with equal
volumes of acetone; the white precipitate was centri¬
fuged, redissolved in 0.1 M phosphate buffer, pH 7.0,
and run on electrophoresis using a normal Cyanogum-+
gel and Tris-NajEDTA-Boric Acid buffer, pH 8.4.
(9)
i

Approximate moleculary
ltration studie
it -
Estimates of molecular sizes of the purified
carotenoproteins (purified by Sephadex G-200 fil¬
tration, DEAE-Cellex 50 Anion Exchange, and dialysis
against water) were obtained from a gel filtration
study.
A column of Sephadex G-200 (2.5 x 60 cm.) was
equilibrated with 0.1 M phosphate buffer, pH 7.0,
(flow rate was 18-20 ml. per hour) and was calibrated
with a series of standard proteins, as explained
by Cheesmane
t al. (1966) and Andrews (1964). Blue
dextran was used to determine the elution volume
of the column. A molecular size estimate was ob¬
plotting log molecular weight against elution
tained by
volume (Andrews, 1964).
o
RESUDIS
Absorption spectra
The absorption spectra of the carotenoproteins
of all three species of isopods under study were
very similar. The spectra of the carotenoproteins,
at various stages of purification, are shown in
Fig. 1, 2, and 3. All display absorption maxima at
(10)
280 nm. and 683 nm. In crude protein preparations,
absorption below 500 nm. was usually so great that
the 280 nm. peak was occluded and corresponding
E200/683 ratios were either high or unobtainable.
With Sephadex G-200 purified proteins, character-
istic free carotenoid peaks were visible between
400-500 nm. and the absorption peaks at 280 nm. and
683 nm. became more clearly defined. At higher
levels of purity, for all the proteins, the E00/683
sios decreased to an even greater degree and the
400-500 nm. absorption was largely removed. It
should be noted that typically as canthaxanthin-
carotenoproteins reach higher stages of purity with
— +--
loss of contaminating proteins, the characteristic
00/683 ratio - the ratio of optical densities at
280 nm. to 683 nm. - is decreased and stabilized
and thus serves as one indicator of carotenoprotein
tio
purity. The observed reduction in the E
therefore may be attributable to removal of other
proteins, including possibly some lipoproteins, from
the crude carotenoprotein sample and the reduction in
free carotenoid absorbance to a removal of lipid and
lipid-dissolved carotenoids. These possibilities
are suggested by the offects of treatment of the
(11)
carotenoproteins with diethyl ether and dialysis
against water. Both of these processes firs
decreased the E/683 ratio and second removed
lipid and dissolved carotenoid, as evidenced by
detection of lipid on the dialysis bags and by the
change in color of the protein solution from green
to blue.
fic
tion
The purity of the carotenoproteins was deter-
1280
mined by absorption spectra (E /683) and electro¬
phoresis, each of which was performed at successive
stages of purification. The five major stages of
purity obtained for each protein are shown in Table 7
with corresponding E
0/683 ratios. As the proteins
were passed through the various purification pro¬
cesses, the E/683 ratios decreased but never
completely stabilized. Noteworthy is the fact that
tereye
only the carotenoprotein of I.
is could
be passed through DEAE-Cellex 50 Anion Exchange
columns; that of I. kirchanskii remained immobile
on the column, while that of I. resecata denatured
on the column.
0
(12)
Eloctroph
resi
The carotenoprotein complexes isolated from
eee
a
sis, I. kirchan

rese
i, and I

I. mont
were remarkably similar electrophoretically as shown
in Fig. 4. At all stages of purification and in
all species three distinct protein bands were present.
These are represented as bands 1, 3, and 4 on Fig. 4.
Band 2 and other intormediary bands probably repre¬
es, as evidenced by the fact that they
sent impur
were colorless and were at least removed upon puri¬
ntereyensis.
fication of the carotenoprotein in I. m
The band which remained at the origin (band 1, Fig. 4),
though originally believed to represent the caroteno¬
ein band, appears, as a result of this investi¬
prot
ation, to be denatured protein or protein held
back by lipid contamination. Evidence indicates
in fact the green carotenoprotein of all three
that
species is represented solely by the two lower bands
(bands 3 and 4, Fig. 4); green color was observed
to move to these positions during the course of
several electrophoretic runs. Upon treatment of the
protein samples with 6 M urea, though a loss of
absorbance at 683 nm. was noted, indicating a change
0
(73)
in carotenoid-protein linkage, there was no change
in the number of bands present, suggesting that
the isopod carotenoproteins are not polymers made
up of different molecular weight subunits as
commonly seen in crustacyanin (Cheesman,
al.,
1966). Similar results were obtained with acetone
denatured protein as well.
Lipocrimson prestain always remained at the
origin and was never observed to migrate down the
ing that the isopod caroteno-
gel, againsug
proteins are not lipoproteins, although they do
ppear to have strong lipophilic properties.
+
eight estim
G-200 liltration
ation -S
eph
Molecu
Preliminary evidence, based on an experiment
in which the carotenoproteins of all three species
were mixed together and observed to move homogene¬
sly through a column of Sephadex G-200 (1.5x
30 cm.), indicated that all have approximately the
same molecular weight.
Sephadex G-200 gel filtration of the purified
nsis carotenoprotein against protein
1.T
tere
standards show it to have a molecular weight
greater than 480,000, perhaps as high as 1,200,000 (Fig:5).
(74)
This work, however, can only be considered as
reliminary and more careful determination must be
made before a definitive statement of molecular
size can be made.
DT
10
D
As a result of this brief investigation the
green carotenoproteins of the three marine idoteid
isopods - Idotea mo
itereyensis (Maloney), Idotea

resecata (Stimpson), and luotea kirchansk
i (Miller
Lee) - were all isolated (that of I. kirchansk
time) in at least a partially purified
for the firs
form and were shown to display remarkable inter-
specific similarities in absorption spectra, elec¬
trophoretic patterns, molecular weight, and stability
to sunlight and heat; in addition, all were shown
to exhibit similar and unique behavior in both lipid
and aqueous environments.
Purification
Purification of the carotenoproteins represented
a major part of the work of this investigation. As
/683 ratios obtained at
evidened by thel
0
(15)
successive stages of purification (Table 1), the
proteins were never completely purified. The
major difficulty seemed to be lipid contamination,
occurring in varying degrees with the respective
species. Lipid and lipid-dissolved carotenoid con¬
tamination was evidonced by the behavior of the
proteins after treatment with diethyl ether and after
dialysis against water. As a result of both of
these procedures, the E/683 ratios of all proteins
decreased (Table 1), indicating a loss of contami¬
ating protein, perhaps lipoprotein in nature, and
the protein solutions tended to change in color
from green to blue or blue-green, the latter implying
that lipid and dissolved carotenoid were being re¬
moved from association with the carotenoprotein.
—
Lipid was, in fact, detected on the dialysis bags
after such treatment.
The levels of lipid contamination varied with
the species. Based on absorption spectra and mobil-
ity on DEAE-Cellex 50 columns, it appeared that the
e
sis contained the
carotenoprotein of 1. monter
least lipid contamination, while those of 1. kircha
ski
resec
a seemed to be more highly contaminated,
and
(76)
as they were both immobile on DEAE-Cellex 50. In
addition, following diethyl ether extraction and
subsequent resaturation and centrifugation with
ammonium sulfate, the carotenoprotein complex of
is formed a solid blue-green precipi¬
I. monte
tate at the bottom of the centrifuge tube, while
har
secata often
those of I.I
i and of I.
formed a green paddy at the air-supernatant inter-
phase as well as a precipitate, indicating a higher
content of lipid.
Although, as a result of the lipid contamination,
completely purified proteins were never obtained,
the finding that lipid contamination of the caroteno-
proteins is present to a considerable degree in all
species implies that these molecules have both
lipophilic and hydrophilic ends. Perhaps this duality
of properties has significance in color change and
other possible functions. It is noteworthy that the

the only species
carotenoprotein of I. monter

capable of undergoing color change (Lee, 1966a),
has the least lipid associated with it. That this
molecule can associate with both lipid and aqueous
environments may have bearing on such a process.
-
Furthermore, such a molecule might even facilitate
(17)
movement of materials between lipid and water
within a biological system. In fact, green caro¬
tenoproteins have been observed to occur in the
prevensis (Lee, personal communica¬
eggs of I.

tion, 1973) as well as in the carapace, suggesting
possible roles in transport or storage of nutrients.
sorptio
bectra
The characteristic 280 nm. and 683 nm. absorption
anthaxanthin-carotenoprotein are
paks of the
likewise present in all species, but with varying
amounts of free carotenoid contamination, depending
on the species and degree of purification (Fig. 1,
2, and 3). This carotenoid-lipid contamination may
in fact be an artifact of the isolation procedure;
within the animal, as noted above, the environment
of the carotenoprotein could be either lipid or
aqueous in nature and therefore the degree of asso¬
ciation with lipid dissolved carotenoid in the animal
night be entirely different than that obtained in
ein extraction.
pro
Tlectroh
sis
Electrophoretic studies resulted in several
(78)
interesting findings. First, the electrophoretic
patterns of the three green carotenoproteins inves¬
tigated are identical (Fig. 4), with the two lower
dark-staining bands (bands 3 and 4 in Fig. 4) repre¬
senting the carotenoprotein complex. The observa¬
tion that green color migrated to the positions
occupied by these two bands implies that this mole-
cule is composed of two subunits. Treatment of the
carotenoprotein with 6 M urea or acetone did not
change the electrophoretic pattern, although some
change in carotenoid-protein linkage was evidenced
in absorption spectra as there was a complete loss
of absorbance at 683 nm.
Furthermore, when samples of protein were
prestained with lipocrimson, the red stain never
grated away from the origin, as did the green color.
This finding suggests that the canthaxanthin-caro-

rchanskii,
e
ansis, I.k
tenoproteins of I. monter
a are not lipoproteins and that the
and I.1
resecat
lipocrimson stain remains attached to lipid-contam¬
inated protein at the ori
in. Perhaps the lipid
found associated with the isopod carotenoproteins
serves in some way to hold the subunits of the
molecule together, which are then quite easily disso¬
ciated upon electrophoresis.
(19)
These findings are in contrast to the situation
found in crustacyanin, an astaxanthin-protein of
large molecular weight which appears as a homoge-
neous band upon electrophoresis under normal condi¬
tions (Cheesman et al., 1966); however, when treated
with a strong denaturing agent such as 6 Murea a
number of bands appear in the electrophoretic pattern,
indicating dissociation into subunits (Cheesman, e
1966). The fact that no such increase in electro¬
phoretic protein bands occurs upon treatment of
isopod carotenoproteins with 6 M urea implies that
erent
the association of subunits is of a much dif
nature than that seen in crustacyanin.
i


to othe
rotenoproteing
1 -
Rel

The present studies therefore indicate quite
close interspecific similarity between the green
canthaxanthin-carotenoproteins of three species
of marine idoteid isopods; these proteins are pre¬
dominantly cuticular in nature, although they occur
in other body regions as well as in the eggs.
In addition to perhaps indicating a similarity in
function of these isopod carotenoproteins, this
relationship is in contrast to that found in the
vertebrate astaxanthin-carotenoproteins in which
0
(20)
close interspecific similarity has been found only
in the egg carotenoproteins (Zagalsky, 1974). No
such relatedness has been observed in the carapace
astaxanthin-carotenoproteins so far studied.
A comparison of the invertebrate canthaxanthin¬
carotenoproteins and the astaxanthin-carotenoproteins
shows there to be some similarity (both classes of
proteins are carotenoid-proteins of high molecular
weight which bleach after varying amounts of time
in sunlight), but the two classes of proteins are
by no means identical (Table 2). Differences are
seen to exist in: 1) Absorption maxima; the typical
isopod carotenoprotein absorbances at 280 nm. and
683 nm. (in the pure form) are in contrast to the
wide variety of absorption maxima seen in the
hin-proteins (for example at 278 nm., 476 nm.,
aal
and 660 nm. for ovoverdin); in addition, the astaxan-
thin-proteins appear to have no lipid-carotenoid
ssociations as do the isopod carotenoproteins in the
impure form as evidenced by a lack of wide absorbance
in the 400-500 nm. range; 2) Color; the canthaxanthin¬
proteins tend to be green to blue-green in color,
depending upon the amount of lipid-carotenoid
association, while the astaxanthin-carotenoproteins
(21)
appear in a wide variety of colors - for example,
crustacyanin is blue, ovoverdin is greon, and
ovorubin red; and 3) Prosthetic group; in addition
to differences in carotenoid prosthetic group,
some astaxanthin-proteins such as ovoverdin are
lipoproteins while present studies indicate the
isopod carotenoproteins to have no lipid as a
prosthetic group; both ovoverdin and ovorubin are
ycoproteins while to date it is not known if the
isopod canthaxanthin-proteins contain a carbohydrate
moiety.
Very preliminary evidence indicates the isopod
carotenoproteins to be of a much greater molecular
size than that of either crustacyanin (MW 380,000)
al., 1966), ovoverdin (MW 300,000)
(Cheesman
(Ceccaldi et al., 1966), or ovorubin (MW 330,000)
(Cheesman, 1958). The evidence suggests the isopod
canthaxanthin-proteins to have a molecular weight in
xoess of 480,000; however, this remains to be con¬
irmed on totally purified material.
This brief investigation of the green caroteno¬

proteins ol luotea monterey
s (Maloney), Idotea

rii (Miller &
ote
ata (Stimpson), and I
Lee) gave results which can only be regarded as
0
(22)
preliminary and much work is yet needed to gain
further insight into the structure and function
of these canthaxanthin-carotenoproteins. However,
these studies do indicate that the invertebrate
canthaxanthin- and astaxanthin-carotenoproteins are
quite different in a number of respects and that
there exists striking interspecific similarities
between the carotenoproteins of three closely
related species of marine idoteid isopods.
0
(23)
T

CKNOL
The author wishes to thank Dr. Welton L. Lee
for initial introduction to the study of caroteno-
sistance
proteins and for providing stimulation and as
throughout the course of this investigation.
0
(24)
ERENGES
ANDREWS P. (1964) Estimation of molecular weights
of proteins by gel filtration. Biochem. J. 91,
222-233.
CECCALDI H. J., CHEESMAN D. F., and ZAGALSKY P. F.
(1966) Quelques proprietes et caracteristiques
de l'ovoverdine. C.r.
Seanc. Soc.
Biol. 160,
587-590.

CHEESMAN D. F. (1958) Ovorubin, a chromoprotein
from the eggs of the gastropod mollusc Pomacea
canaliculata. Proc. R. Soc. B. 1
9, 571-587.
CHEESMAN D. F., LEE W. L., and ZAGALSKY P. F. (1967)
Carotenoproteins in invertebrates. Biol.
Rev. 42.
132-160.

CHEESMAN D. F. and PREBBLE J. (1966) Astaxanthin
ester as a prosthetic group: a carotenoprotein
from the hermit crab. Comp. Biochem. P
siol. 17,
929-936.
CHEESMAN D. F., ZAGALSKY P. F., and CECCALDI H. J.
(1966) Purification and properties of crusta¬
16.
cyanin.
Proc. R. Soc. B.
4, 130-151.
IIGHPT
GILOHRIST B. M. (1968) Distribution and relative
abundance of carotenoid pigments in Anostraca
0
(25)
(Crustacea: Branchiopoda). Comp. Biochem.
4, 123-147.
Physiol.
JENCKS W. P. and BUTEN B. (1964) The denaturation
of crustacyanin. Arcl
hs. Biochem.
phys. 107,
511-520.


KUHN R. and SORENSEN N. A. (1938a), Über astaxanthin
und ovoverdin.
chem. Ges. 71,
dtsch
1879-1888.
LEE W. L. (1966a)
Pigmentation of the marine isopod
eeyel

nsis (Maloney). Comp. Biochem.
Idotea mont:
Phye

ol. 18, 17-36.
LEE W. L. (1972) Chromatophores and their role in color
tereye

ensi
change in the marine isopod Idotea mo
col. 8, 930-947.
exp.!
(Maloney). J
mar. Biol.
LEE W. L. (1973) Personal communication. Hopkins
Marine Station, Stanford University.
A
LEE W. L. and GILUARIST B. M. (1972) Pigmentation,
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isopod ldotea
sata (Stimpson). J. exp. mar.
9, 1-27.
Biol. Ecol. 1
-
MEREJKOWSKY C. DE (1883) Nouvelles recherches sur la
zoonerythrine et autres pigments des animaux.
Bull. Soc. zool. 8, 81-97.
(26)
NORDEN D. A. (1962) Some properties of ovorubin,
a carotenoprotein from the eggs of
macea
it
iculata. Ph.D. Thesi
s, Univers
ity of London.
can
STERN K. G. and SALOMON K. (1937) Ovoverdin, a pig-
ment chemically related to visual purple.
86, 310-311.
e, N
Scier
STERN K. G. and SALOMON K. (1938) On ovoverdin, the
carotenoid-protein of the egg in the lobster.
biol. Chem. 122, 461-72.
WALD G., NATHANSON N., JENCKS W. P., and TARR E.
(1948) Crustacyanin, the blue carotenoid-protein
of the lobster shell.
ol. Bull. mar. biol
o
22, 249-250.
Woo
Hole
ZAGALSKY P. F. (1971) Comparative studies on the
amino acid compositions of some carotenoid¬
ning lipoglycoproteins and a glycoprotein
conta
s and ovaries of certain aquatic
from te
siol. 47
invertebrates. Comp. Biochem
85-395.
IGURE GAPTIONS
Figure 1. Absorption spectrum from 250-750 nm.
of the groen carotenoprotein complex isolated from
Idotea montereyensis (Maloney). (A) represents the
crude extract, (B) extract after being passed through a
column of Sephadex G-200, and (C) Sephadex G-200 purified
ttract after passage through a DEAE-Cellex 50 column.
00/683 - ratio of optical densities at 280 nm. to
683 nm. All spectra were taken of solution in phos¬
phate buffer, pH 7.0.
2. Absorption spectrum from 250-750 nm. of
Figur
— —
the green carotenoprotein complex isolated from idotea
kirchanskii (Miller & Lee). (A) represents the crude
extract, (B) extract after being passed through a
column of Sephadex G-200, and (C) Sephadex G-200
purified extract after being shaken with diethyl ether.
5280,
83 - ratio of optical densities at 280 nm. to
683 nm. All spectra were taken of solution in phos¬
phate buffer, pH 7.0.
e 7
5. Absorption spectrum from 250-750 nm. of
the green carotenoprotein complex isolated from Idotea
resecata (Stimpson). (A) represents the crude extract,
d
(B) extract after being passed through a column of
Sephadex G-200, and (C) Sephadex G-200 purified extract
280/683 -
after being shaken with diethyl ether. E
0
e Captions, cont'd.
Figu
ratio of optical densities at 280 nm. to 683 nm.
All spectra were takon of solution in phosphate
buffer, pH 7.0
Figure
4. Electrophoresis patterns on Cyanogum-

41 gel of the green carotenoprotein complexes of
79.
e
ansis (Maloney), Idotea Kircha

mon

liller & Lee), and Idotea resecata (Stimpson).
(a) - crude extract
(b) - extract after passage through a column of
Sephadex G-200
(c-1) = Sephadex G-200 purified extract after
passage through DEAE-Cellex 50
(c-2) = Sephadex G-200 purified extract after di¬
alysis against distilled water at 5°0. for two days
band (1) - lipid-contaminated protein
band (2) and other unlabelled bands = contaminating
protein
bands (3) and (4) - isopod carotenoprotein
All runs were made on 5% gels w.
ith Tris-NaEDTA-Boric

Acid buller, pH 8.4 for 3-5 hours at 200-300 V.
Prot
ins were stained for with Amido Black 10B.
Figure
ligul 5. Molecular weight determination of the
-
no
e
carotenoprotein of lde
tea mon

ansis (Maloney)
0
Figure
Japtions, cont'd.
Pigure
by gel filtration on Sephadex G-200. The column
was of a length of 60 cm. x 2.5 cm. Flow rate -
18-20 ml./hr. Fraction volume =- 3.0 ml. Samples were
applied in 3.0 ml. solution in a single experiment.
(1) - blue dextran (MW 2,000,o00)
(2) - isopod carotenoprotein (estimated MW 1,200,000)
3) - apoferritin (MW 480,000)
(4) - albumin (bovine) (MW 67,000)
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19.7
IABLE CAPTION
Table 1. The I
/683 ratios of Idoteid caro-
tenoproteins at various stages of purification.
() crude extract
(2) crude extract after being passed through a
column of Sephadex G-200
(3) Sephadex G-200 purified protein after being
dialysed against distilled water for two days at 500.
(4) Sephadex G-200 purified protein after being
shaken with diethyl ether
(5) Sephadex G-200 purified protein after being
passed through a DEAE-Cellex 50 Anion Exchange column
280
/683 - ratio of optical densities at 280 nm. to
683 nm. All spectra were taken of solution in phos-
ohate buffer, pH 7.0.
able 2. A comparison of canthaxanthin-caroteno¬
proteins and as
thin-carotenoproteins on the
bas
is of absorption spectrum, color, prosthetic
groups, stability to light and to heat, and molecular
weight.
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