ABSTRACT
1. On electrophoretic separation, a total of 5 protein bands
were detected in the Balanus nubilis blood.
2. Three of the proteins present in B. nubilis blood showed
peroxidase activity. Because of tests performed on the fastest
of these proteins and absorption spectra obtained for the whole
blodd, this fast protein is assumed to be hemoglobin.
3. Molluscan hemocyanins migrate much slower than Crustacean
hemocyanins and, in some species, split up into electrophoreti¬
respiratory pigments consistently showed strong peroxidase
activity.
14
INTRODUCTION:
The respiratory pigments hemoglobin and hemocyanin are both
found in members of the Arthropod class Crustacea. Hemocyanin
has largely been restricted to the Malacostraca while the dis-
tribution of hemoglobin has been reported as being random and
diverse among the lower Crustacea (Fox, 1957). It is therefore
not surprising that hemoglobin has been observed by Fox in sey-
eral species of parasitic barnacles and by Southward (1963) in
three free living species, including Balanus perforatus, Balanus
crenatus, and Elminius modestus. However Southward's studies
have been conducted primarily on the muscle tissue of these bar-
nacles.
A great deal of success has been achieved by Manwell & Baker
(1963) and subsequent investigators in the separation and iden-
tification of blood respiratory proteins by means of starch gel
electrophoresis. These investigations have been conducted pri-
marily on .he hemocyanins of the Malacostraca. Such methods have
never been used in a study of the respiratory pigments of the lower
Crustacean orders. Accordingly, the barnacle, Balanus nubilis, a
particularly large cirriped found in the intertidal regions of
Monterey Bay, California, was chosen for study. Although the
initial purpose of this study was to determine whether or not
a respiratory pigment was present in the blood of B. nubilis, the
use of Molluscan and Crustacean hemocyanins as well as hunan hemo-
globin as standards made a comparative electrophoretic study of
these sera possible.
Despite preliminary data which implied the presence of hemo-
cyanin, final results strongly suggest that hemoglobin is present
in the blood of B. nubilis.
MATERIALS & METHODS:
B. nubilis was collected from about the Orl foot tidal
level from pilings in Monterey Bay. The blood was collected from
the barnacles by means of a syringe injected into the large blood
sinus located immediately anterior to the mouth, between the body
and the adductor muscle. Ineorder to limit clotting, the syringe
was cooled to 0° C and filled with O.1 cc of a 10% EDTA solution
before the blood was removed from the sinus. Samples from an
average of four animals were collected in this manner and pooled,
vielding 6 cc of blood. This blood was placed in a plastic tube
and centrifuged immediately at 18000g for one hour at 00 C. This
removed any clot that had formed and any tissue or particulate
matter that would enhance further clotting. The supernatant was
either placed immediately in the gel or concentrated with Sephadex
G-200 just prior to electrophoretic separation.
The rock crab, Pachygrapsus crassipes (Randall, 1839), the
black abalone, Haliotus cracherodii (Leach, 1817), and a large
keyhole limpet, Megathura crenulata (Sowerby, 1825), were used as
sources of known hemocyanin while hemoglobin was obtained from
the whole blood of the experimenter. Small amounts of 10% EDTA
were sufficient to prevent massive clotting in the Molluscan and
crab sera and samples could be transferred directly from the ani¬
mals to the gel. Human blood cells were lysed in a homogenizer
and diluted with the same EDTA solution.
Samples were separated on poly-acrylamide gels using Cyano-
gun-41 (Fisher C-388). An E-C vertica gel electrophoresis appa¬
ratus was used throughout as were the procedures outlined in Tech¬
nical Bulletin 41 of the E-C Apparatus Corp. Tris-HOl buffer,
pH 8.9, was used as the gel buffer, while Tris-glycine buffer,
pH 8.3, was used in the chamber. The only variation in the tech-
18
nique as outlined in the E-C bulletin was a modification of the
time necessary for separation of the slower migrating Molluscan
proteins. The gels were run for either 90 or 180 minutes at 400 y.
The gels were stained for total protein in a saturated sol-
ution of Amido black. Peroxidase activity was detected through
staining with ortho-dianisidine, following the procedure of Man-
well and Baker (1963). This method of staining allows the loca-
tion of enzy matic peroxidases and the pseudo-peroxidases, hemo-
globin and hemocyanin. Fifteen to twenty-five minutes after the
gel was placed in the dye solution, approximately 20 drops of
dilute 30% hydrogen peroxide were added. Brown bands began to
appear about  hour where peroxidases had oxidized the dye. Full
color developement was complete 1 hours after the gel was placed
in the dianisidine. The gel was subsequently washed in distilled
water and placed in the Amido black solution for total protein
staining. The dianisidine was stable to any further staining and.
using the abowe procedure, no problems of an over developed back-
ground color or fading of the peroxidase bands were encountered.
Weiser (1965) states that the addition of a 5% solution of
KCN to Crustacean sera containing hemocyanin cleaves the copper
from its protein, resulting in the formation of free apo-hemo-
cyanin and inhibition of peroxidase activity. This apo-hemocyanin
migrates with about  its former speed, joining the free apo-hemo-
cyanin already present in the blood. In the present study the
sera were incubated with equal amounts of KCN at 7° C for at least
2 hours and then compared on the same gel with samples not treated
with KCN. The finished gel was stained for peroxidase and total
protein.
Whittaker's method (1959) of staining hemocyanin containing
gels with rubeanic acid for copper was used; however it did not
144
prove in this case to be sensitive enough for the small quantities
of material separated. The technique was used quite successfully
on whole blood samples.
The absorption spectrum of the B. nubilis blood was obtained
on a Beckman DK-2A Ratio-Recording Spectrophotometer. Deoxy-hemo-
globin was prepared by reduction of the sample with sodium dithio¬
nite (Fox, 1960). The same procedure was applied to the B. nubilis
blood. Sodium hydrosulfite was also used as a reducing agent.
RESULTS:
The blood of B. nubilis was found to be extremely unstable.
Naturally forming clots were massive and the gel that formed tended
to take all of the proetins out of solution. The use of EDTA and
high speed centrifugation was the best of several methods that
were attempted at limiting clot formation, although no method
fully prevented it. This instability was a major problem when-
ever chemical treatment of the centrifuged samples was attempted.
Isolation of specific proteins through differential precipitation
with ammonium sulfate, deoxygenation of the blood with sodium di-
thionite, and most other test reagents added to the blood resulted
in immediate, irreversable clotting or the denaturation of the
blood proteins.
bloed
a absorpticn spectrum of the B. nubiliseshowed the presence
of three major peaks at 418, 460, and 490mu, with relatively heavy
absorption up to 620my. The peak at 418m suggested the presence
of a Soret band and an attempt was made to isolate the protein
responsible for this peak by fractional precipitation, but the
difficulties mentioned earlier made this impossible. Since hemo-
globin was suspected, an attempt was also made to deoxygenate the
blood with sodium dithionite. This would cause the characteristic
spectral shift of the Soret band to 430ma which accompanies the
15
reduction of oxy-hemoglobin to deoxy-hemoglobin. Partial deoxy-
genation appeared to he achieved with the addition of small amounts
of sodium dithionite and a slight shift was observed (Fig. 1).
Further reduction was impossible due again to the instability of
the blood. Other reducing agents had the same effect.
Total protein staining for B. nubilis resulted in the ap-
pearance of five protein bends, hereafter referred to as bands
A, B, C, D, & E, in order of migration from the sample slot (Fig. 2)
Only three bands, A, B & D, appeared consistently. The appearance
of bands C & E was rather random and due apparently to the quality
and freshness of the sample. Human hemoglobin could be seen as a
red band migrating in the gel with a much weaker band in front of
it.
Manwell & Baker (1963) describe the separation of apparently
homogeneous hemocyanins into "fast" and "slow" components as a
result of electrophoretic separation. This effect was observed
in the Pach
grapsus serum with what appeared to be two hemocyanin
components and a weaker, non-hemocyanin protein between them. A
slower apo-hemocyanin protein was also present as was the typically
streaking fibrinogen above it. In agreement with Manwell & Baker's
observation that molluscan hemocyanins migrate slower than Arth-
ropod hemocyanins, the Megathura serum showed two hemocyenin com-
ponents, both migrating about one eighth as fast as Pachygrapsus
hemocyanin. The Haliotus hemocyanin was a single, broad band
with several non-hemocyanin proteins occasionally seen ahead of it.
Peroxidase activity was noted in the B. nubilis proteins at
three points, corresponding with bands A, C, when it appeared,
and exceptionally strong peroxidase activity around band D. A
similarly strong and diffuse band of peroxidase activity was also
noted with human hemoglobin. The dianisidine was oxidized by the
15
Pachygraasus proteins only at the immediate location of the fastest
migrating "fast" hemocyanin. Twenty gels were stained for per-
oxidase with slightly different procedures each time. In all of
these experiments, the Molluscan hemocyanins never showed per-
oxidase activity. Variations in timing in the addition of per-
oxide, concentrations of the dye solutions, light and dark, and
pH of the buffers were employed. Weiser (1565) states that de¬
naturation by heat increased the peroxidase activity of Arthro-
pod hemocyanins. Accordingly gels were incubated at 70° C for
one hour with no positive results. Likewise, chemical denatur-
ation through incubation of the gel in methanol and 8M urea gave
similar results. Tests performed on boiled whole blood for per-
oxidase were also negative. In all of the above experiments, the
peroxidase activity of the B. nubilis blood was present and stable,
at band D.
The addition of KCN to the crab serum produced a quantitive
olvno
shift to apo-hemocyanin, apparentlyxof all three faster migrating
proteins. Some denaturation of the sample could be the reason for
the disappearance of the non-hemocyanin protein. (Fig. 3) The
cyanide effect on molluscan sera was sumewhat different. The two
Megathura bands were unified into a single band that migrated be-
tween them. The Haliotus hemocyanin however appeared to be cleaved
into two proteins, a reaction probably occuring independantly of
the cleavage of copper. This effect was complete for the hemo¬
cyanins at the end of the two hour incubation.
In the B. nubilis and human bloods, the addition of cyanide
caused the gradual inhibition of peroxidase activity, though the
reduction in activity compared with an untreated sample was quite
visible. This effect is due in hemoglobins to the bonding of the
iron and the cyanide ion, blocking the respiratory function of the
6
18
protein. No iron or heme cleavage is known to occur as a result
of this reaction. The B. nubilis blood shows, however, a definite
strengthening of the slower migrating band B corresponding to the
decline in peroxidase activity at band D.
DISCUSSION:
These experiments again emphasize the extreme heterogeneity
of hemocyanins both between the Arthropods and Molluscs, and within
the different Molluscan genera. Completely different protein com-
plexes are indicated for each of the species studied by the dif-
ferences in their migration and the variable occurance of the
"Tast" and "slow" components. The generalizations that are made
concerning the reaction of the cyanide ion with Crustacean hemo-
the
cyanins are not entirely valid in Mollusca, though it is plain
that KCN definitely affects hemocyanin migration. The failure of
Molluscan hemocyanins to show peroxidase activity is yet another
of their unusual properties. No explanation for this phenomenon
can be given on the basis of this study.
A clear spectrum of hemoglobin in D. nubilis with a Soret
band at Alömu and alpha and beta bands at 545mu and 580mu was
never obtained. This was due to the presence of considerable mis¬
cellaneous absorption between 490muand 620m. Furthermore the for-
mation of deoxy-hemoglobin could not be adequately achieved be-
cause of the instability ofthe blood. The following experimental
data does support the presence of hemoglobin in B. nubilis:
1. The absorption spectrum of whole blood has what appears to
be a Soret band, characteristic of the heme group, at 4184.
A partial shift of this peak towards 430mu does occur when attempts
are made to deoxygenate the blood, thus suggesting the formation
of deoxy-hemoglobin. (cf. Fig. 1)
2. According to Pearse (1961), peroxidase activity that is stable
18.
to heat is a property of the pseudo-peroxidases, hemoglobin and
hemocyanin. The peroxidase activity of B. nubilis is stable to
heat as well as other forms of denaturation.
3. The general distribution of respiratory pigments in Crustacea
and the fact that hemoglobin has been found in two Balanus species
would make the presence of hemocyanin in B. nubiliss unlikely.
4. Both the diffuse nature of the peroxidase activity of B. nub-
ilis blood and the gradual and incomplete inhibition of this acti-
vity upon the addition of KCN at band D bears close resemblance to
the behavior of human hemoglobin under similar conditions and none
to that
shown by hemocyanin.
The one test that suggested the presence of hemocyanin in
B. nubilis was the reaction of band D to cyanide treatment. The
corresponding strengthening of band B with the inhibition of per-
oxidase activity at band D would appear initially to be caused by
the fornation of an apo-protein. The hemoglobins of lower Crus-
tacea are known to te very large molecules, approaching the hemo-
cyanins in size and complexity (Waterman, 1960). Therefore it may
be that in a molecule of this sige some spontaneous hydrolysis of
the large protein, band D, releas large protein fragments, band
B. The cyanide anion would then appear to speed up this reaction,
quite independantly of its inhibition of peroxidase activity.
The complexity and size of the Crustacean hemoglobin molecule may
result in the formation of "fast" and "sdow" hemoglobin components
on electrophoretic patterns (possibly bands D & C The probability
is much greater, however, that this bandC, like band A, is a tissue
or enzymatic peroxidase as described by Manwell & Baker (1903).
15
C
REFERENCES
Barka,T. and Anderson,P. (1963). Histochemistry. Harper & Row. N.Y.
Flodin, Gelotte, and Porath (1960). A Method For Concentrating
Solutes of High Molecular Weight. Nature, 188: 493-494.
Fox, H.M. (1957). Hemoglobin in the Crustacea. Nature, 179: 148.
Fox, H.M. and Vevers, G. (1960). The Nature of Animal Colours.
Sidgwick & Jackson Ltd. London.
Manwell, C. and Baker, C.M.A. (1963). Starch Gel Electrophoresis
of Some Marine Arthropods: Studies of the Heterogeneity
of Hemocyanins and on a Ceruloplasm like Protein.
Comp. Biochem.Physiol. 8 (3): 193-208.
Pearse, A.G.E. (1961). Histochemistry. Churchill, London.
Redfield, A.C. (1952) Hemocyanin. In Copper Metabolism. Johns
Hopkins Press, Baltimore, Maryland.
Southward, E.C. (1963). Hemoglobin in Barnacles. Nature, 200: 798.
Waterman, T.H. (1960). The Physiology of Crustacea. Academic Press,
New York.
weiser, .. (1965). Electrophoretic Studies in Blood Proteins in
an Ecological Series of Isopod and Amphipod Species. J. Mar.
Biol. Ass., U.K., 45 (2): 507-523.
Whittaker, J.R. (1959). Location of Hemocyanin on Starch Gel Elec-
trophoretic Patterns. Nature, 184: 193-194.
Fig. 1 : Absorption spectrum for the whole blood of B. nubilis.
The dotted line indicates the partial shift of the peak
at 418m upon attempted deoxygenation of the blood.
A composite gel stained for peroxidase activity and
Fig. 2 :
total protein. Proteins are represented by dark bands;
peroxidase activity is shown by stippling. The sera
shown are: 1. Balanus nubilis, 2. Human hemoglobin,
3. Haliotus cracherodii, 4. Megathura crenulata.
5. Pachygrapsu:
es. Note that peroxidase activity
crassi
is limited to the immedate area occupied by the "fast"
hemocyanin protein in P. crassipes and that no peroxi¬
dase activity is present in the Haliotus and Megathura.
Fig. 3 : A composite gel stained for peroxidase activity and
total protein. Odd numbered slots contain the same
sera and in the same order as Fig. 2. They are:
1. B. nubilis, 2. Hunan hemoglobin, 3. H. cracherodii,
4. M. crenulata, and 5. P. crassipes. Even numbered
slots contain the corresponding sera incubated with KCN.
Protein is again represented by dark bands, peroxidase
by stippling.
18
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