APPLICATION OF DISCONTINUOUS SODIUM DODECYL
SULFATE POLYACRYLAMIDE GEL ELECTROPHORESIS
TO THE BIOCHEMICAL SYSTEMATICS OF
ROCKFISH· (GENUS SEBASTES) HEMOLYZATE
A Thesis
Presented to
the Faculty of the Department of Biology
San Jose State University
In Partial Fulfillment
of the Requirements for the Degree
Master of Arts
By
Thomas P. Jungmann
June, 1982
ABSTI~CT.
-The application of multiphasic sodium dodecyl sulfate
polya-ylamide gel electrophoresis to the biochemical systematics
of
ro~fish
reve~~
(g. Sebastes) hemolyzate was studied.
Results generally
species-specific electropherograms that were usually intra-
speci~cally
invariant.
dist~tion of~·
Experimental findings supported the
chrysomelas and
~·
carnatus as valid species,
which iitherto had not been accomplished on a morphometric or
meristc basis.
Moreover, common protein subunits occurred within
the er-ire genus, and interspecific differences suggested several
biocherical subgroupings.
differ~
These proposed subgroupings generally
from previous classifications based on morphometric and
meristc data.
Some of these differences may ultimately be attri-
buted :.::J pressure adaptive differences in the hemoglobin molecule.
iii
TABLE OF CONTENTS
ABSTRACT . . .
iii
LIST OF TABLES .
v
LIST OF FIGURES
. vi
ACKNOWLEDGEMENTS
vii
INTRODUCTION . . .
1
MATERIALS AND METHODS
9
RESULTS
18
DISCUSSION .
26
LITERATURE CITED .
33
APPENDIX . . . .
36
iv
LIST OF TABLES
Table
1
Page
Date of collection, fork length, and sex for 19 species
of
rockfis~
(Sebastes) from Monterey Bay, California,
from which blood samples were taken for sodium dodecyl
sulfate polyacrylamide gel electrophoresis
2
Protein
s~units
10
present for 12 species of Sebastes
as determL1ed visually from the electropherograms produced
by sodium dodecyl sulfate polyacrylamide gel electrophoresis
of the red blood cell lysate (major molecular weight component
13000, fouod in all Sebastes studied, has been excluded from
this table)
3
. . . . . . . . . . . . . . . . .
24
Comparison of taxonomic groupings of rockfish (genus
Sebastes) based upon morphometries, meristics, and electrophoretic
s~dies
. .
30
v
LIST OF FIGURES
Figure
1
Page
Logarithm of molecular weight versus relative mobility
of standard proteins subjected to sodium dodecyl sulfate
polyacrylamide gel electrophoresis . . . . . . .
2
4
Red blood cell lysate electropherogram for Sebastes
nebulosus,
~-
caurinus
and~-
elongatus showing the three
molecular weight subunits which are common to all 19 species
of rockfish investigated.
Molecular weight: a=65000,
b=33000 and x=l3000
3
19
Red blood cell lysate electropherogram of 6 species of
roc~fish
(Sebastes) demonstrating species-specific
differences and consistency of pattern within a species.
All specimens were collected and identified by Dr. R. Lea
of the California Department of Fish and Game
4
20
Red blood cell lysate electropherogram of 6 species of
rockfish (Sebastes) showing similarity of patterns among
certain species, i.
e.,~-
goodei
and~·
paucispinis
have more proteins in common than with other species
5
22
Similarity diagram for 10 species of Sebastes, depicting
degree of genetic relationships as determined by similarity
indices
. . .
.
. . .. . . .. . . . . .... . . . .
vi
23
ACKNOWLEDGEMENTS
I thank him, the Alpha and Omega, who, through his Spirit, has
given me the strength to complete this work for the glory of his
name, as he has said, "
. . call upon me in the day of trouble; I
will deliver you, and you shall glorify me" and elsewhere, " . . . that
our God may make you worthy of his call, and may fulfill every good
resolve and work of faith by his power, so that the name of our Lord
Jesus may be glorified in you, and you in him, according to the grace
of our God and the Lord Jesus Christ."
My father, mother, and brother
Steven, I also thank, for their love and patience during this
effort.
They were truly a wellspring of encouragement at times when
it was most needed.
Finally, I would like to acknowledge the following
thesis ·advisors for much time spent editing this thesis, and more
specifically; Dr. Glenn A. Funk for the privilege of laboratory space,
and availability of electrophoretic equipment and chemicals, as well
as the suggestion of the SDS technique as a means to increase electrophoretic resolution; Dr. Robert L. Hassur for recommending the blood
as a possible source of genetic variation, and whom I also credit for
the photographs of the electropherograms and graph; Dr. Gregor M.
Cailliet for suggesting Baine's et al. (1975) research that led to an
interpretation of a functional basis of my systematic classification,
and; Dr. Robert N. Lea, by whom many of the specimens were collected
and identified.
This research was not supported by any for@ of grant.
vii
INTRODUCTION
The biochemical genetic information used in evaluating fish
systematics has primarily involved electro-separation techniques
based upon intrinsic charge differences of biological macromolecules.
This has been especially true of studies on rockfishes (genus
Sebastes, family Scorpaenidae, Barrett et al. 1966; Tsuyuki et al.
1968; Tsuyuki and Westrheim, 1970; Johnson et al. 1970a; Johnson
et al. 1970b; Johnson et al. 1972; Johnson et al. 1973; Wishard
et al. 1980).
Dayhoff and Eck (1968), utilizing information of
amino acid sequence data, calculated that only 28.7 percent of
amino acid substitutions occurring via mutations result in amino
acids with different electrical charges.
Therefore, electro-
separations by charge would appear to produce a conservative estimate
of genetic differences.
Moreover, the degree of resolution con-
ferred by charge separations via multiphasic native electrophoretic
systems (biologically active molecules separated by electrophoretic
systems at different pH) may be marginal in some instances.
With the development of sodium dodecyl sulfate polyacrylamide
gel electrophoresis (Shapiro et
~-
1967), it became possible to
electrophoretically separate proteins according to their nolecular
weights.
Specifically, in conjunction with the charge separation
capabilities of polyacrylamide gel electrophoresis, the polyacrylamide
pore exerts a sieving effect.
This aspect was exploited through the
introduction of the "Ferguson plot" in starch gels (Ferguson, 1964)
1
2
and
polyacry:~ide
plot is the
gels (Chrambach and Rodbard, 1971).
~ction
The Ferguson
that describes the linear relation between
relative mobi:ity of a given protein during electrophoresis and
percent gel
c~centration.
That is, as the concentration of gel
increases (abs:issa), the relative mobility of a protein decreases
(ordinate).
T2e slope of this line is a measure of molecular size
(denoted as th: retardation coefficient), whereas they-intercept is
considered the "free
ele~trophoretic
mobility":
i.e., a measure of
mobility due tJ charge in absence of the sieving effect.
Therefore,
statements concerning the relative electrophoretic importance of
charge versus size may be advanced (Hedrick and Smith, 1968).
For
example, if Ferguson plots revealed identical slopes but different
y-intercepts,
~e
could say that electrophoresis based upon charge
separation would provide the greatest resolution, whereas differing
slopes but identical y-intercepts would be indicative of differing
molecular weigtts.
slopes and
If, however, the lines intersected (differing
y-iu~ercepts),
either charge or molecular weight separation
techniques woul:l be valid.
Therefore, to separate molecules strictly on the basis of
molecular
weigh~,
the compounding problems of interfering molecular
charge and conformational anomalies (which would decrease the reso'
lution conferrei by strict molecular weight separation) must be
obviated.
In sodium dodecyl sulfate polyacrylamide gel electro-
phoresis (SDS-PAGE), this is accomplished by adding the anionic
detergent sodiun dodecyl sulfate, which, in conjunction with 100°C
3
and
mercape~~anol,
denatures the proteins into their constituent
polypeptides, and confers a constant amount of negative charge per
unit length of polypeptide chain.
It is believed that the poly-
peptides behave as ellipsoidal rods with the long axis being
proportional to molecular weight (Fish et al. 1970).
Ferguson
plots of SDS-PAGE separations yield lines with different slopes,
but identical y-intercepts.
The precise tenet upon which molecular weight separation by
SDS-PAGE is based, is given by the empirical finding of Shapiro
et al. (1967), Shapiro and Maizel (1969), -and Weber and Osborn
--
(1969), in which the logarithm of molecular weight is inversely
proportional to the relative mobility (Fig. 1).
Therefore, by
forming a standard calibration curve of proteins of known molecular
weight and relative mobility, the molecular weight of an unknown
polypeptide can be determined from its relative mobility value
through linear interpolation.
When the SDS-PAGE separation is
combined with a discontinuous system (stacking and separating gels
at different pH), the resolution of SDS separations is excellent.
Electrophoretic patterns
a~e
thereby more distinct, and bio-
chemical genetic associations can become more obvious.
Figure I.-Logarithm of molecular weight versus relative mobility of standard proteins
subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis.
5
In order to test the utility of SDS-PAGE as a systematic tool,
the rockfishes (family Scorpaenidae) were selected.
They are very
abundant along the California coast and are readily obtainable by
means of party boat fishing or commercial fishing operations.
The
rockfish comprise a very complex genus with approximately 65
species occurring in the eastern north Pacific and have been
studied extensively, but by no means exhaustively.,
It would there-
fore be of much value to determine if this new electrophoretic
technique aids in the understanding of rockfish systematics.
The prime directives, then, of the current research were to:
(1) determine how this electrophoretic technique functions as a
means to understanding problems of a systematic nature, (2) evaluate
the systematic classification of rockfish based upon SDS electrophoresis of the red blood cell lysate, and how its results conform
to the numerous interpretations of previous morphometric and meristic
plus electrophoretic classifications of this taxonomically complex
genus.
For the first objective, a necessary requisite of any systematic
or ecological interpretation, the hypothesis is that this electrochemical method will result in information increase, primarily due to
-the excellent resolution attainable.
If the technique provides no
systematic foundation or results in information decrease (electrophoretic resolution), then any extrapolation of SDS-PAGE tc the
systematics or ecology of rockfish must be rejected.
Therefore, the
evaluation of the utility of this technique is of prime importance.
6
The
syste~tic
comparison (objective two) will primarily
include the morphometric and meristic classification schemes as
proposed by Hubbs and Schultz (1933) as well as the biochemical
systematic studies of Barrett et
~·
and Johnson et
(1972).
~·
(1966), Tsuyuki et
~·
(1968),
Hubbs and Schultz (1933), revising many
of the subgeneric proposals of Jordan and Evermann (1898), considered
relationships
w~thin
amo~g
and
the subgenera Sebastosomus,
Auctospina, Pteropodus, and Primospina.
comprised~-
Sebastosomus
flavidus,
(Hubbs and Schultz, 1933), the
~·
Although the subgenus
and~·
serranoides,
species~·
mystinus
and~·
melanops
entomelas
of subgenera Primospina and Acutomentum respectively, were considered
to be very similar in morphology and meristics to subgenus
Sebastosomus.
Hubbs and Schultz (1933), however, not wishing "to
enter into any attempt to reclassify the multitudinous forms of
Sebastodes" retained the subgenus Sebastosomus as a valid taxonomic
unit.
Moreover, the genus Pteropodus as proposed by Eigenmann and
Beeson (1894) has undergone substantial reclassification.
Hubbs
and Schultz (1933) accepted the Jordan and Evermann (1898) reclassification of genus Pteropodus (~. rastrelliger, ~- caurinus, ~·
vexillaris,
~·
maliger,
~·
gilberti,
~·
carnatus,
~-
chrysomelas,
'
and~- nebulosus), but noted that S. rastrelliger was probably not
closely related to the others and replaced the name ~· gilberti with
S. dalli.
They also added one new species, S. littoris.
The biochemical systematic information of genus Sebastes has
been subject to various interpretations based upon the results of
7
different electrophoretic systems.
The analysis of rockfish hemo-
globin by the starch-gel electrophoretic methods of Barrett et
(1966) resulted in the electrophoretic grouping
s. melanops,
nebulosus,
and~·
and~·
~·
mystinus;
dalli;
Tsuyuki et al. (1968)
and~-
placed~·
of~·
~·
flavidus,
caurinus, S. vexillaris, S.
rosaceous, S. eos,
mystinus
and~-
and~·
constellatus.
melanops in different
biochemical groups, thereby conflicting with Barrett's et al. (1966)
study, but this was primarily on the basis of muscle tissue electrophoresis and only secondarily concerning hemoglobin.
Johnson et al.
(1972), studying six enzymatic systems and muscle proteins, placed
~·
flavidus and
~·
melanops in the same biochemical group, as did
all previous studies.
Moreover, S. caurinus and S. nebulosus have
been shown to demonstrate similar banding patterns by all investigators.
However,
~·
paucispinis which was grouped with S. reedi and S.
crameri by Johnson et
~·
different from Tsuyuki
(1972) separated
~·
combined these two.
~·
(1972), was considered biochemically
~~·
(1968), and whereas Johnson et al.
elongatus and
~-
entomelas, Tsuyuki et al. (1968)
And S. pinniger, considered to be similar to
melanops and S. flavidus by
Tsuyu~i
et al. (1968) and Johnson et al.
(1972), was biochemically differentiated
et al. (1966).
Finally,
~-
with~·
miniatus by Barrett
alutus, thought to be biochemically
~ifferent from all others by Johnson et ~- (1972), was grouped with
S. aleunanus and ~· zacentrus by Tsuyuki ~ al. (1968).
The analysis of the components of the red blood cell and their
function may provide a functional explanation for the proposed
8
hemolyzate systematic classification.
Hemoglobin (reversible oxygen
binder) with a nearly uniform molecular weight in all vertebrates
of approximately 65000 (Riggs, 1970) comprises about 90 percent of
the red blood cell (Lehninger, 1976).
Four polypeptides, two alpha
subunits and two beta subunits, each associated with a reversible
oxygen binding heme group, are joined together by non-covalent
forces to ·form the hemoglobin molecule.
If it is assumed that each
polypeptide is approximately equal in size, this would place their
molecular weight at about 16000.
Carbonic anhydrase is also a
component of the red blood cell and functions to accelerate the
formation and dissociation of carbonic acid.
Its concentration in
the red cell has been shown to be in direct proportion to the degree
of ]lletabolic activity occurring in the cell.
The red cell also
contains 2,3-diphosphoglycerate (allosteric effector of hemoglobin)
and ATP, molecules of comparatively low molecular weight.
MATERIALS AND METHODS
Specimens for study were acquired from party boat returns in
Monterey, California, and from Dr. Robert R. Lea of the California
Department of Fish and Game, who handspeared individuals while SCUBA
diving in Monterey Bay.
collected.
As many different species as possible were
Adult forms were exclusively studied (except 1 juvenile,
s. serranoides) because of known and potential ontogenetic changes
occurring in serum proteins of many fishes (Booke, 1964), and other
factors such as health, sex, season, length, and location were
monitored as closely as possible (Table 1).
Fishes were identified
to species using Miller and Lea (1972) as the key.
Blood was obtained via cardiac punctu+e.
Becton~Dickinson
5-ml-capacity.sterile disposable plastic syringes fitted with
2.2-gauge needles were employed for withdrawing blood.
1~-inch
As a conse-
quence of the independence of intrinsic charge differences of
biological macromolecules with SDS-PAGE separations, the procedure
did not appear to necessitate the immediate removal of blood from
live fish, a definite requisite of multiphasic native electrophoretic
systems because of the ferrous to ferric oxidative changes occurring
within the hemoglobin molecule during storage (Tsuyuki et al. 1968).
Consequently, many of these fish were probably lying on the deck of
-
a party fishing boat for several hours before any blood was actually
withdrawn.
With the specimens obtained from Dr. Lea, a midventra1.
incision was made such that access to the pericardial cavity ·.vas
9
TABLE I.--Date of collection, fork length, and sex for 19 species of rockfish (Sebastes) from
Monterey Bay, California, from which blood samples were taken for sodium dodecyl sulfate
polyacrylamide gel electrophoresis.
Fork
Onte
Collected
June 1981
SEecies
:eaucis:einis
II
II
If
16 June 1981
miniatus
levis
caurinus
flavidus
goodei
II
EaucisEinis
II
II
Einniger
II
II
II
"
caurinus
II
25 June 1981
1 July 1981
nebulosus
chlorostictus
Dato
Length (em)
58
57
58
59
50
?
44
44
36
36
54
49
49
47
46
41
47
45
38
30
25
29
Sex
male
lJlale
female
female
?
female
male
male
female
female
male
female
female
?
male
?
female
female
female
female
male
?
Collected
~-.-...~~··'!"·-:-···-:-:.·"""'
Jlork
Species
Length (em)
28
olongntus
saxicola
28
July 1981
constellatus
27
II
27
II
30
rosaceus
25
23
"
II
23
21 July 1981 chrysomelas
30
II
24
II
24
22
"II
21
carnatus
31
II
26
II
26
atrovirens
?
II
24
If,
23
II
20
melanoEs
28
25
"
mystinus
25
II
31
serranoides
28
Sex
7
female
?
?
female
?
female
?
female
female
female
female
male
female
female
male
male
male
female
male
male
female
male
female
juvenile
......
0
11
possible.
Fer the party boat specimens, in which fillets were first
renoved by deckhands, the pericardial cavity was already exposed.
ruptur~1g
After
heart.
the pericardium, the needle was directed into the
~aunt
The
of blood available varied from a few drops to
perhaps over one milliliter and appeared to be a direct function of
the size of t1e heart.
with blood
(~~ically,
In some specimens, the heart was engorged
the large fish), whereas in others, the heart
was very constr.icted, and the small volume of blood obtained was
unquestionably contaminated with pericardial fluid.
Blood samples
were immediately placed in Becton-Dickinson 5-ml heparinized
vacutainers, and gently inverted several times in order to ensure
mixing of con-::ents.
Samples were then placed on ice and ultimately
refrigerated until further analysis in the laboratory.
Isolation of red blood cell components prior to electrophoresis
involved washing the red blood cells with a 1.3% saline solution.
Initially the blood was centrifuged in a Becton-Dickinson Serofuge
for one minute, and serum was then pipetted out and discarded.
Two
milliliters oi 1.3% saline solution were then added to the pellet of
red blood cells, centrifuged for one minute, and the resultant supernatant discarded.
This step was repeated a total of three times.
Lysis of the red blood cells was achieved by adding a volume of
distilled, deionized water (approximately twice the volume of the
red blood cell pack) to the red blood cell pellet.
The components
of the red blood cell, now released from the cell, were isolated
c
~rom the cell stroma by centrifugation.
was frozen.
At this point the sample
13
laid with water utilizing a Buchler Polystaltic pump.
The prewetted
glass wall (from separating gel addition) allows water to flow do\m
the glass wall in a gentle stream, and thereby precludes the possibility of gel distortions caused by large drops rlishing down the side
when the force of gravity overcomes capillary action on a dry glass
wall.
The Buchler Polystaltic pump was preferred over a syringe or
pipette because of the precise ability to control'water flow.
After
perhaps thirty minutes, a sharp water-gel interface was observed,
indicating that polymerization had taken place.
The stacking gel, a 4%T 2.7%C formulation, was prepared by
adding 2.66 ml of monomer solution, 5 ml of stacking gel buffer, 0.2
ml of 10% SDS, 12.2 ml of H 0, 100 microliters of ammonilli~ persulfate,
2
1 1
and 10 microliters of N,N,N ,N -tetramethylethylenediamine (TEMED)
to a 100-ml
erler~eyer
flask (Hoefer Scientific Instruments, 1980).
The water layer in the electrophoretic unit was decanted and the inner
glass sandwiches repeatedly rinsed with one to two milliliters of
stacking gel solution.
Finally, the stacking gel was poured into the
unit almost to the top and the plastic combs were inserted while
ascertaining that no bubbles were trapped beneath the teeth.
Poly-
merization occurred in approximately one-half hour, at which point
the teflon ~ombs were gently removed.
Solubilization and reduction of protein samples was effected
combining equal volumes of red blood cell lysate sample with equal
volumes of 2x treatment buffer (Appendix A) and subjecting this to a
boiling water bath for 90 seconds.
Th e samp 1 e was t h en store d on 1ce
·
or frozen until the electrophoretic run.
Sodium dodecyl sulfate
14
polyacrylamiC.e gel electrophoresis low molecular weight standards
(SDS-PAGE-LMri) were acquired from Bio-Rad (Richmond, California) to
be used as markers to determine the molecular weights of some chosen
protein subunits of the red blood cell lysate.
A 1:20 dilution of
these standards was made by adding 5 microliters of SDS-PAGE-LMW to
45 microliters of water and SO microliters of 2x treatment buffer.
The sample was then subjected to heat treatment as described abov~.
-~ter
placement of the electrophoresis unit in the lower buffer
chamber, tank buffer was poured directly into both lower and upper
buffer chambers.
Ten microliters of each protein sample in question
was gently injected into the separate polyacrylamide wells with a
Hamilton microsyringe.
A drop of bromphenol blue was added to at
least one sample in each slab to function as the tracking dye.
Additionally, one well per slab was allocated for the LMW standard.
At this point, the electrodes were connected to a Beckman Duostat
and a constant current of 25 milliamperes was applied to each slab.
Voltage was monitored as a function of time.
The run was allowed to
continue until the tracking dye approached the lower edge of the gel
(slightly longer than an hour), at which point the run was terminated
and the unit disassembled.
Glass sandwiches were separated by gently
prying the sides apart, allowing the gel to adhere to only one glass
Finally, the leading edge of the tracking dye was marked with
a razor blade for future relative mobility measurements, and the gel,
now situated on one glass plate, was placed in a photo tray for overstaining with 0.125% Coomassie Blue.
Excess stain was eluted
decanting the staining solution, rinsing the gel with distilled
15
water, an3 then immersing in destain Solution I.
When the destain
solution became opaque, it was poured off and fresh destain I added.
This procedure was repeated until the protein bands were delineated
against a clear gel background.
At this point, the gel was stored in
destain II.
Relative mobility measurements (defined as the distance the
leading edge of a given band migrated, divided by the tracking dye
distance) were calculated at this stage.
The molecular weight of
specified polypeptides was then determined through linear interpolation
when their relative mobility was compared to the calibration curve of
the standard proteins of known molecular weight and relative mobilities.
A permanent record was formed by transferring the gel to .a piece
of filter paper.
Gels were then placed on a slab gel dryer (Hoefer
model SE 500), and dried for approximately 1~ to 2 hours.
The presence or absence of certain protein subunits was strictly
visually analyzed.
Although spectrophotometric scanning of gels would
theoretically provide the greatest experimental objectivity, in reality,
the baseline interference level of the instrument ("noise") and presence
of very faint bands, precluded the possibility of quantitating these
visually faint bands.
Therefore, th~s procedure was limited to those
protein subunits which were deemed significant visually.
Moreover, the
relative mobilities of many molecular weight components were distinguished
only 0.1 centimeters, and with a standard deviation of approximately
0 · 3 centimeter, it is easily seen how two molecular weight components,
n -·
v.l centimeters apart, used to distinguish species, could easily be
considered the same molecular weight within the accepted range of
16
experimental error.
Assumption of molecular distinction then, was
primarily nediated through the visually unique banding patterns of a
given species' electropherogram.
The calculation and presentation of
molecular weights must be considered within these limitations.
Similarity indices, defined as the ratio of protein subunits
common to the species under consideration to the total number of
protein subunits common and different, were then calculated.
The index
could range from a value of zero to unity, a zero value indicated no
bands were common to the species comparison (genetic similarity= 0),
while a value of unity indicated all bands were common (complete
genetic similarity).
This similarity index, then, represents that
proportion of bands that are common to the species group under
consideration.
However, because electrophoretic distinctions among
morphological species were often attributed to single, very faint
polYPeptide units, a similarity index of unity is not indicative that
they are identical species, only that immediate differences were not
readily apparent (perhaps because of reduced protein concentrations
in some tracts).
It is believed that these molecular weight differences
would become more obvious if protein loads "ere increased.
A similarity diagram based upon these similarity indices was
accomplished by calculating the group similarity index for al* fishes
in which relative mobility measurements were available, taking the
progressively smaller subsets which displayed a cumulatively greater
of common protein subunits, and calculating their similarity
indices.
The number associated with the horizontal lines of the
diagram represents the similarity index uniting those
17
fishes to the left of the point of divergence (vertical lines).
Therefore as the group of fish showing a certain degree of genetic
similarity is gradually reduced in size, the similarity index uniting
them correspondingly increases.
RESULTS
Common to all rockfish hemolyzate electropherograrns investigated
were three protein subunits (Fig. 2), 65000 (a), 33000 (b), and
13000 (x). These molecular weight values were calculated via linear
interpolation from the known molecular weight and relative mobility
of standard proteins presented in this figure.
The rate of protein
migration as a function of molecular weight can be visually observed
in this 'electropherogram, as phosphorylase B (molecular weight 92500)
has the lowest relative mobility, whereas lysozyme with a molecular
weight of approximately 14400 has the greatest relative mobility.
Variation in protein subunits was generally found among morphological species and a high degree of consistency characterized most
intraspecific electrophoretic patterns (Fig. 3).
melanops
and~·
mystinus can be distinguished by the polypeptide with
a molecular weight of 66000 (m), and similarly,
~·
-For example, S.
~-
atrovirens and
mela.nops can be differentiated by protein subunit 46000 (i).
These
ty~es
of differences can be found for all morphological species
in this electropherograrn (except
even for
~·
~·
mystinus and
~-
serranoides) and
chrysomelas and S. carnatus two recognized species
between which no morphological or meristic differences are obvious.
ly, the ~ndividual variation found within morphological species
is not reflected in this figure, because all intraspecific electropatterns were observed to be consistent.
The presence of common bands among certain morphological species
ed delineation of certain subgroupings based upon similarity
18
Figure 2.--Red blood cell lysate electropherogram for Sebastes nebulosus, ~· caurinus, and
S. elongatus showing the three molecular weight subunits which are common to all species of
rockfish investigated. Molecular weight: a=65000, b=33000, and x=l3000.
Figure 3.-Red blood cell lysate electropherogram of 6 species of rockfish (Sebastes)
demonstrating species-specific differences and consistency of pattern within a species. All
specimens were collected and identified by Dr. R. Lea of the California Department of Fish
and Game.
N
0
21
indices (Fig. 4).
Specifically,~-
paucispinis and S. goodei share
the greatest number of bands and are therefore grouped together by a
high similarity index of 0.9.
Likewise, the similarity index calculated
for other species groups was presented in Figure 5 as derived from
Table 2.
The electropherogram patterns of six species
s. carnatus,
~·
were similar, with a
.
S) .
Flg.
~·
atrovirens,
melanops,
simil~rity
(~.
~· mysti~us
chrysomelas,
and
~·
serranoides)
index of 0.6 (Fig. 3, Table 2, and
W1"thin this ~£rouping, in addition to the three components
typical in all Sebastes, there was the presence of molecular weight
subunit 49000 (h) and 26000.
and
s.
Specific differences between
carnatus included the presence in
~·
~-
chrysomelas
carnatus of molecular weight
subunit 46000 (i), which was completely absent
in~·
chrysomelas.
Sebastes carnatus and S. atrovirens also shared similar patterns,
including the 46000 (i) molecular weight subunit mentioned ahove;
however, a molecular weight subunit of 66000 (j) was present in
~·
atrovirens and lacking
a similarity index of 0.6,
.9 similar.
in~-
carnatus.
while~·
These latter 3 fishes had
carnatus and S. atrovirens were
Sebastes mystinus and ~· melanops both had no molecular
subunit 46000 (i), but had molecular weight subunit 50000 (m),
Finally, ~: serranoides (pattern is somewhat obscured, most
because of a problem created by a sealing compound contamination
at
edge of the spacer) was very difficult to differentiate from
Figure 4.--Red blood cell lysate electropherogram of 6 species of rockfish (Sebastes)
showing similarity of patterns among certain species, i.e., S. goodei ahd ~· paucispinis
have more proteins in common than with other species.
5. -Similarity diagram .for 10 species of Sebastes depicting degree of genetic
lationships as determined by similarity indices.
0.9
goodei
paucispinis
1.• mystinus
0.9
Sebastes 0.2
.
serranoides
melanops
0.6
chrysomelas
0.3
0.9
carnatus
atrovirens
0.9
caurinus
nebulosus
Genetic Similarity
0.1
0.2
r-----~-------
N
LH
---
24
TABLE 2. -Protein subunits present for 12 species of Sebastes as _
determinec visually from th: electropherograms produced by sodium
dodecyl sulfate polyacrylamide gel electrophoresis of the red blood
cell lysate (major molecular weight component 13000, found in all
Sebastes studied, has been excluded from this table).
0.46 46000
OAS 43000
0.50
0.52
0
0
31000--------
- ----- -23000
--
25
S. mystinus, and therefore has a similarity index of unity with
s.
mystinus.
The electrophoreogram
of~-
caurinus
resulted in a similarity index of 0.9.
and~-
nebulosus (Fig. 2)
Again, both had bands common
to genus Sebastes, but additional protein subunits 48500 (f),
31000, and 24000 (g) unified them.
a polypeptide
fra~nent
Differences included
above the 24000 (g) subunit of S. caurinus which
-
of~-
was obvious in only one of the two specimens
caurinus, but barely
detectable because of a reduced protein load.
Electropherograms of
al~ost
~·
paucispinis and
identical (similarity index= 0.9).
~-
goodei (Fig. 4) were
They were distinguished
the presence of a faint nolecular weight subunit
the 33000 (b) subunit
of~-
paucispinis, absent
Relative mobility measurements
for~-
irnm~diately
in~-
below
goodei.
chlorostictus,
~-
saxicola,
S. levis, -S. flavidus, -S. miniatus, -S. rosaceus, and -S. constellatus
were not available and therefore were not included in the proposed
schematic (Fig. 5).
DISCUSSION
Systematic studies using the SDS technique overcome many of the
difficulties encountered in classical meristic and morphometric
studies.
For example, the gradation of characters (morphological
continuity) typically observed among rockfish often questions the
validity of natural distinctions among proposed subgenera.
Chen (1971)
states, "However, because of the existence of intermediate species,
subgroupings within Pacific Sebastes have not been widely accepted."
individual variation, Chen (1971) also remarked, "Individual
in morphological characters within populations of the
of Sebastomus are obvious upon examining the morphometric
scatter-diagrams . . . Such variations may obscure specific morphodifferences."
mo1~hological
And speaking of a covariance analysis of
differences between conspecific samples as well as
samples of different species, he said, "With the exception
depth, geographic variation was observed in all characters
" With SDS--PAGE, these conventional problems may be nonElectropherograms between species may show similar patterns,
relative mobilities, or completely different patterns
terms of additional or deleted bands.
Therefore, patterns are
and interspecific separation becomes apparent.
This does not
a biochemical gradation may not exist, but that species
is mediated by presence or a b sence concepts (e.g., protein
' and not limited by determining means and modes (realm of
along a continuum of morphological or meristic characters.
26
27
An example is the ~· chrysomelas and ~- carnatus complex, two
. 1 s pec 1·es which ' to this author's knowledge, have not previously
nom1na
been subjected to electrophoresis.
Hubbs and Schultz (1933) commented
regarding t h.ese t wo forms, "A detailed and critical study of this pair
forms,as well as other similar pairs of variable species, would
appear to be very promising."
In 1957, Phillips stated of these two
species, "In the present study, no characters were found that would
adequately separate these two .
" and "It is not unlikely that the
two are simply extreme variations of the same species . . . "
However,
the technique of SDS-PAGE, the intraspecific electrophoretic
patterns were consistent and S. chrysomelas and
~-
carnatus (Fig. 3)
were easily distinguished by molecula; weight subunit 46000 (i).
difference suggests that the two are valid species.
Thus, what
and morphometric studies have been unable to accomplish, may
been achieved through high resolution SDS-PAGE.
The excellent resolution of SDS-PAGE results in information
concerning the systematics of the genus Sebastes.
For example,
as was previously established, the molecular weight components 65000,
>and 13000 were encountered in all 19 species of rockfish collected,
information was not revealed by the native electrophoretic data
et ~· (1966) and Tsuyuki et ~· (1968).
The precise re~son may be related to the (1) complexing nature of
or the (2) conservative estimate of genetic difference as
charge separation techniques (Shaw, 1970).
Specifically,
with the 1-gene-1 polypeptide concept (Markert and Whitt,
28
k
196 8) , eac.•
band of the SDS-PAGE electropherogram is one polypeptide
and therefore represents a specific gene locus.
However, from perhaps
the constituent set of polypeptides within an organism, many permutations
occur
50
as to create a massive gradation of proteins of slightly
charges, molecular weights, and so on.
Native electrophoretic.
would reflect these manifold numbers of combinations as large
;
bands.
This would represent information decrease concerning
electrophoretic systematic studies.
Secondly, 2..11d by definition, these diffuse bands of native
electrophoretic systems reflect proteins of very similar charge.
This
is in accordance with the postulated conservative estimate of genetic
prod~ced
by native electrophoretic systems.
That is,
the native technique only.allows for the expression of approxi28.7%
of the genetic difference, the rest of the proteins (70%)
of very similar charge (but perhaps very different molecular weights,
, size, and so on) .are contained within these bands, and
the bands become very diffuse.
Not only does this represent
loss, but the fundamental design of the system (charge
autorra.tically limits the degree of resolution possible.
SDS-PAGE these proteins are denatured (non-complexed) into
constituent set of polypeptides of specific genes, and would be
represented as distinct bands of unique molecular
(therefore overcoming conservative estimate?).
The blurring
(resolution decrease) and loss of information may thereby be
These differences in resolution (information) can be
seen by co mpar2ng
a typical SDS electropherogram with one
29
. ve
from a na t).
states,
e-, ""ctrophoretic
--
system.
In fact, Tsuyuki et _al. (1968)
"An exanination of the comparative electropherograms in Fig. 2
1 quickly point out the confusion that would follow any attempt to
lish generic or intrageneric breakdown of rockfish on the basis
hemoglobins c.lone."
However, this statement contradicts the current
electrophoretic study, because not only was generic classification
possibly established on the basis of the hemolyzate (Fig. 2 with the
molecul~r weigh~ subunits 65000, 33000, and 13000 unifying genus
----·
s.
, but subgroupings were also readily apparent
(~.
goodei -
paucispinis and S. caurinus- S. nebulosus associations).
Therefore,
resolutior; SDS-PAGE has accomplished what previous native electrostudies have had difficulty doing.
The syste:na.tic determinations resulting from SDS-PAGE (Table 3)
not correspond well with the morphological and meristic classificascheme put forth by Hubbs and Schultz (1933), and only partially
previous electrophoretic studies.
More precisely, fish of
subgenus Sebastosomus (Hubbs and Schultz, 1933),
~·
serranoides and
S. melanops, had similarity indices of 0.9 (~. flavidus excluded from
because no relative mobility measurements were available).
the intrageneric similarity comparison of ~· melanops and
S. serranoides was very high, the intergeneric comparison of ~- mystinus
S. serrancides yielded the greatest biochemical relationship,
all three fishes were highly similar (0.9).
Previous biochemical
et ~- 1966) found "similar" electrophoretic patterns
S. melanops, ~- mystinus, and S. flavidus (Table 3).
This
30
t•~onomic
TABLE 3.--Comparison of
groupings of rockfish (genus Sebastes) based upon morphometries,
erist.ics,, and electrophoretic studies~
Hubbs ;; Schul t:x
Barrett et: a!.
(1933)
(1%!1)
1111
Meristics
~
Electrophoresis
Tsuyuki et al.
(1968)
--
El ec1:rophoresis
Johnson et al.
(1972)
--
'in ectrophoresis
Current Study
Electrophoresis
Hoghomet:rics
Su genus
GROUP I
Seba.:stosomus
lavidus
ael:anops
serranoides
[ colUI!IlbLanus
dalli
.
Subgenus Pteropodus
rastrelliger
caurinus
vexillaris
nebulas is
dalli
carnatus
chrysoselas
ciliatus
jordani
aystinus
mystinus
rr
[m~ni~tus
,polyspinis
LP1nn1ger
GROUP II
aurinus
ubgenus Auct:ospina
a:uriculat.us
G
hvidus
•elanops
[
ve.xillaris
nebulosus
elonga1:us
en tome las
dalli
GROUP III
aurora
rosac:eous
GROUP IV
~
eos
constellatus
No Association Shown
paucisipinis
goodei
oval is
"""liger
YUf05
littoris
hopi:insi
""'c.donaldi
levis
auriculatus
saxicola
ruhri vinct:us
elongatu.s
rhcdochloris
chlorosticl:us
serriceps
lavidus
elanops
G
inniger
ieutianus
alut:us
G
zac ent l"\15
rcaurinus
[Eebulosus
No AssociaLion Shown
brevispinis
crameri
diploproa
helvomaculat.us
m.a.liger
paucispinis
proriger
reedi
n.Jherrimus
rubri vinct:us
saxicola
vilsoni
elonga-c:us I
entooaelas II
aurora III
chloros-c:ic~us IV
leul:ianus V
z.acen'trus
U
~
ruberriaus
n.sbrivinctus
:~§f~g:::
~
carnatus
at.rovirens
[Caurinus
l_!lebulosus
caurinus VI
jpaucispinis
maliger
l_¥oodei
No Association
auriculatus
diploproa VII
helvomaculatus VIII
saxicola IX
~
~~:!~;:X
pinniger
proriger
caen.aema t. icus
variegal:us XI
alutus XII
ra.meri XIII
paucispinis
reedi
levis XIV
G
Inferred
flavidus
saxicola
chloros-c:ictus
pinniger
elongatus
levis
miniat.us
rosaceous
cons1:ella1:us
31
is in agreement with SDS-PAGE results, again with the exception of
S. flavid~, for which relative mobility measurements were not
Other biochemical genetic data (Tsuyuki et
available.
~·
1968;
Johnson et al. 1972) in'clude the electrophoretic association of
s.
s flavidus ' and S. pinniger.
melanous, _.
On this basis it would be
to validate the subdivision of S. mystinus,
s.
~-
melanops,
serranoides, and ~· flavidus into separate subgenera.
Designation subgenus Pteropodus (Hubbs and Schultz, 1933) does
not appear to be justified on the basis of SDS-PAGE.
Although~-
S. carnatus, and S. atrovirens were united by a relatively
similarity index of 0.6 and the S. caurinus - S. nebulosus
index of 0.9, the intrageneric comparison of these 5 •fishes
a similarity index of only 0. 3.
Therefore, this morphological
does not display high biochemical intrageneric coherency.
this intrageneric conflict, plus the fact that all fish
col
of subgenera Pteropodus, Sebastosomus, and Primospina (except
• caurinus
and~-
nebulosus) had a relatively high similarity index
0.6, indicates that these morphological subgenera may not be valid
subgenera.
The very high similarity index (0.9) of~· paucispinis and S.
(differentiated by a minor band, difficult to see, immediately
33000 protein subunit in~- paucispinis, absent in S. goodei)
a natural biochemical relationship and thereby supports subThis concurs with Jordan's et al. (1930) arrangement of
but, conflicts with the Barrett~~- (1966) and
!!~- (1968) electrophoretic studies, which showed no bio-
32
chemical assocl. ation between these two fishes.
It is possible that some of these electrophoretic differences
may be explained by the association between bathymetric distribution
activity and the Root effect found by Baines (1975).
This associa-
was postulated to be possibly due to multiple hemoglobins, which
are of special significance to this study, because
the electrophoretic
should be able to detect this molecule.
A few species of
in this research were the same as studied by Baines
(1975), but
comparison of these electrophoretic results with Baine's Root
information is impossible since this technique produced one
band in the molecular weight range of hemoglobin polyPeptides.
ecologically similar fishes did tend to be electrophoretically
For example, the midwater fishes, ~- mystinus, ~- serranoides,
S. melanops, had similar electropherograms.
Moreover,
~- Eaucispinis
S. goodei, both deep-dwelling demersal species, also had very similar
, which were quite different than those of the shallower,
water species.
Further research into the detailed structure of
molecules in these ecologically different species is
to resolve this question.
------------------------~~~~
~
•••~v•.a
Junamann. Tho
·-···-
Annlir.:dinn nf ..u ...........~-··-
LITERATURE CITED
Baines, G. K.
1975. Blood pH effects in eight fishes from the
family Scorpaenidae. Comp. Biochem. Physiol.,
5l(A): 833-843.
teleos~ean
Barrett, I., J. Joseph, and G. Moser. 1966. Electrophoretic analysis
of herrJglobins of California rockfish (genus Sebastodes).
Copeia, 3: 489-494.
Booke, H. E. 1964. A review of variations found in fish serum
proteL1S. New Y9rk Fish and Game J., 2(1): 47-57.
Chen, Lo-Chai. 1971. Systematics, variation, distribution, and
biology· of rockfishes of the subgenus Sebastomus (Pisces,
Scorpaenidae, Sebastes). Bull. Scripps Inst. Oceanography,
No. 18, Univ. of California Press, Berkeley, pp. 115.
Chrambach, A., and D. Rodbard. 1971. Polyacrylamide gel electrophoresis. Science, 172: 440-450.
Dayhoff, M. 0., and R. V. Eck. 1968. Atlas of Protein Sequence and
Struct:rre. McGregor and Werner, Inc., pp. 356.
Eigenmann, C. H., and C. H. Beeson. 1894. Revision of the fishes of
the s~family Sebastinae of the Pacific Coast of America. Proc.
U.S. ~at. Mus., 17(1009): 375-407.
Ferguson, K. A.
1964.
Metabolism, 13: 985.
Fish, W. W., J. A. Reynolds, and C. Tanford. 1970. Gel chromatography
of proLeins in denaturing solvents. J. Biol. Chern., 245(19):
5166-5168.
Hedrick, J. L., and A. J. Smith. 1968. Size and charge isomer
separation and estimation of molecular weights of proteins by
disc gel electrophoresis. Arch. Biochem. Biophys., 126: 155-164.
Hubbs, C. L., and L. P. Sc~ultz. 1933. Descriptions of two new
American species referable to the rockfish genus Sebastodes,
with notes on related species. Univ. Wash. Publ. Biol., 2(2):
15-44.
Johnson, A. G., F. M. Utter, and H. 0. Hodgins. 1970a. Electrophoretic
variants of 1-alpha-glycerophosphate dehydrogenase in Pacific
ocean perch (Sebastodes alutus). J. Fish. Res. Bd. Canada,
27 (5): 943-945.
-----1970b. Interspecific variation of tetrazolium oxidase in
Sebastodes (rockfish). Camp. Biochem. Physiol., 37: 281-285.
33
34
----1972. Electrophoretic investigation of the family Scorpaenidae.
Fish. Bull., 70(2): 403-413.
----1973. Estimate of genetic polymorphism and heterozygosity in
three species of rockfish (genus Sebastes). Comp. Biochem.
Physiol., 44(B): 397-406.
Jordan, D. S., and B. W. Evermann. 1898. The fishes of north and
middle America. Bull. U.S. Nat. Mus., 47(2): 1771-1827.
Jordan, D. S., B. W. Evermann, and H. W. Clark. 1930. Checklist of
the fishes and fishlike vertebrates of north and middle America
north of the northern border of Colombia and Venezuela. Rep.
U. S. Fish. Comm. 1928, App. (10): pp. 670.
Lehninger, A. L. 1976. Biochemistry.
New York, pp. 1104.
Markert, C. L., and G. S. Whitt.
Experientia, 24: 977-1088.
1968.
Worth Publishers, Inc.,
Molecular varieties of isozymes.
Miller, D. J., and R.N. Lea. 1972. Guide to coastal marine fishes
of California. Calif. Fish and Game~ Fish Bull., 157. pp. 249.
Phillips, J. B. 1957. A review of the rockfishes of California
(family Scorpaenidae). Calif. Dept. Fish and Game. Fish Bull.,
104 : pp: 15 8 .
Riggs, A. 1970. Properties of fish hemoglobins. Chapter 6 In;
Hoar, W. S., and D. J. Randal (EDS), Fish Physiology, Vol. 4
Academic Press, New York, pp. 209-252.
Shapiro, A. L., E. Vinuela, and J. V. Maizel, Jr. 1967. Molecular
weight estimation of polypeptide chains by electrophoresis in
SDS-polyacrylamide gels. Biochem. Biophys. Res. Commun., 28(5):
815-820.
Shapiro, S. L., and J. V. Maizel, Jr. 1969. Molecular weight estimation of polypeptides by SDS-polyacrylamide gel electrophoresis:
-further data concerning resolving power and general considerations.
Anal. Biochem., 29: 505-514.
Shaw, R. C.
1~70.
How marty genes evolve?
Biochem. Genet., 4: 275-283.
Tsuyuki, H., E. Roberts, R. H. Lowes, and W. Hadaway. 1968. Contribution
of protein electrophoresis to rockfish (Scorpaenidae) systematics.
J. Fish. Res. Bd. Canada, 25(11): 2477-2501.
Tsuyuki, H., and S. J. Westrheim. 1970. Analysis of the Sebastes
aleutianus-S. melanostomus complex, and description of a new
scorpaenid species, Sebastes caenaematicus, in the northeast
Pacific ocean. J. Fish. Res. Bd. Canada, 27(12): 2233-2250.
35
Weber, K., and M. Osborn. 1969. The reliability of molecular weight
determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem., 244(16): 4406-4412.
Wishard, L. N., F. M. Utter, and D. R. Gunderson. 1980. Stock
separation of five rockfish species using naturally occurring
biochemical genetic markers. Marine Fish. Rev., 42(3-4): 64-73.
APPENDIX A
Fornulas for Preparing Electrophoretic Solutions
Taken from Hoefer Scientific Instruments Catalog (1980)
Stock Solutions
(All solutions should be filtered)
1.
Monomer Solution (30%T 2.7%C)
Acrylamide
Bis
H 0
2
58.4 g
1.6g
to 200 ml
is
neurotoxic and
should be handled
with care.)
~Acrylamide
Store at 4°C in the dark.
2.
Running Gel Buffer (1. 5 M tris-Cl pH 8. 8)
36.3 g Adjust to pH 8.8 with HCl
to 200 ml
3.
Stacking Gel Buffer (0.5 M tris-Cl pH 6.8)
Tris
H 0
2
4.
10% SDS
SDS
H 0
2
5.
50 g
to 500 ml
Initiator (10% ammonium persulfate)
Ammonium persulfate
H o
2
6.
3.0 g Adjust to pH 6.8 with HCl
to 50 ml
0.5 g
to 500 ml
Running Gel Overlay (0. 375 M tris-Cl pH 8.8, 0.1% SDS)
25 ml Solution (2)
l.O ml Solution (4)
to 100 ml
7.
2X Treatment Buffer (0.25 M tris-Cl 6.8, 4% SDS, 20% glycerol,
10% 2-mercaptoethanol)
Tr:ls
SDS
2-mercaptoethanol
H 0
2
2.5 ml Solution (3)
4.0 Solution ~4)
1.0 ml
to 10.0 ml
Divide in aliquots and freeze
36
37
8.
Tank Buffer (0.25 M tris pH 8.3, 0.192 M glycine, 0.1% SDS)
Tris
Glycine
SDS
H20
9.
12 g
57.6 g
40 rnl Solution (4)
to 4.0 liters
Stain Stock (1% Coornassie Blue R-250)
Coornassie Blue R-250
H 0
2
2.0 g
to 200 rnl
Stir and filter
10.
Stain (0.125% Coornassie Blue R-250, SO%
Coornassie Blue R-250
Methanol
Acetic acid
H 0
2
11.
10% acetic acid)
62.5 rnl Stain Stock (9)
250 rnl
50 rnl
to 500 rnl
Destaining Solution I (50% methanol, 10% acetic acid)
Methanol
Acetic• acid
H 0
2
12.
rne~hanol,
500 rnl
100 rnl
to 1.0 liter
Destaining Solution II (7% acetic acid, 5% methanol)
Acetic acid
Methanol
H 0
2
700 rnl
500 rnl
to 10.0 liters
38
Working Solutions
TABLE :l.
Separating
Gel
Stacki_ng
Gel
10%T 2.7%C
4%T 2.7%C
30%T z.-%C(l)
20 ml
2.66 ml
Buffer (2)
15 ml
5.0 m1
Buffer ::3)
10% SDS (4)
H 0
2
-~oniun
persulfate (5)
TEMED
0.6 ml
0.2 ml
24.1 ml
12.2 rnl
300 ul
100 ul
20 ul
10 ul
Separat2ng Gel
To 20 rnl of the 30%T 2.7%C stock solution #1, add 15 ml of
buffer t2,0.6 ml of 10% SDS (#4), 24.1 ml of H o, 300 ul of ammonium
2
persulfa~e
solution (#5), and 20 ul of TEMED.
Stacking Gel
To 2.66 m1 of the 30%T 2.7%C stock solution #1,
buffer
#~,0.2
add 5 ml of
ml of 10% SDS (#4), 12.2 ml of H 0, 100 u1 of ammonium
2
persulfare (#5), and 10 ul of TEMED.