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J. Embryol. exp. Morph. Vol. 19, 1, pp. 95-101, February 1968
With 2 plates
Printed in Great Britain
95
Ultracentrifugal and
electrophoretic analysis of the water-soluble
fraction of chick embryo yolk
By P. CARINCI & L. MANZOLI-GUIDOTTI1
From the Institute of Histology and General Embryology,
University of Bologna
INTRODUCTION
Water-soluble proteins, mainly livetins (a-, /?-, y-), are present in hen egg yolk
(Martin, Vandegaer & Cook, 1957). They represent 5 % of fresh yolk solids
(Saito, Martin & Cook, 1965). During the later stages of incubation the watersoluble proteins (water-soluble fraction, WSF) undergo a marked increase in
relative proportion; after 15 days of incubation they form over 15% and at
18 days over 40% of yolk-residual solids (Saito et ah 1965).
This proportional increase of the WSF is tentatively explained by the passage
of egg-white proteins into yolk (Mclndoe, 1960; Saito et al. 1965). Indeed some
egg-white proteins (ovalbumin, conalbumin and lysozyme) have been found in
the yolk from 14 to 15 days of incubation (Saito & Martin, 1966; Carinci,
Wegelin & Manzoli-Guidotti, 1966). At these stages ovalbumin is present in the
yolk in such a great quantity that it is difficult to detect qualitative and quantitative changes of other proteins without resorting to further fractionations.
We have studied these changes by ultracentrifugal and electrophoretic analysis
of the WSF, of its globulin components and of the egg-white proteins separated
by the same procedures.
MATERIAL AND METHODS
We have used White Leghorn hen fertile eggs provided by the Corticella
agricultural station (Bologna). Two independent sets of experiments were
performed on non-incubated eggs and on eggs after 10, 15 and 21 days of
incubation (38-39 °C).
Yolk was obtained by puncturing the vitelline membrane or yolk sac and was
then diluted with 0-16 M-NaCl (1:2, w/w). Care was taken to avoid contamination of the yolk with egg-white and, for the incubated eggs, with embryonic
fluids from the developing embryo.
1
Authors' address: Institute of Histology and General Embryology, University of Bologna,
Bologna, Italy.
96
P. CARINCI & L. MANZOLI-GUIDOTTI
The WSF was prepared according to Martin et al. as described previously
(Carinci et al. 1966) and then was exhaustively dialysed at 4 °C against 0-16 MNaCl.
Albumen was removed in toto, diluted with 0-16M-NaCl (1:2, w/w), and
carefully homogenized with a Waring Blender.
To separate globulin, aliquots of WSF and albumen were mixed with a
saturated solution of ammonium sulphate to 45-50 % of saturation. The procedure was repeated four times.
The final precipitate was dissolved in M-NaCl (these fractions will be referred
as WSFp and Ap) and dialysed exhaustively against M-NaCl at 4 °C for ultracentrifugal and electrophoretic investigation. In this way we obtained a material
from non-incubated egg albumen free from albumin contaminations (electrophoretic analysis) and with sedimentation properties comparable to those
described by Kaminski (1954).
The yolk and albumen fractions (WSF, WSF P and Ap) obtained at all
stages of incubation were examined, in M-NaCl (pH 6-5) and at 1 % protein
concentration (micro-Kjeldahl), in a Phywe model U 50 L analytical ultracentrifuge at 20 °C and 167 241 g. Calculated sedimentation coefficients (in
Svedbergs, S), referred to 1 % protein concentration, were corrected for temperature, viscosity and partial specific volume. Corrections were not made for
Johnston-Ogston effect or differences in the refractive index increments. The
relative peak areas were estimated from enlarged tracings (x 15).
Electrophoretic analysis was carried out using cellulose polyacetate (Gelmann Sepraphore III, 1 x 6 | in. strips) in tris barbital-sodium barbital buffer,
pH 8-8, ionic strength [i = 0-05, 300 V. The electrophoregrams were stained
with Amido Schwartz (1 % in methanol:acetic acid: water, 45:45:10, v/v) and
then scanned, in some cases, with a Chromoscan densitometer (Joyce-Loebl).
RESULTS
Water-soluble fraction
The ultracentrifugal examination of WSF of non-incubated egg (WSF0)
and at 10 days of incubation (WSF10) yields two peaks after 35 min centrifugation
(Plate 1, fig. a). The sedimentation coefficients permitted us to identify the
faster peak as y-livetin and the slower one as a-+/Mivetin (Saito et al. 1965).
The electrophoretic patterns of WSF0 and WSF10 show the three well-known
zones produced by a-,/?-,y-livetin; in addition we have noticed a fourth zone
at low concentration and with a mobility similar to that of ovalbumin, by
comparison with egg-white (Plate 2, fig. c).
The ultracentrifugal examination of WSF15 also shows two peaks of the same
sedimentation coefficient as those of WSF0 and WSF10, but with much less of
the fast component (see Table 1).
The electrophoretic pattern of WSF15 is more complex. There are 7-8 zones
PLATE 1
/. Embryol. exp. Morph., Vol. 19, Part 1
\
J
A
v
K
Sedimentation patterns of water-soluble fraction (a, b, c), water-soluble fraction precipitate
(e, /»g), albumen precipitate (d), and water-soluble fraction + albumen precipitate (h). See text
for experimental details.
P. CARINCI & L. MANZOLI-GUIDOTTJ
facing p. 96
J. Embryo/, exp. Morph., Vol. 19, Part 1
PLATE 2
Electrophoretic patterns of water-soluble fraction (c, e), water-soluble fraction precipitate (d, f), egg-white (a), egg-white precipitate (b), and water-soluble fraction + albumen
(g). See text for experimental details.
P. CARINCI & L. MANZOLI-GUIDOTTI
Analysis of embryonic yolk
97
with anodic mobihty and one irregular zone with cathodic mobility. This pattern
is qualitatively similar to that of total egg-white (Plate 2, fig. a). Among the
anodic zones, a-livetin and ovalbumin, which is divided into three components
(Fevold, 1951), can be definitely identified. Comparison of the electrophoretic
pattern of the WSF15 with that of egg-white shows that the other anodic zones
have very similar mobilities to those of ovomucoid, G3, G2 and conalbumin,
while comparison with that of WSF0 shows that the 5th band has a mobility
very similar to that of y#-livetin and the 7th band to that of y-livetin.
Table 1. Rate coefficients and relative proportions of protein components
Incubation (days)
WSF
(a)
S
%
(b)
S
%
WSF P
(fl)
S
%
s
%
(c)
0
10
15
21
2-6
79
2-8
80
2-7
95
2-6
100
6-9
21
6-9
20
6-9
5
—
—
2-4
21
30
26
2-7
34
2-9
30
7-6
79
—
—
7-4
74
—
—
71
57
16-2
9
7-5
59
15-4
11
31
95
16-3
5
30
87
15-7
13
3-3
87
16-2
13
—
—
—
A
S
%
(6)
%
S = Svedbergs at 20 °C and 1 % protein concentration; proportion from uncorrected areas of
Schlieren peaks, (a), (b), (c) iindicate individual peaks of the various fractions examined
Analytical ultracentrifugation of WSF21 (1 % protein concentration) shows a
single peak, even after 90min centrifugation (Plate 1, fig. e). A second fast
sedimenting component can be detected in low amounts at 2-5% protein concentration. The electrophoretic pattern of WSF21 is, however, quite similar to
that at 15 days.
Water-soluble fraction precipitate
The WSFp0 and WSF pl0 show two peaks after 30 min centrifugation (Plate 1,
fig. e). Their sedimentation coefficients correspond to those of fast and slow
7
JEEM 19
98
P. CARINCI & L. MANZOLI-GUIDOTTI
sedimenting components of the total WSF, but the slow component is in a
lower proportion (see Table 1).
Electrophoretically there is evidence of /?- and y-livetin, the latter divided
into two bands (Mok & Common, 1964), in the following proportions: /?livetin 15%, y-livetin 31 %, y-livetin 50%. a-livetin was not demonstrable.
We have therefore in WSF P a loss of a-livetin and a strong reduction in the
concentration of /?-livetin. This is due to the solubility properties of these
proteins at the (NH^SC^ concentration employed by us (Gorini & Lanzavecchia, 1955; Williams, 1962). This explains the diminution of the relative
proportion of the slow component in the ultracentrifugal pattern.
WSFp at 15 and 21 days yields the same ultracentrifugal pattern. On reaching
speed, two peaks appear; the slower peak divides into two peaks after 15 min
centrifugation (Plate 1, figs. / , g). Rate coefficients and relative proproportion
of the three components are given in Table 1.
Electrophoretic examination of both WSF pl5 and WSF p21 shows two anodic
migration bands and, inconsistently, one cathodic migration band (Plate 2,fig./ ) .
The cathodic migration component has a mobility similar to that of lysozyme.
One anodic migration component moves as y-livetin and the other moves a bit
faster; a- and /?-livetin are not present.
Albumen precipitate
The albumen precipitate in non-incubated eggs and at 10 and 15 days incubation exhibits two peaks by ultracentrifugal analysis (Plate 1, fig. d). The
sedimentation rate and relative proportions are given in Table 1.
Electrophoretic analysis demonstrates two zones of moderate anodic migration, which presumably correspond to the G 3 and G4 fractions of albumen. A
cathodic migration zone is not consistently found (Plate 2, fig. b).
Water-soluble fraction + total albumen
WSF0 was combined with non-incubated egg total albumen to give an ovalbumin final concentration of 40 % of total proteins (Carinci et al. 1966).
The electrophoretic pattern of this combination (WSF0 + Ao) is quite similar
to that of WSF15 and WSF21 (7-8 zones of anodic migration) Plate 2, fig. g).
With analytical ultracentrifugation only one peak (S = 2-9) is observed, as
for WSF21.
The precipitate obtained from this mixture (WSFQ + A,,) using (NH4)2SO4
dissolved in M-NaCl, shows two peaks after 14 min centrifugation. The slower
peak later splits into two peaks (Plate 1, fig. h). Sedimentation coefficients of
these components are respectively S = 2-2, 4 8 % ; S = 7-3, 4 4 % ; S = 15-1,
8%. These sedimentation coefficients are in close agreement with those of
WSFp at 15 and 21 days incubation. Electrophoresis gives three anodic migration bands on staining with Amido Schwartz.
Analysis of embryonic yolk
99
DISCUSSION
In the first incubation period the qualitative and relative quantitative protein
composition of chick embryo yolk WSF remains unchanged. Indeed, the
electrophoretic and ultracentrifugal patterns of WSF10 are very similar to those
of WSF0 with regard to both total WSF and the globulin fractions.
These are in agreement with previous observations (Biagi, 1964; Saito &
Martin, 1966; Carinci et ah 1966). However, the electropherogram presence of
a component of WSF0 migrating as ovalbumin, heretofore unreported, is to be
noted.
From 15 to 21 days incubation the yolk protein composition undergoes
marked changes. By electrophoresis on cellulose acetate WSF shows 4-5 new
zones with anodic mobility .The second, third and fourth zones are due to the
presence of ovalbumin readsorbed from albumen.
The examination of the precipitate obtained by (NHJaSC^ treatment (globulins) further clarifies the WSF composition during the second period of incubation.
These components thus separated have different ultracentrifugal properties
from those of WSF0 globulins. A new component is present (S = 15-16), not
previously shown, with a sedimentation coefficient value in the range of albumen
macroglobulin. It is very likely that the macroglobulin is reabsorbed from eggwhite into the yolk but it cannot be excluded that this may be an aggregate of
yolk protein. Another component is y-livetin (5 = 7). Finally, the slow sedimentation component (S = 2-3) in the last period of incubation does not
correspond to /?-livetin, which it did in the early phase of incubation as demonstrated by electrophoresis.
In summary, the changes in the yolk WSF observed in the final phase of incubation are due partly to the selective utilization of yolk proteins (disappearance
of /#-livetin) and partly to the appearance of new proteins absorbed from eggwhite.
Almost all egg-white albumins and globulins are found in the yolk. These
represent the major proportion of yolk protein in the last phase of incubation as
demonstrated by the relative quantitative values.
It is therefore probable that a large part of the reserve albumen protein is
reabsorbed by the chick embryo after passage into the yolk. Since yolk is rich
in soluble enzymes (see Bellairs, 1964), it is possible that the reserve proteins are
broken down into simple units, especially amino acids. Indeed, the active
absorption of amino acids by yolk-sac entodermic cells is well established
(Holdsworth & Hastings Wilson, 1967).
Since small quantities of albumen proteins are found in the circulating fluids
of the developing chick embryo, it is possible that their use occurs in distinct
ways in response to embryonic requirements.
7-2
100
P. CARINCI & L. MANZOLI-GUIDOTTI
SUMMARY
1. Ultracentrifugal and electrophoretic analysis of chick embryo yolk watersoluble fraction (WSF) has been carried out using fertile non-incubated eggs and
at 10, 15 and 21 days incubation.
2. Ultracentrifugal and electrophoretic analysis of WSF globulins has been
carried out at the same incubation stages.
3. The ultracentrifugal and electrophoretic patterns of both the WSF and its
globulin components at 10 days incubation are very similar to those observed in
non-incubated eggs.
4. Ultracentrifugal and electrophoretic patterns at 15 and 21 days incubation undergo marked changes due to the appearance of egg-white proteins
in the yolk.
5. The significance of these findings in relation to embryogenesis is discussed.
RIASSUNTO
Ricerche sul vitello delVembrione di polio: analisi alVultracentrifuga
ed alVelettroforesi della frazione idrosolubile
1. E' stata studiata, mediante analisi all'ultracentrifuga analitica ed all'elettroforesi, la composizione della frazione idrosolubile del vitello dell'uovo di
polio non incubato ed al 10°, 15° e 21° giorno di incubazione.
2. E' stata esaminata inoltre, con le medesime techniche, la composizione
delle globuline della frazione idrosolubile per i medesimi stadi di incubazione.
3. II quadro sedografico ed elettroforetico della frazione idrosolubile in
toto e delle sue componenti globuliniche in 10° giorno di incubazione e sovrapponibile a quello osservato nell'uovo non incubato.
4. In 15° e 21° giorno il quadro sedografico ed elettroforetico e profondamente modificato in parte per la comparsa di proteine provenienti dall'albume.
5. II significato di tali dati ai fini delPembriogenesi viene discusso.
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Analysis of embryonic yolk
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(Manuscript received 3 August 1967)