Changes in the protein secondary structure of hen’s egg yolk determined
by Fourier transform infrared spectroscopy during the first eight
days of incubation
Sabrina Lilienthal, Astrid M. Drotleff,1 and Waldemar Ternes
Institute of Food Toxicology and Analytical Chemistry, Department of Analytical Chemistry, University of
Veterinary Medicine Hannover Foundation, Bischofsholer Damm 15/ Building 123, 30173 Hannover, Germany
able changes in secondary structure. However, it is unlikely that these changes were provoked by structure
changes of the proteins originally present in plasma;
instead, the physiological influx of albumen into the
yolk sac was expected to play an important role in
the protein modifications of egg yolk, as was shown
both by FTIR and IEF of the water-soluble egg yolk
proteins. Moreover, FTIR was used to determine the
naturally occurring proportions (%) of the secondary
structure elements in egg yolk and its 3 fractions on
d 0 of incubation. The granules fraction mainly consisted of a mixture of inter- and intramolecular β -sheets
(57.04% ± 0.39%). The plasma fraction was found to
consist mainly of α-helices (43.23% ± 0.27%), whereas
LDL was composed almost exclusively of intramolecular
β -sheets (67.36% ± 0.56%) or β -turns, or both. On
the other hand, whole egg yolk was mainly composed
of intermolecular β -sheets (39.77% ± 0.48%), potentially indicating molecular interchanges between the
individual fractions.
ABSTRACT In this study, incubation-induced alterations in the protein secondary structures of egg yolk
and its major fractions (granules, plasma, and lowdensity lipoproteins [LDL]) were monitored during the
first 8 d of embryogenesis using Fourier transform infrared spectroscopy (FTIR) and isoelectric focusing
(IEF). Two factors potentially connected with egg
yolk protein secondary structure changes were evaluated, i.e., the pH value of incubated egg yolk, and
phosvitin, an important egg yolk protein assumed to
play an important role in hematopoiesis as the iron
carrier during early embryogenesis. However, neither
the significant increase in pH value (6.07 to 6.92) of
egg yolk during incubation of fertilized eggs, nor the
release of iron from phosvitin were found to be directly related to the changes in protein secondary structure in egg yolk and its fractions. FTIR showed that
the protein conformation in whole egg yolk, granules,
and LDL was stable during incubation, but separate
evaluation of the plasma fraction revealed consider-
Key words: Fourier transform infrared spectroscopy (FTIR), egg yolk fractions, protein secondary structure,
incubation
2015 Poultry Science 94:68–79
http://dx.doi.org/10.3382/ps/peu051
INTRODUCTION
is composed of approx. 48% water, 33% lipids, and
17% proteins. The 2 egg yolk fractions—plasma and
granules—contain the major egg yolk proteins and
lipoproteins. The plasma contains the water-soluble
fraction, namely the livetins, whereas the granules
are mainly comprised of the lipovitellins (as parts
of high-density lipoprotein, HDL), phosvitins, and
a small amount of low-density lipoproteins (LDL).
Egg yolk provides the developing embryo with lipids,
proteins, vitamins, minerals, and the maternal antibodies that are indispensable for the first immune
defense of the embryo, as reviewed by Burley and
Vadehra (1989). During incubation, the yolk, its major fractions, and the individual proteins necessarily
have to change both qualitatively and quantitatively
due to specialized remodeling processes during embryonic development. Mann and Mann (2008) recently
identified 119 proteins from chicken egg yolk, some
of which derived from egg white, and evaluated their
An avian egg is the largest cell that contains in addition to genetic information all proteins needed as a
source of amino acids for the growth of the developing
embryo. This growth takes place outside of the maternal body, protected only by a hard shell and supported
by the needed exchange of gases, thermal energy, and
occasional turns. To elucidate the functions of albumen
proteins in embryogenesis, during past decades, there
has been great interest in changes in albumen composition during embryonic development (Moran, 2007;
Sugimoto et al., 1999). Similarly, egg yolk has been
studied very closely under various aspects. Egg yolk
C 2015 Poultry Science Association Inc.
Received June 19, 2014.
Accepted October 21, 2014.
1
Corresponding author: [email protected]
68
YOLK PROTEIN STRUCTURE CHANGES DURING INCUBATION
distribution between the plasma and the granules fractions. Among the diversity of yolk proteins, the phosphoprotein phosvitin is of particular interest because
it forms a protein-like complex with iron, which is utilized for hemoglobin formation by the chick embryo.
This process is already apparent at the d 2 of incubation and is macroscopically visible in the vascularization of egg yolk with red blood vessels (Bruns and
Ingram, 1973). Phosvitin naturally binds up to 95% of
the iron present in yolk (Greengard et al., 1964; Saito
et al., 1965) and evidently represents an important iron
reserve for the growing embryo (Burley and Vadehra,
1989). Although many biological and chemical mechanisms proceeding during incubation have been elucidated, little is yet known about the early phase of embryonic development with regard to protein changes on
a molecular level in the remaining egg yolk.
Fourier transform infrared spectroscopy (FTIR) is
a useful instrument for studying the secondary structure of proteins by analyzing the amide I band (1,700
to 1,600 cm−1 ), which is particularly affected during
conformational changes in protein secondary structure
(Barth and Zscherp, 2002). Recently, the secondary
structures of native and differently treated egg yolk
samples were investigated by Ge et al. (2011) and
Blume et al. (2015) using the FTIR technique. FTIR
was shown to be suited for monitoring modifications
of the protein secondary structure of egg yolk caused
by increasing temperature, pasteurization, freeze- and
spray-drying, or alternating ionic strength. However, to
the best of our knowledge, FTIR has thus far not been
applied to investigate structure alterations of egg yolk
and its 3 major fractions during the early phase of embryonic development.
It has been proposed in numerous spectroscopic studies that the egg yolk protein phosvitin has different conformations when dissolved at neutral pH and
under varying conditions, such as pH values, thermal influences, or the release of iron, as reviewed by
Samaraweera et al. (2011). However, there is no study
available comparing the secondary structure of ironbound and iron-free phosvitin by means of FTIR that
would clarify whether phosvitin changes its conformation when performing its expected role as the iron carrier of egg yolk during embryonic development.
A promising approach for a better understanding of
the changes in the protein secondary structure during embryonic development is to combine the results
of FTIR with those from the conventional method of
isoelectric focusing (IEF). As demonstrated by Ternes
(1989), the water-soluble proteins of egg yolk (only
livetins without lipoproteins) can be determined using
the IEF technique. Considerable research has been conducted on many albumen proteins in the course of incubation (Qiu et al., 2012, 2013), but no results have
been reported thus far on the isoelectric focusing of
the water-soluble proteins of egg yolk during embryonic
development. During early incubation, changes in egg
yolk proteins may occur that may affect the isoelectric
69
points of egg yolk proteins. Because IEF is known to
separate and focus proteins on the basis of differences
in their isoelectric points into highly resolved bands,
as reviewed by Sommer and Hatch (2009), we chose
the IEF technique as a method for confirming concomitantly our results obtained by FTIR.
The aim of the study was to evaluate for the first
time the ongoing incubation-induced changes in egg
yolk, its 3 major fractions, and the associated proteins during the early phase of avian embryonic development. It should be emphasized that this study
was concerned with protein changes on a molecular
level rather than with embryogenesis itself. To reach
the aims of the study, 1) we determined the pH values
of yolk from fertilized and unfertilized eggs during an
8-d incubation, and the protein secondary structure of
egg yolk samples on various days of incubation to reveal a potential relation; 2) we investigated the release
of iron from phosvitin and its effect on the secondary
structure of phosvitin to evaluate whether the apparent phosvitin-involving hematopoiesis (evident during
early embryogenesis) is accompanied by protein conformation changes; and 3) we also explored the changes
in protein secondary structure of the 3 major fractions
of whole egg yolk during early embryonic development
using FTIR. These results, then, were put in relation
to those from a conventional method for the analytical evaluation of the isoelectric point (pI) of proteins
(IEF). The findings are expected to further elucidate
the changes in egg yolk proteins of fertilized eggs during embryogenesis and provide a better understanding
of the molecular mechanisms of protein modification in
the egg development process.
MATERIALS AND METHODS
Chemicals and Reagents
Distilled water (pH 6.70) was prepared with a
TKA-GenPure system (TKA, Niederelbert, Germany).
Freeze-dried phosvitin (from chicken egg yolk), ovalbumin, ovotransferrin, and lysozyme (all from hen
egg white) were obtained from Sigma-Aldrich (Hamburg, Germany) and used without further purification. Nitric acid (Rotipuran Supra 69%), perchloric acid (Rotipuran Supra 70%), trichloroacetic acid
(20%), ethylenediaminetetraacetic acid disodium salt
dihydrate (EDTA), and iron standard solution for
AAS (Single-Element AAS-Standard Eisen) were purchased from Carl Roth (Karlsruhe, Germany). The
electrophoretic material was acquired from Serva Electrophoresis GmbH (Heidelberg, Germany). All solvents
were of analytical reagent grade.
Sample Preparation
Egg yolk. Unfertilized table eggs (determined by egg
candling, when indicated; quality category A, medium
size) were purchased from a local supermarket and
70
LILIENTHAL ET AL.
stored in a cooling chamber at 6◦ C until use, for no
longer than 1 wk.
Fertilized eggs (VALO-SPF, Lohmann Tierzucht,
Cuxhaven, Germany) were incubated at 37.5◦ C and at
60% humidity at the Clinic for Poultry, University of
Veterinary Medicine Hannover, Foundation, Germany
for up to 8 d. On d 0, 3, 4, 5, and 8, we took 3 eggs
from each group and stored them in a cooling chamber
at 6◦ C for 24 h to terminate the embryos’ lives prior to
sampling. The unfertilized table eggs were then manually broken and the egg yolks separated. Each yolk was
carefully washed with distilled water and rolled on a filter paper to remove albumen and the chalazae adhering
to the vitelline membrane. After the vitelline membrane
was punctured with a cannula, the yolk was collected
and pooled in a beaker cooled with iced water (table
egg sample).
For the sample preparation of the fertilized eggs,
the procedure changed after d 0 of incubation, due
to the fragility of the vitelline membrane. From d 3
on, the shell of eggs was broken on top and discarded
together with the embryo. Egg yolk and albumen were
separately poured carefully into a beaker cooled with
iced water (fertilized egg sample). The egg yolk samples
were then freeze-dried (FD) as described by (Jaekel
et al., 2008) (table eggs FD and fertilized eggs FD samples). The sample size was n = 3 for each day.
Granules, plasma, LDL. Granules, plasma, and
LDL were separated from egg yolk according to the procedure of Ulrichs and Ternes (2010) but with a modified
centrifugation rate and time (20,000 × g for 30 min at
4◦ C) for a higher purity of the 3 fractions. The sediments, referred to as granules, were washed 3 times
with distilled water. After each washing, the granules
were centrifuged as above. The supernatants, referred
to as plasma, were collected in another beaker until further processing for the separation of LDL. Aliquots of
the 3 fractions were immediately used for FTIR spectroscopy; the remains were freeze-dried for preservation
as above. The sample size was n = 4 for each day.
Plasma + albumen. Egg yolk plasma samples of unfertilized, nonincubated table eggs were fractionated as
described above. Fresh albumen was added in different
concentrations (5, 10, and 20%) to the plasma samples
and homogenized. Plasma without the addition of albumen was used as a reference. The samples were investigated spectroscopically (see below). The sample size
was n = 4 for each experiment.
pH Measurements
Unfertilized table eggs (table egg sample) and fertilized eggs (VALO-SPF, Lohmann Tierzucht, Cuxhaven,
Germany) (fertilized egg sample) were incubated as described above. The pH of the egg yolks (table egg and
fertilized egg sample) was measured using a Knick 766
Calimatic pH meter with a SE 102 N pH sensor (Knick
GmbH, Berlin, Germany).
Phosvitin Dialysis
Phosvitin (25 mg/500 μL distilled water) was dialyzed according to the method of Taborsky (1963) with
the following modifications (phosvitin dialysate sample): QuixSep microdialysis capsules (Carl Roth, Karlsruhe, Germany) were used, and the dialysis was carried
out with stirring over a period of 3 d at 4◦ C.
Atomic Absorption Spectroscopy
For determination of the iron concentration,
phosvitin (25 mg/500 μL distilled water) was weighed
into Teflon cups and placed in a drying cabinet at
103 ± 2◦ C overnight. All phosvitin samples (phosvitin
reference and phosvitin dialysate sample) were wet
ashed for further analysis with flame atomic absorption spectroscopy (F-AAS) (PU9100x, Philips GmbH,
Hamburg, Germany) according to the procedure of
Bäckermann and Ternes (2008).
For calibration, the standard addition technique was
used. The calibration range for iron determination was
from 0.2 to 3.6 mg/L. The samples were all within the
linear calibration range.
FTIR Spectroscopy
For FTIR analysis, 200 mg of each native (not
freeze-dried) sample, except albumen and plasma, were
dispersed in 800 mg distilled water. Albumen and
plasma were analyzed without further dilution (granules, plasma, albumen samples). Freeze-dried samples
of egg yolk and LDL were reconstituted to the same
degree of dilution as the native samples (fertilized egg
yolk FD and fertilized LDL FD samples). A preliminary test revealed no differences between freeze-dried
and native egg yolk samples (data not shown).
FTIR was performed similarly to the method of
Matheus et al. (2006) using a Tensor 27 spectrometer (Bruker Optik GmbH, Ettlingen, Germany) with a
BioATR (Attenuated Total Reflectance) II cell (Bruker
Optik GmbH) for spectra recording and measurement
of dissolved samples. The cell was purged with dry air
and connected to a Haake thermostat (Thermo Electron GmbH, Karlsruhe, Germany). The temperature of
the samples was adjusted to 25◦ C. For each sample, 5
replicates were automatically taken, averaged from 128
scans at 4 cm−1 resolution (zero filling factor: 2). Distilled water was measured as a reference.
OPUS Software Version 6 (Bruker Optik GmbH) was
used for spectra evaluation. Every sample measurement
was replicated 4 times, unless stated otherwise. The
spectra were atmospheric vapor compensated and vector normalized prior to averaging. Vector normalization
and Fourier self-deconvolution were applied to the average of each sample. The second derivative was used to
ascertain positions of the absorbance peaks located in
the amide I region. To reveal differences in proportions
71
YOLK PROTEIN STRUCTURE CHANGES DURING INCUBATION
of the secondary structure elements in the major egg
yolk fractions, the peak areas of the second-derivative
spectra were determined by integration. The integration was carried out from maximum to maximum of
each peak. The total area of all integrated peaks from
each spectrum was defined to be 100%. The individual
peak areas were expressed as proportions (%) of the
corresponding secondary structure elements.
Isoelectric Focusing
Isoelectric focusing was carried out according to the
method of Ternes (1989) with the following modifications: freeze-dried egg yolk samples (fertilized egg
yolk FD sample), freeze-dried albumen samples (albumen FD sample), and reference substances of albumen
proteins (ovalbumin, ovotransferrin, lysozyme sample)
were stirred with distilled water for 30 min and centrifuged at 14,000 × g for 5 min at 4◦ C. Electrophoresis was conducted at 5◦ C with a voltage gradient from
500 to 2,000 V within 5 h; this voltage was held until the amperage reached a value of 1 mA (final focusing). The gels (Servalyt precotes wide-range pH 3–10,
SERVA Electrophoresis GmbH, Heidelberg, Germany)
were stained with Coomassie blue (Serva blue W solution, SERVA Electrophoresis GmbH, Heidelberg, Germany) according to the manufacturer’s instructions.
The pH gradient was measured with a standard protein
marker (IEF Marker 3–10, Liquid Mix, SERVA Electrophoresis GmbH, Heidelberg, Germany), of known individual pI values.
Statistical Analysis
Statistical analysis was performed using GraphPad
Prism version 6.04 (GraphPad Software Inc., San Diego,
CA). All reported values represent the mean ± SEM,
calculated with Prism or Excel 2010 (Microsoft Corporation, Redmond, WA). These values were subjected to
a 2-way ANOVA followed by the Tukey post-hoc test in
the case of the pH measurements or a 1-way ANOVA
followed by the Tukey post-hoc test in the case of the
FTIR measurements. Where there were only 2 groups
to be compared, an unpaired t-test was performed.
Values were considered to be statistically different at
P < 0.05.
RESULTS AND DISCUSSION
pH Measurements
The effect of incubation time on the pH value of yolk
from unfertilized table eggs compared to that of fertilized eggs is shown in Table 1. Irrespective of the condition of the eggs, the pH values of table eggs and fertilized eggs were not significantly different on d 0 (P >
0.01). The pH of both types of yolk increased during d 0
to 8 of incubation. The pH of egg yolk from unfertilized
table eggs stayed slightly acidic (6.04 to 6.20), whereas
the pH of egg yolk from fertilized eggs increased toward
neutrality (6.07 to 6.92). In table eggs, changes in pH
values were non significant. In the case of the fertilized eggs, there were several highly significant changes
in pH values over time. Clearly, incubation of fertilized eggs resulted in an average yolk pH significantly
higher than that of table eggs from d 3 on (P < 0.01).
On the remaining days, the same level of significance
was found between yolk pH of fertilized eggs and yolk
pH of table eggs (P < 0.0001). Considering the wellknown influence of differing pH values on the secondary
structure of proteins (Hu and Du, 2000; Taborsky, 1968;
Yasui et al., 1990), it was important for the subsequent
FTIR studies to recognize the significance of increasing
pH value in the yolk of the fertilized hen’s egg in comparison to the pH value of yolk from table eggs during
incubation.
In good accordance with reported results on yolk
from incubated fertilized and nonfertilized eggs (Hatta
et al., 2001), our findings demonstrate that the pH of
egg yolk from table eggs stayed slightly acidic, whereas
that of egg yolk from fertilized eggs gradually went from
its original acid state to a neutral state from d 0 to 8
of incubation. The pH value changes were presumably
due to the albumen of incubated eggs, which is alkaline (Hatta et al. 2001) and has been reported to be
taken up into the yolk sac in an early stage of incubation (Yoshizaki et al., 2002). Babiker and Baggott
(1992) and Simkiss (1980) described the migration of
Table 1. pH values of yolk from table eggs compared to pH values of yolk from fertilized
eggs as a function of incubation time.
Day
Sample
Table eggs
Fertilized eggs
P
0
3
4
5
8
6.04 ± 0.017
6.03a ± 0.03
NS
6.16 ± 0.015
6.42b ± 0.024
6.15 ± 0.009
6.46b ± 0.016
6.17 ± 0.014
6.51b ± 0.059
6.20 ± 0.019
6.92c ± 0.077
∗∗
∗∗∗∗
∗∗∗∗
∗∗∗∗
Mean ± SEM of 4 independent observations of each group at each corresponding day. Within a
row, values with different superscripts differ significantly. Within a column, significant differences
(P < 0.01; P < 0.0001) are marked with asterisks (∗ ).
∗∗
= P < 0.01.
∗∗∗∗
= P < 0.0001.
NS = not significant.
72
LILIENTHAL ET AL.
water from albumen toward the yolk sac during the
first 4 to 6 d of incubation. According to those authors, this shift leads to a significant increase in the
volume of the yolk sac contents, and it is also accompanied by an increase in the pH value of yolk caused
by an alkaline liquid component of the albumen and
possibly a small amount of albumen itself. The liquid
follows the osmotic gradient due to the previous movement of sodium ions from the albumen toward the yolk
sac, as reported by Stern (1991). We thus support the
hypothesis that the observed increase in the pH of the
fertilized eggs was related to the migration of minerals
(i.e., sodium ions), small albumen proteins, and water
from the slightly alkaline albumen to the yolk. In the
context of the abovementioned information, our findings regarding the general relation between pH value,
incubation time, and condition of eggs appeared plausible and thus had to be taken into consideration in the
following FTIR studies on factors influencing protein
secondary structure.
Phosvitin Dialysis
Because phosvitin is known to bind almost 95% of
the iron present in egg yolk (Greengard et al., 1964;
Taborsky, 1980), it is considered to be of great importance in embryonic development for hemoglobin formation by the chick (Osaki et al., 1975). Hence, it was
of particular interest for the next FTIR experiments
to discover whether the release of iron from phosvitin
could have a visible effect on the protein secondary
structure of egg yolk and its major fractions. However,
as no method for the purification of phosvitin from incubated eggs has been developed thus far, we decided to
use a phosvitin standard substance for the following experiment. Dialysis of phosvitin reduced its iron content
from 1.98 ± 0.061 to 0.114 ± 0.032 μg/mg, resulting
in a total reduction of almost 95%, significantly lower
than before dialysis (P < 0.0001). In accordance with
the literature (Taborsky, 1963), our results clearly show
that the iron content of phosvitin can be significantly
reduced by dialysis with 0.01 M EDTA. Compared to
earlier results obtained by Albright et al. (1984) and
Taborsky (1963), it is clear that the dialysis conducted
by us led to an even greater release of iron than that
achieved by those authors, resulting in the almost ironfree phosvitin status, which was particularly essential
for subsequent FTIR measurements.
FTIR Spectroscopy
Protein secondary structures in phosvitin reference samples and iron-free phosvitin samples.
The most informative part of the FTIR spectrum
regarding the secondary structure of proteins is the
amide I region between the wave numbers 1,600 and
1,700 cm−1 . Figure 1 shows the second-derivative FTIR
spectra of phosvitin in aqueous (H2 O) solution be-
Figure 1. Second-derivative FTIR spectra (25◦ C) of phosvitin before (phosvitin reference sample, dotted line) and after dialysis with
0.01 mol/L EDTA (phosvitin dialysate sample, black line). All samples
dispersed in distilled water. ∗ , asymmetric shoulder, which indicates an
increased proportion of intramolecular β -sheets compared to phosvitin
reference.
fore (phosvitin reference sample) and after dialysis
(phosvitin dialysate sample) with 0.01 M EDTA. The
peaks’ positions are displayed. The reference spectrum
showed one distinct peak at 1,648 cm−1 , which was assigned to random-coil or α-helical structures, and one
other additional component at 1,674 cm−1 , which was
assigned to β -turns (Kong and Yu, 2007). No major
changes in secondary structure took place after dialysis,
but a shift was observed in the absorbance units from
1,648 to 1,654 cm−1 , indicating random coils that partly
converted into α-helical structures. It is also obvious
that an asymmetric shoulder (marked by an asterisk
[∗ ]) appeared on the low frequency side of the spectrum,
indicating an increased proportion of intramolecular β sheets compared to the phosvitin reference.
As long known from the literature, phosvitin binds
ferric ions (Fe3+ ) strongly and extensively but also liberates iron when incubated with chelating agents such
as EDTA (Taborsky, 1963) or during hemoglobin formation by the chick in embryonic development (Osaki
et al., 1975). However, as can be seen from a recent
review by Samaraweera et al. (2011), the literature on
the protein secondary structure of phosvitin is somewhat contradictory. Renugopalakrishnan et al. (1985)
reported that the structure of phosvitin consisted of αhelices, β -sheets, and β -turns, but because their measurements were made by low-resolution FTIR, those results may not be as accurate as ours. On the other hand,
Prescott et al. (1986) described the secondary structure
of phosvitin determined by laser Raman spectroscopy
to be deficient in both α-helix and conventional β sheet conformations and thus assumed it to exist as an
unordered conformation. Our findings suggest that in
H2 O solution at neutral pH, the predominant structures
of phosvitin were random coils or α-helices, or both.
73
YOLK PROTEIN STRUCTURE CHANGES DURING INCUBATION
Figure 2. Second-derivative FTIR spectra (25◦ C) of freeze-dried,
reconstituted egg yolk samples from fertilized eggs in H2 O solution
drawn at different stages of incubation (d 0, 3, 4, 5, 8). The black and
stippled bold lines show the spectrum at the beginning (d 0) and the
end (d 8) of incubation, respectively. Day 4 is shown as a dotted line.
The intermediate spectra are displayed as thin lines. Black lines are
drawn above the integrated peak areas for clarification of the calculation for the secondary structure proportions (%). (n = 3 for each
day).
Additional components, such as β -turns (1,674 cm−1 )
and intramolecular β -sheets (marked by an asterisk
[∗ ]) were observed, but these contributed to only a
small portion of the protein secondary structures. As
known from the literature (Pelton and Mclean, 2000),
it is difficult to differentiate between α-helices and
random coils in the region around 1,650 cm−1 in nondeuterated solutions by FTIR measurement, as used in
the present study. In a study of phosvitin in D2 O by
Losso et al. (1993), those authors explicitly assigned
the components at 1,648 cm−1 to unordered conformations because in D2 O, it is possible to distinguish
clearly between random-coil and α-helical structures
(Byler and Susi, 1986; Surewicz and Mantsch, 1988).
Hence, we consider our assumption to be plausible that
phosvitin was mainly composed of random coils and
β -turns in solution at neutral pH before dialysis. Supportive evidence is provided by the asymmetric shoulder on the low frequency side of the spectrum, which
appeared after the dialysis while a shift to higher wave
numbers was observed, suggesting a partial conversion
from random coils to intramolecular β -sheets and αhelices (Figure 1). This could be due to the fact that
a protein in a random-coil structure is highly dynamic
with no secondary or tertiary preference. After the release of iron, the stabilization of the secondary structure by metal ion complexation is lost, resulting in a
partial conversion into β -sheets and α-helices as reported by Yasui et al. (1990). Because phosvitin is part
of it, the importance of these findings is further discussed below in the section dealing with the granules
fraction.
Protein secondary structures of egg yolk samples from fertilized eggs at different stages of incubation. Figure 2 shows the second-derivative FTIR
spectra of freeze-dried egg yolk from fertilized eggs at
various stages of incubation (0, 3, 4, 5, 8 d) in H2 O
solution. Most informative in terms of protein changes
during incubation were the peaks’ wave numbers, correlated with specific secondary structure content, as the
numbers were in good agreement with those of Blume
et al. (2015). All spectra showed distinct peaks at 3 independent wave numbers, indicating different types of
protein secondary structures, as shown in Table 2. The
peak at 1,621 cm−1 was assigned to intermolecular β sheets, whereas, according to Matheus et al. (2006), the
peak at 1,652 cm−1 was found to correspond to α-helical
and random-coil structures. The peak in the spectral
region around 1,685 cm−1 was assumed to arise from
β -turns (1,685 ± 2.0 cm−1 ) located within this area,
as reviewed by Kong and Yu (2007). No shift in wave
numbers or reformation of peaks was observed during
incubation, and no significant changes were registered
in comparison to the other days of incubation, as determined by integration of the peak areas of the bands at
1,621, 1,652, and 1,685 cm−1 . It should be emphasized
that the differences in intensities and absorbance units
between the individual days of incubation did not appear randomly but were detected in the same sequence
for each individual experiment (n = 3).
Because no significant differences in wave numbers or
peak areas were recorded in whole egg yolk, and given
that egg yolk is a highly complex medium composed
of various proteins (Anton, 2013), we decided to study
its different fractions individually, namely, the granular
and plasma fractions. In this way, we expected to visualize potential changes in secondary structure during
Table 2. Proportions (%) and band positions of egg yolk and its 3 major fractions determined on d 0 of
incubation by means of FTIR and integration of the peak areas of the second-derivative spectra.
Proportions% ±% (band position [cm−1 ])
α -helices
Egg yolk
Granules
Plasma
LDL
37.14 ± 0.23 (1,652)
25.61 ± 0.27 (1,654)
43.23 ± 0.27 (1,652)
nd
Intermolecular β -sheets
Intramolecular β -sheets
39.77 ± 0.48 (1,621)
nd
57.04 ± 0.39 (1,625)
29.15 ± 0.34 (1,616)
nd
0.49 ± 0.17 (1,616)
67.36 ± 0.56 (1,635)
Mean ± SEM of 4 independent observations of each egg yolk fraction.
nd = not detected.
β -sheets or β -turns, or both
23.09
17.36
27.62
32.15
±
±
±
±
0.38
0.19
0.14
0.48
(1,685)
(1,689)
(1,683)
(1,687)
74
LILIENTHAL ET AL.
incubation that could have previously been masked by
the complexity of whole egg yolk.
Protein secondary structures in the granules
fraction of egg yolk from fertilized eggs at different stages of incubation. To further characterize the
potential changes in protein secondary structure during incubation, the granular fraction of egg yolk from
fertilized eggs was investigated by FTIR at different
stages of incubation (0, 3, 4, 5, 8 d), as shown in
Figure 3. The second-derivative FTIR spectra showed
distinct peaks at 3 independent wave numbers, as
shown in Table 2. According to Matheus et al. (2006),
the intermolecular β -sheet structures in the amide I
band range from 1,615 to 1,625 cm−1 , whereas the intramolecular β -sheet structures range from 1,612 to
1,642 cm−1 . Even though the second-derivative procedure enhanced the possibility of resolving the overlapping secondary structural components contributing
to the amide I band, it was difficult to clearly assign
the peak at 1,625 cm−1 to a single secondary structure element. It may thus originate from a mixture of
inter- and intramolecular β -sheets, whereas the peak at
1,654 cm−1 was found to correspond to α-helices and
random coils. The peak in the spectral region around
1,689 cm−1 was considered to arise from more than one
secondary structure component, since it was asymmetric in shape and clearly showed the formation of a slight
shoulder on the low frequency side of the spectrum.
Hence, this peak was presumably composed of β -turns
(1,685 ± 2.0 cm−1 ) and β -sheets (1,691 ± 2.0 cm−1 ),
both of which are located within this area, as reviewed
by Kong and Yu (2007). During incubation, no shift
was observed in wave numbers or reformation of peaks.
Figure 3. Second-derivative FTIR spectra (25◦ C) of granule samples (not freeze-dried) from egg yolk of fertilized eggs in H2 O solution
drawn at different stages of incubation (d 0, 3, 4, 5, 8). The black and
stippled bold lines show the spectrum at the beginning (d 0) and the
end (d 8) of incubation, respectively. Day 4 is shown as a dotted line.
The intermediate spectra are displayed as thin lines. (n = 4 for each
day).
As known from the literature, the granular fraction
is utilized during incubation most rapidly and completely of all 3 egg yolk fractions (Saito et al., 1965).
Moreover, granules consist of 16% phosvitin, comprising the majority of phosvitin, a phosphoprotein that
strongly binds up to 95% of the iron present in egg yolk
(Greengard et al., 1964; Taborsky, 1980). This mineral has thus been assumed to be of great importance for early embryonic development (Burley and
Vadehra, 1989), and this hypothesis has been supported by Bäckermann and Ternes (2008), who reported statistically significant F-AAS results indicating a 21% lower iron content in the granular
fraction of fertilized 5-d incubated eggs than that
of unfertilized eggs. We thus support the assumption that the iron bound to phosvitin plays an
important role in hematopoiesis during the early developmental stages of the embryo because it has been reported in the literature (Bruns and Ingram, 1973) that
the first hemoglobinized red blood cells become visible
between 26 and 38 h of incubation in the blood islands.
It is striking that the protein secondary structure of
granules remained unchanged in FTIR measurements,
indicating the stability of the remaining, unconsumed,
granules during the first 8 d of incubation. Similar results were described by Bäckermann and Ternes (2008),
who found no major differences by electron microscopy
in the yolk granules of native and 5-d embryonated
eggs. From this observation and further experiments,
they concluded that the digestion and proteolysis of
the granules takes place intracellularly, leading to the
digestion of the granules in the endodermal cells of the
yolk sac membrane, not in the yolk sac. This assumption is thus in good agreement with our findings that
FTIR measurement showed no change in the secondary
structure of the granules. Consequently, our results are
supported by the findings of Yoshizaki et al. (2004) that
Japanese quail yolk granules appear to be of a solid
composition and that digestion during incubation occurs step by step, leaving several parts undegraded until final consumption by the embryo. Moreover, one has
to keep in mind that the minor changes observed in the
phosvitin dialysate sample (see above) might not be noticeable in the examination of whole egg yolk because
phosvitin accounts for only approx. 4% of the protein in
total egg yolk (Ternes et al., 1994) and that only about
21% of the iron in egg yolk is released during the first
5 d of incubation (Bäckermann and Ternes, 2008).
Protein secondary structures in the plasma fraction of egg yolk from fertilized eggs at different
stages of incubation. The second-derivative FTIR
spectra of the plasma fraction of egg yolk from fertilized eggs during incubation (0, 3, 4, 5, 8 d) are shown
in Figure 4. The peaks’ positions and their assignments
to secondary structures are displayed and are in agreement with those of Pelton and Mclean (2000). The spectra showed distinct peaks at 3 different wave numbers.
The peak at 1,616 cm−1 was assigned to intermolecular
β -sheets, whereas the peak at 1,652 cm−1 was found to
YOLK PROTEIN STRUCTURE CHANGES DURING INCUBATION
Figure 4. Second-derivative FTIR spectra (25◦ C) of plasma samples (not freeze-dried) from egg yolk of fertilized eggs drawn at different
stages of incubation (d 0, 3, 4, 5, 8). The black and stippled bold lines
show the spectrum at the beginning (d 0) and the end (d 8) of incubation, respectively. Day 4 is shown as a dotted line. The intermediate
spectra are displayed as thin lines, (n = 4 for each day). All samples
were not further diluted. ∗ , asymmetric shoulder, which indicates the
formation of intramolecular β -sheet structures.
correspond to α-helices and random coil structures. The
peak in the spectral region around 1,683 cm−1 could
possibly arise from β -turns, which are located within
this area. Strikingly, on d 3 and 4 of incubation, an
asymmetric shoulder appeared on the high frequency
side of the intermolecular β -sheets (marked by an asterisk [∗ ]), indicating the formation of intramolecular
β -sheet structures, thus leading to a significant increase
in the absorbance units (1,631 cm−1 ). This shoulder was
absent on all other days of incubation tested (0, 5, 8).
In the literature, the route of how albumen is taken
up by embryonic tissues, transported, and digested during embryonic development has long been debated. Today, it is generally accepted that an accumulation of
fluid in the yolk cell occurs during the first 4 to 6 d
of incubation due to the movement of albumen through
the blastoderm (Babiker and Baggott, 1995; Latter and
Baggott, 2002). In a study by Yoshizaki et al. (2002) on
eggs of the Japanese quail, the authors suggested that
the main route of transporting albumen was from the
albumen sac to the extraembryonic cavity, to the amniotic cavity, to the intestine, and to the yolk cell system. Additionally, an early uptake of a small amount
of albumen by receptor-mediated endocytosis into the
ectodermal cells of the yolk sac was observed on d
4 to 7.
Taking this knowledge into account, we hypothesized
that the appearance of the shoulder on d 3 and 4 of incubation might be due to an influx of albumen into the
yolk sac. To verify our assumption, we conducted a further experiment. Figure 5 shows the second-derivative
FTIR spectra recorded of native albumen (albumen
sample), fractionated table egg yolk plasma (plasma
75
Figure 5. Second-derivative FTIR spectra (25◦ C) of plasma samples (not freeze-dried) from egg yolk of unfertilized, nonincubated eggs
with the addition of different amounts of unfertilized, nonincubated albumen (5, 10, and 20%). A plasma sample without additives served as
a reference. To clarify the changes in the secondary structure elements
in the spectral region around 1,631 cm−1 (formation of the shoulder),
a supplementary spectrum of undiluted albumen is imaged (dotted
line), (n = 4 for each experiment). ∗ , increasing formation of a shoulder, depending on the concentration of albumen added to the plasma
samples.
sample), and plasma samples with the addition of 5,
10, and 20% native albumen (plasma sample + 5% albumen, plasma sample + 10% albumen, plasma sample + 20% albumen). The peaks’ positions and their
assignments to secondary structures of albumen are in
agreement with those of Synytsya et al. (2013) and Ji
et al. (2013). The peak at 1,635 cm−1 was assigned to intramolecular β -sheets, whereas the peak at 1,654 cm−1
was found to correspond to α-helical and random-coil
structures. The signals appearing in the spectral region around 1,681 cm−1 were presumably composed of
β -turns. It is obvious that the reference spectrum of
plasma from table eggs did not differ from that of d 0
embryonated eggs. With regard to the other spectra,
the increasing formation of a shoulder was observed
at 1,631 cm−1 (marked by an asterisk [∗ ]), clearly depending on the concentration of albumen added to the
plasma samples. Evidently, the main peak of albumen
arose exactly in the spectral region where the formation of the shoulder occurred in both the plasma samples of table eggs and in embryonated eggs. Thus, our
results indicate that the origin of the shoulder on d 3
and 4 of incubation was less likely provoked by structure changes of the proteins originally present in plasma
than by the influx of albumen into the yolk sac during incubation, attributing the characteristic secondary
structure elements of albumen to plasma. These results
from our study are supported by those of Latter and
Baggott (2002), who found that a certain amount of albumen enters the yolk sac in the first days of incubation
but moves thereafter to other compartments, e.g., the
allantoic cavity, the amniotic cavity, and the growing
76
LILIENTHAL ET AL.
tissues (Simkiss, 1980). However, we consider this movement to be a possible reason for the observation in our
study that there was no more appearance of a shoulder on the high frequency side of the intramolecular
β -sheet structures after d 4, indicating the absence of
albumen or albumen proteins (Figure 4); this is further
discussed in the section dealing with the IEF. Based on
our experiment, we estimate that the proportion of albumen proteins in the plasma fraction on d 3 and 4 of
incubation had increased by approx. the same amount
as indicated by the addition of 5% albumen (Figures 4
and 5). This assumption would in turn be in good accordance with the results of Hatta et al., 2001, who
found that about 10% of the egg white proteins were
transferred to the yolk after 10 d of incubation.
Since the formation of a shoulder, obviously arising
from inflowing albumen, was observed exclusively in
the plasma fraction, we hypothesize that the differences
in physico-chemical properties between the plasma and
granular fractions of egg yolk were the reason for the
different susceptibility to inflowing albumen during incubation. As to the granular fraction, we assume that
the phenomenon described above was not observed because granules are particulate structures (Ternes et al.,
1994), which thus would scarcely absorb added substances or those entering from the outside.
Protein secondary structures in the LDL fraction of egg yolk from fertilized eggs at different
stages of incubation. For the sake of completeness,
the freeze-dried LDL fraction of egg yolk from fertilized eggs was examined at d 0 and 8 of incubation,
as shown in Figure 6, where the peaks’ positions are
in agreement with those of Pelton and Mclean (2000).
For comparison, a second-derivative spectrum of native (not fertilized, not freeze-dried) LDL is also imaged. The second-derivative FTIR spectra of the LDL
samples from fertilized eggs showed distinct peaks at
3 independent wave numbers. The peak at 1,616 cm−1
was assigned to intermolecular β -sheets, whereas the
peak at 1,652 cm−1 was found to correspond to α-helical
and random-coil structures. An asymmetric shoulder
at 1,633 cm−1 was observed (marked by an asterisk [∗ ])
on the low frequency side of this secondary structure
element, indicating the presence of intramolecular β sheets contributing to the spectrum. The peak in the
spectral region around 1,683 cm−1 was considered to
arise from β -turns, which are located within this area.
Obviously, no shift in wave numbers of peaks was observed between d 0 and 8 of incubation in the freezedried LDL samples. However, we cannot predict how
non-freeze-dried, incubated LDL samples would appear
at d 0 and 8 of incubation, as the observed freeze-drying
effect may have masked the potential structural changes
in the non-freeze-dried, incubated LDL samples. Therefore, this topic has to be part of future studies.
In the native LDL sample, only 2 peaks were detected. The main peak was located at 1,635 cm−1 and
was assigned to intramolecular β -sheets, whereas the
peak at 1,687 cm−1 was asymmetric and showed an
Figure 6. Second-derivative FTIR spectra (25◦ C) of freeze-dried,
reconstituted LDL samples from egg yolk of fertilized eggs in H2 O
solution drawn at different stages of incubation (d 0, 8) and secondderivative FTIR spectrum (25◦ C) of native LDL samples in H2 O solution. The black and stippled bold lines show the spectrum at the
beginning (d 0) and the end (d 8) of incubation, respectively. The native LDL sample is shown as a dotted line. (n = 1 for freeze-dried
LDL samples, n = 4 for native LDL samples). ∗ , asymmetric shoulder, indicating the presence of intramolecular β -sheets contributing to
the spectrum. ∗∗ , asymmetric shoulder, indicating the presence of an
additional, not further specified structure element contributing to the
β -turns peak (1,687 cm−1 ).
asymmetric shoulder on the low frequency side of the
spectrum at 1,675 ± 1 cm−1 (marked by two asterisks
[∗∗ ]). This peak may thus have been composed of β turns (1,685 ± 2.0 cm−1 ), which are located within this
area, as reviewed by (Kong and Yu, 2007). Apparently,
a huge shift in wave numbers occurred from 1,635 to
1,652 cm−1 , between the native and freeze-dried LDL
samples, which can be explained by ongoing structure
changes caused by freeze-drying, as reported in detail
by Blume et al. (2015).
Evaluation of the secondary structure elements
(proportions [%]) from different egg yolk fractions
at d 0. To the best of our knowledge, this is the first
report on proportions (%) of the secondary structure
elements of egg yolk and its 3 major fractions determined by FTIR. Table 2 contains the results of the
integrated peak areas of second-derivative structure
elements and corresponding wave numbers from the
egg yolk fractions shown in Figures 2, 3, 4, and 6.
The secondary structure elements were present in varying proportions in egg yolk and its 3 major fractions.
Egg yolk was mainly (39.77% ± 0.48%) composed of
intermolecular β -sheets, closely followed by α-helices,
and with minor proportions of β -sheets or β -turns, or
both. The granules fraction was shown to be composed
predominantly (57.04% ± 0.39%) of β -sheet structures that originated from a mixture of inter- and intramolecular β -sheets with some α-helices and β -sheets
or β -turns, or both. The plasma fraction was mainly
(43.23% ± 0.27%) composed of α-helices, followed by
YOLK PROTEIN STRUCTURE CHANGES DURING INCUBATION
almost equal ratios of first, intermolecular β -sheets
(1,616 cm−1 ), and second, β -sheets or β -turns, or both
(1,683 cm−1 ). However, there were no α-helical structures whatsoever in the LDL fraction. Interestingly,
this fraction was overwhelmingly (67.36% ± 0.56%)
composed of intramolecular β -sheets and minor proportions of β -sheets or β -turns, or both. When comparing the different proportions of protein secondary
structures contributing to the individual egg yolk fractions, it became clear that the FTIR spectra of the
granules, plasma, and LDL fractions did not form the
sum of whole egg yolk. In fact, each fraction represented an individual single spectrum, which differed
significantly from the spectra of the other fractions.
This was most apparent when the egg yolk plasma
and LDL fractions were compared with each other.
As the plasma fraction was mainly composed of αhelices (43.23% ± 0.27%), it is particularly striking
that the LDL fraction did not show any α-helical structures at all, even though the LDL fraction accounts
for 90% of the plasma fraction. On the other hand,
the LDL fraction was chiefly composed of intramolecular β -sheets (67.36% ± 0.56%), none of which were
detected in the plasma fraction. Therefore, the high
proportions of intermolecular interactions in whole egg
77
yolk may have other sources than a contribution of the
individual fractions. The source could rather be the
molecular interchanges between the proteins of LDL
and the water-soluble proteins of egg yolk, namely the
livetins, which have been suggested as playing a crucial
role in the formation of intermolecular forces (Blume
et al., 2015; Ulrichs et al., 2015) and hence manifest themselves in the different shape of the plasma
spectrum.
Isoelectric Focusing of the Water-soluble
Proteins of Yolk from Incubated Eggs,
Albumen, and Reference Substances of
Albumen Proteins
The pI values of the water-soluble proteins from
freeze-dried egg yolk of fertilized eggs (d 0 through
8) and freeze-dried albumen (d 0) were determined by
means of IEF, as shown in Figure 7. Comparison with
3 reference substances of individual hen albumen proteins made possible identification of the major protein
bands. In a previous experiment (data not shown), it
was confirmed that native egg yolk samples displayed
the same bands in the IEF as the freeze-dried egg yolk
Figure 7. Isoelectric focusing of the water-soluble proteins of freeze-dried egg yolk from fertilized, incubated eggs (d 0 through 8), freeze-dried
albumen (d 0), and reference substances of albumen proteins (Coomassie blue staining). Bands characteristic for albumen proteins (K, L, M, N)
increased in intensity at d 3, d 4, and d 5 (D, E, F), indicating influx of albumen proteins into the yolk sac on d 3–5 of incubation. pI, isoelectric
point.
78
LILIENTHAL ET AL.
samples. The pI values of the standard proteins used
here, ovalbumin, ovotransferrin, and lysozyme, were 4.4
to 4.6, 5.6 to 6.2, and 10.7, respectively, which is in
good accordance with the literature (Holen and Elsayed, 1990). Ovalbumin (54%), ovotransferrin (13%),
and lysozyme (3.5%) are among the major proteins in
albumen (Ternes et al., 1994) detected at the corresponding pI in the albumen samples from d 0. Two
further bands were detected but could not be assigned
to the standard proteins. In the freeze-dried egg yolk
samples, it was possible to distinguish between approx.
10 protein bands in the pH range of 4.5 to 6.4. Of
these, 4 could clearly be identified as identical to the
standard proteins. These bands, we suggest, arose from
ovalbumin (about 4.5), which has 3 isoforms due to the
different degrees of phosphorylation and ovotransferrin
(5.6, 5.9, 6.4). These were considered to be of further
interest due to an increase in intensity during incubation that started on d 3 and reached its maximum on
d 4 of incubation (Figure 7, columns D, E, F). From
d 5 on, there was a decrease in intensities that continued until d 8. Because no extra bands were identified during incubation to indicate the emergence of new
proteins, we assumed the increase in intensities during
incubation originated from the abovementioned influx
of albumen proteins into the yolk sac. When the IEF
results are compared with our FTIR measurements in
the plasma samples of egg yolk from fertilized eggs and
in the plasma samples with the addition of albumen, it
is clear that the 2 experiments corroborate each other.
Because the increase in protein band intensities was detected on the same days as the formation of the shoulder in the plasma samples (d 3 and 4), the IEF results
confirm our FTIR findings. However, no lysozyme was
detected on any day during incubation in the freezedried egg yolk samples, but as it was present in such
lower concentrations in albumen than the other reference substances, it seems likely that lysozyme was not
detectable in the amounts applied.
In conclusion, we characterized for the first time on a
molecular level the incubation-induced changes in egg
yolk from fertilized eggs, its 3 major fractions, and the
associated proteins during the early phase of embryonic development. Neither the significant changes in
pH value of fertilized egg yolk during incubation nor
the slight alterations in the protein secondary structure of phosvitin after the release of iron showed a detectable effect on the protein secondary structure of
whole egg yolk and its major fractions in the course
of incubation. Nevertheless, the FTIR method was
found to be a useful tool for characterizing alterations
in the secondary structure of complex protein compositions: LDL was found to be the only egg yolk
fraction without α-helical structure components, and
an influx of albumen into the plasma fraction during
early embryonic development could clearly be visualized. Furthermore, the conventional method of isoelectric focusing was shown to be suitable for confirming
the alterations in protein composition detected during
early incubation. These findings give insight into the
changes in egg yolk proteins of fertilized eggs during
embryogenesis and provide better understanding of the
molecular mechanisms of protein modification in the
egg development process.
ACKNOWLEDGMENT
The authors wish to thank the Clinic for Poultry,
University of Veterinary Medicine Hannover, Foundation, Germany for kindly incubating the eggs.
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