Comp. Biochem. Physiol. Vol. 100B, No. 1, pp. 1-9, 1991 0305-0491/91 $3.00 + 0.

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MINI-REVIEW

EGG WHITE PROTEINS

LEWISSTEVENS
Department of Biological and Molecular Sciences, University of Stifling, Stirling FK9 4LA, Scotland, UK
(Tel: 0786 73171); (Fax: 0786 64994)

(Received 25 March 1991)

Abstract--1. Egg white proteins are the principal solutes present in egg white, making up approximately
10% of its weight.
2. They are globular proteins and most have acidic isoeiectric points.
3. Many are glycoproteins with carbohydrate contents ranging from 2 to 58%.
4. Of the major egg white proteins, lysozym¢ is the only one having catalytic activity, but many have
specific binding sites, e.g. for vitamins such as biotin, riboflavin and thiamin, or for metal ions such as
Fe Ill.
5. A major group are those showing proteinas¢ inhibitory activity, and they include ovomucoid,
ovoinhibitor, cystatin and ovostatin.
6. The synthesis of egg white protein occurs in the oviduct, and is hormonally controlled either by
oestrogens or progesterone.
7. Extensive studies have been carried out in the genes coding for egg white proteins

INTRODUCTION incubation (Winter et aL, 1967). The total number of

Egg white or albumen is deposited by the tubular
glands around the developing oocyte during its
passage through the oviduct, and this is followed by
the deposition of shell by the shell gland. In a typical
egg of the domestic fowl, the egg white makes up
58% by volume, and contains ~ 50% of the total egg
protein (Gilbert, 1971). During the development of
the embryo, the albumen or egg white gradually
becomes taken up into the aruniotic fluid, and then
into the embryo itself, so at the time of hatching none
remains. The egg white thus provides the aqueous
environment in which the embryo develops, and it
also provides some of the nutrient. Egg white is
composed of 88.5% water, 10.5% protein, 0.5%
carbohydrate and the remainder of other solutes
(Gilbert, 1971). Egg white is relatively homogeneous,
containing very little particulate material and most of
the solutes are proteins; this together with its ready
availability has made egg white proteins some of the
most studied by biochemists. Nevertheless, in spite of
extensive studies the physiological roles of many of
the proteins are not yet understood. Some proteins
may serve principally as nutrients, but if so, this does
not explain why many of those studied have con-
served amino acid sequences. If they simply served as
a balanced source of amino acids, then conservation
of the sequences would seem to be less important.
Some of the proteins have distinctive properties that
suggest functions, e.g. proteinase inhibitors, binding.
proteins etc., but the physiological significance of
these properties is not always clear. The essential role
of one protein, namely the riboflavin binding protein
(RfBP), has been demonstrated, since its absence
in a mutant is generally lethal at about 13 days'
egg-white proteins is not known; Gilbert (1971)
suggested that it was in excess of 40. Since that time
more of the minor constituents have been identified.
With some of the major proteins, their structures,
their genes, and the regulation of their synthesis have
been studied in great detail. In fact much information
on the structures has been obtained since the reviews
of Baker (1968) and Osuga and Feeney (1977). This
review therefore considers the structure and proper-
ties of those proteins that have been purified from the
egg white of the domestic fowl, and makes compari-
sons with those of other avian species where infor-
mation is available. Some of the properties of the
proteins are summarized in Table 1.
OVALBUMIN
Ovalbumin is the most abundant of egg white
proteins, comprising 54% of the total proteins. It is
a glycoprotein and has an isoelvctric point of 4.5. Its
complete sequence of 385 amino acids has been
determined (Nisbet et al., 1981). It has four cysteine
residues and a single cystine disulphide bridge. When
egg white proteins are separated by electrophoresis,
three ovalbumin bands appear (Lush, 1961); these
correspond to the dephosphorylated, mono- and
di-phosphorylated forms, and the sites of phos-
phorylation have been identified as serines 68 and
344. In addition ovalbumin has two further sites of
modification: the N-terminus is acetylated, and the
carbohydrate moiety is linked through asparagine
292. There are two types of oligosaccharide referred
to as high mannose-type and hybrid-type, either of
which is linked through the single asparaglne residue

¢~ ~oo~-~ |
 LEW~S STEVENS
Table I.Physicalpropertiesof egg whiteproteins
Amount in
egg white
Protein (%) M,
Ovalbumin 54 45,000
Ovotransferrin 12 76,600
Ovomucin 1.5 • = 210,000
~ = 720,000
Ovomucoid 11 28,000
Ovoinhibitor 1.50 49,000
Cystatin 0.01 13,100
Ovostatin 0.5 780,000
Lysozyme 3.4 14,300
OvoglobulinG2 1.0 47,000
OvoglobulinG3 1.0 50,000
Riboflavinbindingprotein 1.0 29,200
Avidin 0.5 68,300
Thiamin bindingprotein 38,000
(Ishihara et al., 1981). In addition, two polymorphic
forms of ovalbumin arc known, ovalbumin A and
ovalbumin B, and these differ in having asparagine
and aspartic acid respectively at position 311. Most
secreted proteins have an N-terminal signal sequence
of hydrophobic amino acids, to enable them to
cross the endoplasmic reticulum. In the case of
ovalbumin the signal sequence is in the middle of the
polypeptide chain at residues 234-252 (Lingappa
et al., 1979).
An interesting feature of the structure of oval-
bumin that became evident from its sequence, is its
homology with a group of proteinasc inhibitors
known as serpins. It was found to have 30% sequence
homology with the archetype member of the family
• ~-antitrypsin (Hunt and Dayhoff, 1980). Most mem-
bers of the serpin family have what is described as a
stressed (S) and a relaxed (R) conformation. Proteo-
lytic cleavage converts them from the S to the R
conformation. This conformational change can be
detected by spectroscopic methods such as circular
dichroism (Bruch et al., 1988). It is also found that
the S and R forms exhibit different heat stabilities.
Ovalbumin is also susceptible to proteolysis; when
treated with subtilisin it is cleaved at residues 346 and
352, releasing a hexapeptide and the major fragment
which comprises 346 amino acid residues. However
the cleaved product does not show a conformational
change (Tewkesbury and Carrell, 1989) or a differ-
ence in heat stability (Stein et al., 1989).
Although members of the serpin family possessing
proteinase inhibitory activity have the S and R
conformation, this appears to be lost in members
such as ovalbumin that do not have proteinase
inhibitory activities. Stein et aL (1989) suggest that in
ovalbumin the S to R transition served no useful
purpose and so has been lost during the course of
evolution.
Although preliminary X-ray diffraction studies
have been made on ovalbumin crystals (Miller et al.,
1983) a detailed 3-dimensional structure has not been
proposed. The ovalbumin gene has the distinction of
being the first split gene to be discovered (Breathnaeh
et aL, 1977, see under "Gene organization and
expression of egg white proteins"). The amino acid
sequence derived from the nueleotide sequence of the
Japanese quail shows 42 amino acid substitutions and
three deletions (Mueha et al., 1990), when compared
to that of the domestic fowl.
% of
carbohydrate pl Possible function if known
3.05 4.5 ?
2.6 6.06 Iron transport
13 4.5-5.0 Structural
58 Structural
16.5-32.6 4.1 Proteinase inhibitor
5-9.6 5.1 Proteinase inhibitor
0 5.6 and 6.5 Thiolproteinaseinhibitor
5.8 4.9 Proteinaseinhibitor
0 10.7 Enzyme
? 4.9-5.3 ?
? 4.8 ?
11 4.0 Riboflavin transport
7 10.0 ?
0 ? Thiamin transport
OVOTRANSFERRIN
Ovotransferrin is a giycoprotein which occurs in
egg white, egg yolk and in plasma. The proteins from
all three sources have the same amino acid sequence,
but there are slight differences in the glycosylation
(Williams, 1968). The protein has a M, of 80,000 and
is made up of two domains with a short linking region
(Williams, 1982). The two domains can be separated
after proteolysis of the linking region. Each domain
has a very strong binding site for Fem (K~i~ ~ 10-24).
The two domains are referred to as the N-domain and
the C-domain. The protein is rich in disulphide
bridges, having six in the N-domain and nine in the
C-domain, giving the protein high stability. There is
about 40% homology in the sequences of the two
domains, which are believed to have arisen from gene
duplication 500 million years ago (Williams, 1982).
There is no convincing evidence for the existence of
a simpler monomeric form in the plasma of present-
day vertebrates. Most of these disulphide bridges are
internal and are not reduced by 2-mercaptoethanol
without prior denaturation. There is however one in
the C-domain linking residues 478 to 671 that is more
readily reduced and is assumed to be more exposed
(Williams et aI., 1985). A similar one does not exist
in duck ovotransferrin. Both the complete amino acid
sequence and the nucleotidc sequence were published
in 1982 (Williams et aI., 1982; Jcltsch and Chambon,
1982). Although its 3-dimensional structure has not
yet been published, those of two very closely related
proteins have, namely human lactofcrrin (Anderson
et al., 1987) and rabbit serum transfcrrin (Bailey
et aI., 1988).
The function of ovotransferrin is generally
accepted as that of iron transport. It binds two atoms
of Fe m, one in each domain. The order of iron
binding is p H dependent; at p H 6.0 itbinds firstto the
C-domain, but at p H 8.5 it first binds to tbe N-
domain. For effective iron binding, the sites require
synergistic anion binding (Oe et al., 1989). The
transferrin molecule interacts with cell surface recep-
tors to transfer iron into cells. The mechanism and
requirements for this process have been studied in
detail using chick-embryo red blood cells and ovo-
transferrin fragments (Oratore et al., 1989). When
ovotransferrin is cleaved between the two domains
using proteinases, the two separate domains N and
C will reassociate non-covalently. Williams and
 Egg white proteins
Moreton (1988) have shown, by removal of the
C-terminal fragments of both the N- and C-domains,
that the two domains no longer reassociate. Specifi-
cally, residues 320-332 of the N-domain and residues
683-686 of the C-domain are required for reassoci-
ation. Using either N- or C-domains separately, no
binding to the red cell membranes occurs. However,
if fragments of both N- and C-domains are used
together then binding to the red blood cells occurs,
whether or not the fragments are able to dimerize
with one another or not. Both domains are thus
required for effective binding to cell surface receptors
and hence uptake.
OVOMUCIN
Ovomucin makes up 1.5% of the total protein of
egg white. It is very largely responsible for conferring
the high viscosity of egg white. It therefore plays a
role in maintaining the structure of egg white, and it
has been shown that the thick egg white, which makes
up the outer 50% of the volume has a higher ovo-
mucin content than that of the thin egg white. Interest
in its physical properties has centred on the loss of
viscosity of egg white on storage, which is assumed to
be largely due to changes in the properties of ovo-
mucin, and also on the foaming properties of egg
white which also arise from the ovomucin content.
The latter properties are of particular interest for the
food industry. For many years the main difficulty in
carrying out biochemical studies on ovomucin, was
that of obtaining soluble preparations. Robinson
and coworkers (see Robinson, 1987) succeeded in
obtaining soluble preparations after reduction of
the disulphide bridges. Robinson and Monsey
(1971, 1975) fractionated reduced ovomucin into two
components designated ~- and fl-ovomucin. The
~-ovomucin has M, of 210,000 and contains about
13% by weight carbohydrate. The fl-ovomucin, in
contrast, has a much higher carbohydrate content
(~58%) and a M, of 720,000, which is an aggregate
of smaller units of 112,000. fl-Ovomucin is substan-
tially responsible for the gelatinous properties of
ovomucin, and this is largely due to its high carbo-
hydrate content, which comprises N-acetylgalac-
tosamine, galactose and sialic acid. Some of the
carbohydrate residues are sulphated. The polypeptide
has a very high content of the amino acids sedne
(13%) and threonine (16%), and it is to these that the
carbohydrate residues are linked. Ovomucin not only
has the ability to self-associate, forming large aggre-
gates, but can also associate with other proteins
present in egg white. The physical properties of
ovomucins have been investigated more recently by
Kato et al. (1985) and Rabouille et al. (1990). Kato
et al. (1985) obtained a soluble ovomucin preparation
having Mr 8.3 x 106, which on sonication is reduced
to 1.1 x 106, and further reduced with mercapto-
ethanol to 2.3 x 105. They showed that the foaming
properties reduced as M, decreased, but the emulsify-
ing properties which relate to surface hydrophobicity
increased. Rabouille et al. (1990) used sonication
followed by gel filtration to purify ovomucin. Their
purified material appears to correspond to that of the
fl-ovomucin prepared by Robinson and Monsey
(1971). It has a Mr of >40 x 106 but can be degraded
by reduction using dithiothreitol to a M, value of
3 x 10~. Physical methods such as light scattering and
electron microscopy suggest that it is a flexible linear
molecule.
PROTE~ASE INHIBITORS
A major group of proteins present in egg white are
proteinase inhibitors. Of these, ovomucoid is the
most abundant and most extensively studied, but
recently other less abundant inhibitors have been
studied. Although the functions of these are not
known for certain, it is believed they may have a
protective role against bacterial proteinases. Protein-
ases can be classified according to the nature of their
catalytic site into serine proteinases, thiol proteinases,
acid protcinases (having aspartic acid at the active
site) and proteinases requiring metal ions (Hartley,
1960). Of these the serine proteinases are most wide-
spread. Ovomucoid and ovoinhibitor are both inhibi-
tots of serine proteinases, cystatin is an inhibitor of
thiol proteinases, and ovostatin inhibits a variety of
proteinases.
Ovomucoid
Ovomucoid makes up 10% of the protein in egg
white. It is a heat stable glycoprotein of 185 amino
acid residues and nine disulphide bridges. It is the
latter that account for its heat stability. The sequence
comprises three homologous tandem domains which
are believed to have arisen through two gene dupli-
cations (Kato et al., 1978). Domains I and II are
referred to as a-type domains and show greater
similarity to each other than to domain III, which is
known as a b-type domain. Each domain has three
intradomain disulphide bridges. The b-type domain
differs from the a-type domain in that it is shorter
between the first and second cysteines which form
halves of the first and second disulphide bridges
(Laskowski and Kato, 1980). There are three poten-
tial proteinase inhibitor sites, one in each domain. In
ovomucoid from domestic fowl only one domain
shows proteinase inhibitory properties, whereas in
the turkey, two domains inhibit, and in the duck all
three domains inhibit. In addition to the number of
inhibitory domains, there is also the specificity of
these domains. Most ovomucoids are tested against
the proteinases trypsin and chymotrypsin. The inhi-
bition patterns depend on the sequence in a limited
region of each domain. The mechanism of inhibition
occurs in two steps. First the inhibitor (I) is bound by
the enzyme (E), and this is followed by cleavage of a
single peptide bond to form a modified inhibitor (I*).
A stable inhibitory complex (C) is formed which only
dissociates very slowly, due to the low rate constants,
ko~ and ko~..
k~ ko~o
E+I=C=E+I*.
k~ k~o
It depends on the particular amino acid on
the carboxyl side of the peptide bond that is
cleaved, whether that domain inhibits t r ~ s i n or
chymotrypsin. It is possible to cleave ovomucoid
between the second and third domain using V-8
proteinase (Empie and Laskowski, 1982) and then
4 LEW~SSTEVENS
purify isolated third domains. Laskowski and
coworkers (Laskowski et al., 1987, 1990) have made
a very extensive comparative study of the amino acid
sequences of over 125 different species of birds. From
these it is possible to show that the part of the
sequences that shows most variation corresponds
with the region of enzyme-inhibitor contact. From
X-ray crystallographic studies it is clear that 8 out of
11 enzyme-inhibitor contact positions are strongly
hypervariable (Laskowski et al., 1987). This results in
the ovomucoids from closely related species, e.g.
domestic fowl, turkey, golden pheasant, showing
different patterns of inhibitory activity. It has been
suggested that the significance of this type of variabil-
ity is to enable the proteinase inhibitors to adapt to
inhibiting a changing range of bacterial proteinases.
O~oinhibitor
Ovoinhibitor is also an inhibitor of serine protein-
ases, and is similar to ovomucoid in its properties. It
is larger than ovomucoid, having an M, value of
49,000, and comprising seven domains, each having
a similar arrangement of disulphide bridges to that of
ovomucoid. Six of the domains are of the a-type, and
the seventh, which occupies the C-terminus, is a
b-type. Ovoinhibitor from domestic fowl is able to
inhibit trypsin, ~-chymotrypsin, subtilisin and Asper-
gillus oryzae alkaline proteinase (Liu et al., 1971).
One molecule of ovoinhibitor is able to inhibit two
molecules of trypsin and two of chymotrypsin, each
binding to different domains. Subtilisin, on the other
hand competes with ~-chymotrypsin for the same
sites. There is evidence that the two trypsin binding
sites are not equivalent (Zahnley, 1974). A possible
advantage of having a number of domains, each of
which inhibits proteinases is that a wider range of
proteinases may be inhibited, and so afford a greater
measure of protection from microorganisms.
Cystatin
Cystatin was first isolated in small quantities from
egg white by Fossum and Whitaker (1968) and
known as ficin inhibitor on account of its properties.
The name cystatin was proposed by Barrett (1981). It
occurs at concentrations of about 12.5/~g/ml in egg
white and also in lower concentrations (1/~g/ml) in
serum (Anastasi et al., 1983). There are two major
forms of cystatin having pI values of 6.5 and 5.6
referred to as A and B (Turk et al., 1983) and 1 and
2 (Anastasi et al., 1983). Each of the two forms exists
in short and long forms, the former lacking the first
eight amino acid residues present in the 116 residue
polypeptide chain of the latter. The two major forms
are immunologically identical and neither contains
any carbohydrate. Cystatin inhibits a number of
cysteine proteinases including ficin, papain, cathepsin
B, cathepsin H, cathepsin L and dipeptidyl peptidase
I, but not clostipain or streptococcal proteinase, and
it only weakly inhibits bromelain (Anastasi et al.,
1983). Cystatin has been sequenced (Schwabe et al.,
1984; Turk et al., 1983) and its three-dimensional
structure determined by X-ray crystallography at
2.0 A resolution (Bode et al., 1988). It contains an
s-helical region and a five stranded/~-pleated sheet
and two intrachain disulphide bridges. The region
binding to proteinases has been identified.
Ovostatin
Ovostatin (formerly known as ovomaeroglobulin)
is a large molecule having a tetrameric structure (M,
780,000 = 4 × 195,000). It inhibits a wide range of
endoproteinases including thermolysin (a metal-ion
requiring proteinase) and collagenase (Nagase et aL,
1983). Its structure and mechanism of action is like
that of the serum proteinase inhibitor, ~2-macro-
globulin (Nagase et al., 1983). The proteinases first
cleave a bond within ovostatin, which then undergoes
a conformational change so as to hinder the access of
large, but not small substrate molecules to the cata-
lytic site. Thus ovostatin inhibits proteinase activity
when measured using protein substrates, but not
when using low Mr substrates such as peptide nitro-
anilides. Ovostatin shows 40% homology with ~2-
macroglobulin, but differs from it in being insensitive
to methylamine, having distinct immunogenicity, and
a distinct peptide map after cyanogen bromide treat-
ment. Ovostatin from both the domestic fowl and the
duck have been studied, the latter inhibits both
metalloproteinases and serine proteinases, whereas
the former inhibits metalloproteinases only. An
elegant model for the mechanism of inhibition by
duck ovostatin, has been proposed by Ruben et al.
(1988) based on a series of electron micrograph
studies.
LYSOZYME
Of all the proteins in egg white, iysozyme is the one
which has been most thoroughly investigated at the
molecular level. This is primarily due to a combi-
nation of factors: it is fairly abundant and easily
purified, it is a relatively small sized protein, it was the
first structure for which an atomic resolution X-ray
structure was published (Phillips, 1967) and a mech-
anism proposed based on the structural information
(see Imoto et al., 1972; Hammes, 1982).
Lysozyme is unusual among the major egg white
proteins in having an alkaline pI, which means that
it can form complexes with ovomucin, ovalbumin
and ovotransferrin. It has a total of 129 amino acid
residues and contains four disulphide bridges. Its
enzyme activity is that it is able to cleave peptido-
glycans, such as are found in the cell walls of bacteria.
Its role in egg white may be that of protection from
invading bacteria. Its mechanism of action has been
studied in great detail, and the precise amino acid
residues involved in the catalysis, e.g. aspartate-52
and glutamate-35, are well known. Their importance
in catalysis is evident, since when either is replaced by
the corresponding amide, asparagine or glutamine,
using site directed mutagenesis, catalytic activity is
reduced to 5% and 0.1% respectively, of that of the
unmodified enzyme (Malcolm et al., 1989). X-ray
crystallography of the complex between lysozyme
and one of its substrates reveals the spatial disposi-
tion of enzyme and substrate. X-ray crystallography
gives a static picture of the interaction. More recently
a more dynamic picture is given by spectroscopic
methods which can be applied in solution as opposed
to crystals. Using N M R spectroscopy it is now
possible to identify resonances for 126 of the 129
amino acid residues (Redfield and Dobson, 1988).
 Egg white proteins
From this, a dynamic picture of the changes during
catalysis will be realized. It has been shown that one
of the four disulphide bridges can be selectively
reduced, and the secondary and tertiary structure
retained, although the catalytic activity is reduced by
40% and the heat stability reduced (Radford et al.,
1991).
A number of comparative studies have been made
on lysozymes from different avian sources. There are
two basic types: c-type (chicken) and g-type (goose).
The latter has a higher M, of 21,000 compared with
14,000. There are substantial differences in sequence,
but also similarities in the three-dimensional structure
which led Weaver et al. (1985) to suggest that both
types have diverged from a common evolutionary
precursor. Joll6s et al. (1979) compared the sequences
of a number of lysozymes, mainly of the c-type,
including those from pheasants, quails, turkey,
partridge and ducks. Between 10 and 14 substitutions
occur within the phasianoid lysozymes, and more
between the phasianoids and the anatid birds.
OVOGLOBULINS
Ovoglobulins were originally divided into three
classes G1, G2 and G3 on the basis of their
separation by moving boundary electrophoresis
(Longsworth et al., 1940). Subsequent work showed
that G1 was lysozyme. Ovoglobulins G2 and G3 have
been least thoroughly investigated from the bio-
chemical standpoint. Although they have been puri-
fied (Feeney et al., 1963; Stevens and Duncan, 1988)
and their Mrs have been determined, no structural
studies have been carried out on them. No catalytic
or binding properties have yet been attributed to
them. Their main point of interest to date has been
the presence of a large number of genetic variants
(Baker et al., 1970). The differences between two of
these have been investigated by peptide mapping
(Stevens and Duncan, 1988). Ovoglobulins G2A and
G2B differ in their pI and also in their sensitivities to
chymotrypsin and V8 proteinase.
VITAMIN BINDING PROTEINS
The vitamins present in the fertilized egg are
needed to satisfy the growth requirements of the
developing embryo until the time of hatching. A
number of these are present in both the egg white
and yolk bound to their respective binding proteins.
Vitamin binding proteins have been identified for
riboflavin, biotin, thiamin, cyanocobalamin, retinol
and cholecalciferol, those for the last two being
present principally in egg yolk (white, 1987). The
best characterized are those for riboflavin, biotin and
thiamin and will be discussed in the next subsections.
The role of the binding proteins appears to be that of
ensuring the uptake of the vitamins into the develop-
ing oocyte. This is most clearly demonstrated in the
case of the mutant deficient in riboflavin binding
protein. The developing embryos die on or near
13 days' incubation of riboflavin deficiency. The
deficiency cannot be overcome by feeding the hens a
diet supplemented with riboflavin, but only by direct
injection of riboflavin into the eggs (white and
Merrill, 1988). There are however many unanswered
questions concerning the vitamin binding proteins
(White, 1987).
Riboflavin binding protein (RfBP)
Riboflavin binding protein is the most abundant
vitamin binding protein in egg white, making up
approximately 1% of the protein content. The pro-
tein has been purified from egg white from domestic
fowl and sequenced (Norioka et al., 1985), although
its three-dimensional structure has not yet been
determined (White and Merrill, 1988). It has nine
disulphide bridges, and this probably accounts in part
for its high thermal stability. Solutions of RfBP can
be boiled for 30 min without denaturation. From its
circular dichroic spectra it is estimated that RfBP
from domestic fowl has approximately 24% ~-helix
and 39% ~-sheet (Walker et al., 1991). The protein
has a total of eight phosphate groups which together
with the acidic amino acid residues and sialic
acid account for its low pI of 4.0. It has two
oligosaccharide groups attached to asparagine 36 and
147. Unlike other flavin binding proteins it does not
show a high degree of specificity in the flavins to
which it is able to bind (Becvar and Palmer, 1982).
The phosphate groups and the sialic acid residues are
important for the uptake of RfBP into oocytes, since
their removal leads to a marked decrease in uptake
(Miller et al., 1982). The riboflavin:RfBP complex
lacks the fluorescence characteristic of riboflavin
(Rhodes et al., 1959), and this is a useful property to
measure the extent of binding.
Avidin
Avidin is much less abundant in egg white than
RfBP, the former making up only 0.05% of the total
protein. Nevertheless it is the most studied of the
vitamin binding proteins. It first came to light in the
form of a nutritional syndrome caused by consuming
uncooked egg white. The latter contained avidin
which has a very high affinity for biotin, making the
dietary source of the latter unavailable. It is this
extremely high affinity for biotin (K~-~0.6 x 10-15 M),
that has resulted in numerous applications which
make use of this strong binding. Avidin is a glyco-
protein comprising four subunits, each having a
single binding site for biotin. The subunits comprise
128 amino acids and the M, based on the sequence is
15,600. The very strong binding of avidin to biotin,
equivalent to a free energy change of 21 kcal/mol
is remarkably high for a non-covalent interaction
involving an organic ligand. It is unclear what advan-
tages the very strong binding has in relation to the
possible physiological roles of the protein. It is, for
example, 1000-fold higher than that of the biotin
binding protein in egg yolk (Green, 1990). It seems
unlikely that its role is in the transport of biotin, since
a high proportion of the circulating avidin does not
have biotin attached. It seems more likely that it may
act in order to protect from bacterial attack.
Its properties were comprehensively reviewed in
1975 (Green, 1975) and since then the emphasis has
been very largely on the application of biotin-avidin
technology, such that a complete volume of Methoda
in Enzymology is devoted to this (Wilchek and Bayer,
1990). Avidin has a pI = 10.5 and a single disulphide
bridge. It has proved difficult to obtain suitable
6 L~v~s STEVENS
crystals of avidin for X-ray diffraction studies, poss-
ibly because it is a glycoprotein, although a closely
related compound, streptavidin, which is not glyco-
sylated has been studied. The oligosaccharide
residues are not important for binding since they can
be cleaved without significantly affecting the binding.
The four biotin binding sites on avidin are grouped
in two pairs on opposite faces of the molecule. A
number of chemical modification studies have been
carded out to ascertain which amino acid residues are
involved in the binding domain. A drastic reduction
in binding occurs when four tryptophan residues in
each subunit are modified by N-bromosuccimide.
Biotin protects the four tryptophans from modifi-
cation. Dinitrophenylation of one lysine residue
also prevents biotin binding (see Green, 1990). The
tryptophan residues and the lysine residue are thus
implicated in the binding.
Avidin-biotin technology has wide applications,
including affinity chromatography, cytochemistry,
gene probes and drug delivery (see Wilchek and
Bayer, 1990). It utilizes the strong binding of biotin
to avidin. The carboxyl group on biotin, together
with the low reactivity of its fused ring system, means
that it can be covalently linked through the carboxyl
group to a variety of compounds e.g. binders or
probes. Once biotinylated, the compounds will inter-
act strongly to avidin, or conjugated avidin. Since
avidin has four binding sites it can also be used as a
crosslinker.
Thiamin binding protein
Thiamin binding protein has been purified from
egg white by affinity chromatography using thiamin
pyrophosphate coupled to aminoethyl-Sepharose
(Munniyappa and Adiga, 1979). It has M, 38,000 and
is not a glycoprotein, and has a much lower affinity
for the vitamin (Kd 0.3/tM) than in the case of RfBP
or avidin. A similar protein has been purified from
egg yolk (Munniyappa and Adiga, 1981) which cross
reacts with monospecific antiserum to egg white
thiamin binding protein, suggesting that both are
products of the same gene, although they may differ
in posttranslational modification.
GENE ORGANIZATION AND EXPRESSION OF EGG
WHITE PROTEINS
The organization of the genes for most of the major
egg white proteins has been studied: these include
ovalbumin, ovotransferrin, ovomucoid, ovoinhibitor,
lysozyme and avidin. The syntheses of each of these
proteins are hormonally induced in the oviduct of the
domestic fowl. For ovalbumin, ovotransferdn, ovo-
mucoid, and lysozyme induction is by oestrogens,
and for avidin it is by progesterone (O'Malley et aL,
1979). The synthesis of RfBP has also been shown to
be under steroid hormone control (Clagett et aL,
1970) although the organization of its gene has not
been studied. An important historical discovery was
made during the investigation of the ovalbumin gene
structure, namely the first detection of intervening
sequences in the structure of a gene (Breathnach et
al., 1977). The ovalbumin gene comprises eight exons
and seven introns (McReynolds et al., 1978) and
much detailed information is now available concern-
ing promoter sequence for the ovalbumin gene (Tsai
et al., 1988). Within a 46 kb region which includes the
ovalbumin gene, two additional genes X and Y of
unknown function, having sequence homology with
the ovalbumin gene, have been found (Royal et al.,
1979). These genes are under steroid hormone control
and also have seven introns, but are transcribed at a
much lower level than ovalbumin mRNA (LeMeur
et al., 1981). Ovalbumin mRNA may represent as
much as 50% of the polyA-containing mRNA in
hormonally stimulated tissue.
The gene for ovotransferrin is the largest of those
studied for egg white proteins, having an overall
length of 10.5 kb and including 16 introns (Cochet
et al., 1979). Oestradiol causes a more rapid increase
in the transcription of the ovotransferdn gene than
the ovalbumin gene, and this may be due to different
flanking regions that affect promoter efficiency. The
ovomucoid gene has been completely sequenced.
Each of the three domains of ovomucoid are coded
for by two exons (Stein et al., 1980) and the domain
boundaries correspond to the intron--exon junctions.
Ovoinhibitor is believed to have evolved from a
common single domain ancestor (Scott et al., 1987)
common to both ovomucoid and ovoinhibitor and
this is born out by the similar organization of
their gene structure. Ovoinhibitor comprises seven
domains: its gene is about 10.3 kb and consists of 16
exons, each domain having two exons, and like
ovomucoid the domain boundaries coincide with
intron-exon junctions.
The gene for lysozyme and the eDNA for lysozyme
mRNA have been cloned (Jung et al., 1980). It
comprises four exons of which the first codes for the
translation signals, the signal peptide and the first 28
amino acid residues. The eDNA for avidin has been
cloned (Gope et al., 1987) but the complete organiz-
ation of the gene has not yet been resolved.
UPTAKE OF EGG WHITE PROTEINS BY DEVELOPING
EMBRYOS
During the development of the embryo the volume
of the egg white diminishes, and disappears
altogether by the time of hatching. The growing yolk
sac transports fluid from the egg white to the yolk.
Up to 8-9 days' incubation the egg white thickens as
its volume decreases. At 11 days the seroamniotic
suture ruptures leaving an opening between the
albumen sac and the amniotic cavity, allowing the
former to pass through. A few studies have been
made of the fate of proteins from the egg white. A
question which has not been answered is whether or
not there is a selective uptake of particular proteins
from the egg white. A selective uptake might suggest
that certain proteins were needed at particular stages
of development. Baintner and Feher (1974) studied
the uptake of trypsin inhibitory activity from egg
white. They detected uptake of trypsin inhibitory
activity into the amniotic cavity and then orally
into the chick embryo. Sugimoto et al. (1984) have
identified the inhibitory activity in both egg white and
egg yolk as ovomucoid, further supporting the idea of
transport from egg white to egg yolk. This has also
been demonstrated by Western blot analysis, for
ovalbumin, and ovotransferrin, and by measurement
 Egg white proteins

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Gope M. L., Kcinanem R. A., Kristo P. A., Coneeely O. M.,
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