Comp. Biochem. Physiol. Vol. 100B, No. 1, pp. 1-9, 1991

0305-0491/91 $3.00 + 0.00 © 1991 Pergamon Press pic

Printed in Great Britain

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

incubation (Winter et aL, 1967). The total number of 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 properties of those proteins that have been purified from the egg white of the domestic fowl, and makes comparisons with those of other avian species where information is available. Some of the properties of the proteins are summarized in Table 1.

INTRODUCTION 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 conserved 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' ¢~

~oo~-~

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 phosphorylation 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 |

LEW~S STEVENS Table I.Physicalpropertiesof egg whiteproteins Amount in egg white % of Protein (%) M, carbohydrate pl Ovalbumin 54 45,000 3.05 4.5 Ovotransferrin 12 76,600 2.6 6.06 Ovomucin 1.5 • = 210,000 13 4.5-5.0 ~ = 720,000 58 Ovomucoid 11 28,000 16.5-32.6 4.1 Ovoinhibitor 1.50 49,000 5-9.6 5.1 Cystatin 0.01 13,100 0 5.6 and 6.5 Ovostatin 0.5 780,000 5.8 4.9 Lysozyme 3.4 14,300 0 10.7 OvoglobulinG2 1.0 47,000 ? 4.9-5.3 OvoglobulinG3 1.0 50,000 ? 4.8 Riboflavinbindingprotein 1.0 29,200 11 4.0 Avidin 0.5 68,300 7 10.0 Thiamin bindingprotein 38,000 0 ? (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 ovalbumin 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 members of the serpin family have what is described as a stressed (S) and a relaxed (R) conformation. Proteolytic 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 difference 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.

Possible function if known ? Iron transport Structural Structural Proteinase inhibitor Proteinase inhibitor Thiolproteinaseinhibitor Proteinaseinhibitor Enzyme ? ? Riboflavin transport ? 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 presentday 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 Ndomain. For effective iron binding, the sites require synergistic anion binding (Oe et al., 1989). The transferrin molecule interacts with cell surface receptors to transfer iron into cells. The mechanism and requirements for this process have been studied in detail using chick-embryo red blood cells and ovotransferrin 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. Specifically, residues 320-332 of the N-domain and residues 683-686 of the C-domain are required for reassociation. 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 ovomucin 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 ovomucin, 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 substantially responsible for the gelatinous properties of ovomucin, and this is largely due to its high carbohydrate content, which comprises N-acetylgalactosamine, 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 aggregates, 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 mercaptoethanol to 2.3 x 105. They showed that the foaming properties reduced as M, decreased, but the emulsifying 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. Proteinases 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 widespread. Ovomucoid and ovoinhibitor are both inhibitots 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 duplications (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 potential 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 inhibition 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 variability 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 proteinases, 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 Aspergillus 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-macroglobulin (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 catalytic site. Thus ovostatin inhibits proteinase activity when measured using protein substrates, but not when using low Mr substrates such as peptide nitroanilides. Ovostatin shows 40% homology with ~2macroglobulin, but differs from it in being insensitive to methylamine, having distinct immunogenicity, and a distinct peptide map after cyanogen bromide treatment. 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 combination 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 mechanism 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 peptidoglycans, 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 disposition 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 biochemical standpoint. Although they have been purified (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 developing 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 protein 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 glycoprotein 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 advantages 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, possibly because it is a glycoprotein, although a closely related compound, streptavidin, which is not glycosylated 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 modification. 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 interact 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, ovomucoid, 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 organization 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 Gilbert A. B. (1971) The egg: its physical and chemical aspects. In Physiology and Biochemistry of the Domestic Fowl (Edited by Bell D. 3. and Freeman B. M.), Vol. 3, pp. 1379-1399. Academic Press, New York. Gope M. L., Kcinanem R. A., Kristo P. A., Coneeely O. M., Beattie W., Zarucki G., Schulz T., O'Malley B. W. and Kulomaa M. S. (1987) Molecular cloning of the chicken avidin gene. Nucleic Acid~ Res. 15, 3595-3606. Green N. M. (1975) Avkfin. Adv. Prot. Chem. 29, 85-133. REFERENCF~ Gren N. M. (1990) Avidin and Streptavidin. Meth. Enzymol. 184, 51-67. Anastasi A., Brown M. A., Kembbavi A. A., Nicklin M. J. H., Sayers C. A., Sunter D. C. and Barrett A. J. Hammer C. HI., Buss E. G. and Clagett C. O. (1973) Avian riboflavinuria. 8. The fate of the riboflavin-binding pro(1983) Cystatin, a protein inhibitor of cysteine proteintein-riboflavin complex during incubation of ben's eggs. ases. Biochem. J. 211, 129-138. Poultry Sci. 52, 520-530. Anderson B. F., Baker H. M., Dodson E. J., Norris G. E., Rumball S. V., Waters J. M. and Baker E. N. (1987) Hammes G. G. (1982) Enzyme Catalysis and Regulation, pp. 132-143. Academic Press, New York. Structure of human lactoferrin at 3.2 A resolution. Proc. I-Iartley B. S. (1960) Proteolytic enzymes. A. Rev. Biochem. natn. Acad. Sci. USA 84, 1769-1773. 29, 45-72. Bailey S., Evans R. E., Garratt R. C., Gorinsky B., Hasnain S., Horsburgh C., 3hott H., Lindley P. F., Mydin A., Hunt L. T. and Dayhoff M. O. (1980) A surprising new protein superfamily containing ovalbumin, antithrombinSarra R. and Watson J. L. (1988) Molecular structure of III, and alpha:proteinase inhibitor. Biochem. biophys. serum transferrin at 3.3 A resolution. Biochemistry 27, Res. Commun. 95, 864-871. 5804-5812. Baintner K. and Feher G. (1974) Fate of egg white trypsin Imoto T., 3ohnson L. N., North A. C. T., Phillips D. C. and Rupley J. A. (1972) Vertebrate lysozymes. In The inhibitor and start of proteolysis in developing chick Enzymes (Edited by Boyer P. B.), 3rd edn, Vol. 7, embryo and newly hatched chick. Devl. Biol. 36, pp. 666-868. Academic Press, New York. 272-278. Baker C. M. A. (1968) The proteins of egg white. In Egg Ishihara H., Takahashi N., Takenchi E. and Tejima S. (1981) Either high mannose-type or hybrid-type Quality, a Study of Hen's Eggs (Edited by Carter T. C.), oligosaccharide is linked to the same asparagine pp. 67-108. Oliver and Boyd, Edinburgh. residue of ovalbumin. Biochim. biophys. Acta 669, Baker C. M. A., Crozier G., Stratil A. and Manwell C. 216-221. (1970) Identity and nomenclature of some protein polymorphisms of chicken eggs and sera. Adv. Genetics 15, Jeltsch 3. M. and Chambon P. (1982) Complete nucieotide sequence of the chicken ovotransferrin mRNA. Eur. J. 211-226. Biochem. 122, 291-295. Barrett A. J. (1981) Cystatin, the egg white inhibitor of Joll~s J., Ibrahim I. M., prager E. M., Scboentgen F., Jolles cysteine proteinases. Meth. Enzymol. 80, 771-778. P. and Wilson A. C. (1979) Amino acid sequence of Becvar J. and Palmer G. (1982) The binding of flavin pheasant lysozyme. Evolutionary change affecting proderivatives to the riboflavin-binding protein of egg white. cessing of prelysozyme. Biochemistry 18, 2744-2751. J. biol. Chem. 257, 5607-5617. Bode W., Engh R., Musil D., Thiele U., Huber R., Jung A., Sippei A. E., Grez M. and Schutz G. Karshikov A., Brzin J., Kos J. and Turk V. (1988) The (1980) Exons encode functional and structural units of chicken lysozyme. Proc. nam. Acad. Sci. USA 77, 2.0 A X-ray crystal structure of chicken egg white cystatin and its possible mode of interaction with cysteine protein5759-5763. Kato I., Kohr W. J. and Laskowski M. (1978) Evolution ases. EMBO J. 7, 2593-2599. Breathnach R., Mandel J. L. and Chambon P. (1977) of ovomucoids. Proc. llth FEBS Meeting, Vol. 47, pp. 197-206. Pergamon Press, Oxford. Ovalbumin gene is split in chicken DNA. Nature 270, Kato A., Oda S., Yamaka Y., Matsudomi N. and 314-319. Kobayashi K. (1985) Functional and structural properties Bruch M., Weiss V. and Engel 3. (1988) Plasma serine proteinase inhibitors (serpins) exhibit major conforof ovomucin. Agri. biol. Chem. 49, 3501-3504. mational changes and a large increase in conformational Laskowski M. and Kato I. (1980) protein inhibitors of stability upon cleavage at their reactive sites. J. biol. proteinases. A. Rev. Biochem. 49, 593-626. Laskowski M., Kato I., Ardeit W., Cook J., Denton A., Chem. 263, 16626-16630. Empie M. W., Kohr W. J., Park S. J., Parks K., Schatzley Clagett C. O., Buss E. G., Saylor E. M. and Grish S. J. B. L., Schoenbcrger O. L., Tashiro M., Vichot G., (1970) The nature of the biochemical lesion in avian renal Whatley H. E., Wieczorek A. and Wieczorek M. (1987) riboflavinuria: 6. Hormone induction of the riboflavinbinding protein in roosters and young chicks. Poultry Sci. Ovomucoid third domain from 100 avian species: 49, 1468-1472. isolation, sequences, and hypervariability of enzymeCochet M., Gannon F., Hen R., Marteaux L., Perrin F. and inhibitor contact residues. Biochemistry 26, 202-221. Chambon P. (1979) Organization and sequence studies of Laskowski M., Apostol I., Ardelt W., Cook J., Giletto A., the 17-piece chicken conalbumin gene. Nature 282, Kelly C. A., Lu W., Park S. J., Qasim M. A., Whatley H. E. et al. (1990) Amino acid sequences of ovomucoid 567-574. Empie M. W. and Laskowski M. (1982) Thermodynamics third domain from 25 additional spoAes of birds. J. prot. Chem. 9, 715-726. and kinetics of single residue replacements in avian ovomucoid third domains: effect on inhibitor inter- LeMeur M., Glanville N., Mandel J. L., Gerlinger P., actions with serine proteinases. Biochemistry 21, Palmiter R. and Chambon P. (1981) The ovalbumin gene family: Hormonal control of X and Y gene transcription 2274-2284. and mRNA accumulation. Cell 23, 561-571. Feeney R. E., Abplanalp H., Clary J. J.,Edwards D. L. and Clark J. R. (1963) A geneticallyvarying minor protein Lingappa V. R., Lingappa J. R. and Blobel G. (1979) constituent of chicken egg white. J. biol. Chem. 238, Chicken ovalbumin contains an internal signal sequence. Nature 281, 117-121. 1732-1736. Fossum K. and Whitaker J. R. 0968) Ficin and papain Liu W.-H., Means G. E. and Feeney R. E. (1971) inhibitorfrom chicken egg white.Arch. Biochem. Biophys. The inhibitory properties of avian ovoinhibitors against 125, 367-375. proteolytic enzymes. Biochim. biophys. Acta 229, 176-185.

o f enzyme activity for lysozyme (Sugimoto et al., 1989). It appears therefore that for all four proteins there is a transfer o f egg white proteins into the yolk sac. The RfBP has also been shown t o d i s a p p e a r from the egg white by 14 days' incubation ( H a m m e r et al., 1973).

8

LEW~ STEVENS

Longsworth L. G., Cannon R. K. and Maclnnes D. A. (1940) Electrophoretic study of the proteins of egg white. J. Am. chem. Soc. 62, 2580-2590. Lush I. (1961) Genetic polymorphisms in egg albumen proteins of the domestic fowl. Nature 189, 981-984. Malcolm B. A., Rosenberg S., Corey M. J., Allen J. S., Da Baetselter A. and Kirsh J. F. (1989) Site-directed mutagenesis of catalytic residues Asp-52 and Glu-35 of chicken egg white lysozyme. Proc. natn. Acad. Sci. USA 116, 133-137. McReynolds L., O'Malley B. W., Nisbet A. D., Fothergill J. E., Givol D., Fields S., Robertson M. and Brownlee G. G. (1978) Sequence of chicken ovalbumin mRNA. Nature 273, 723-728. Miller M. S., Benmore-Parsons M. and White H. B. (1982) Dephosphorylation of chicken riboflavin-binding protein and phosvitin decreases their uptake by oocytes. J. biol. Chem. 257, 6818-6824. Miller M., Weinstein J. N. and Wlodawer A. (1983) Preliminary X-ray analysis of single crystals of ovalbumin and plakalbumin. J. biol. Chem. 258, 5864-5866. Mucha J., Klaudiny J., Klaudinyova V., Hanes J. and Simuth J. (1990) The sequence of Japanese quail ovalbumin cDNA. Nucleic Acids Res. 18, 5553. Munniyappa K. and Adiga P. R. (1979) Isolation and characterization of thiamin-binding protein from chicken egg white. Biochem. J. 177, 887-894. Munniyappa K. and Adiga P. R. (1981) Nature of the thiamin-binding protein from chicken egg yolk. Biochem. J. 193, 679-685. Nagase H., Harris E. D., Woessner J. F. and Brew K. (1983) Ovostatin: a novel proteinase inhibitor from chicken egg white. 1. Purification, physicoehemical properties, and tissue distribution of ovostatin. J. biol. Chem. 258, 7481-7489. Nisbet A. D., Saundry R. M., Moir A. J. G., Fothergill L. A. and Fothergill J. E. (1981) The complete amino-acid sequence of hen ovalbumin. Eur. J. Biochem. 115, 335-345. Norioka N., Okada T., Hamazume Y., Mega T. and Ikenaka T. (1985) Comparison of the amino acid sequences of hen plasma-, yolk-, and white-riboflavin binding proteins. J. Biochem. 97, 19-28. Oe H., Takahashi N., Doi E. and Hirose M. (1989) Effects of anion binding on the conformations of the two domains of ovotransferrin. J. Biochem. 106, 858-863. O'Malley B. W., Roop D. R., Lai E. C., Nordstrom J. L., Catterall J. F., Swaneck G. E., Colbert D. A., Tsai M.-J., Dugaiczyk A. and Woo S. L. C. (1979) The ovalbumin gene: organization, structure, transcription and regulation. Rec. Prog. Hormone. Res. 35, 1-46. Oratore A., D'Andrea G., Moreton K. and Williams J. (1989) Binding of various ovotransferrin fragments to chick-embryo red cells. Biochem. J. 257, 301-304. Osuga D. T. and Feen6y R. E. (1977) Egg proteins. In Food Proteins (Edited by Whitacher J. R. and Tannenbaum S. R.), pp. 209-267. Avi Publishing, Westport, Ct. Phillips D. C. (1967) The hen egg-white lysozyme molecule. Proc. natn. Acad. Sci. USA 57, 484-495. Rabouille C., Aon M. A., Muller (3, Cartaud J. and Thomas D. (1990) The supramolecular organization of ovomucin: biophysical and morphological studies. Biochem. J. 266, 697-706. Radford S. E., Woolfson D. N., Martin S. R., Lowe G. and Dobson C. M. (1991) A three-disulphide derivative of hen lysozyrne. Biochem. J. 273, 211-217. Rediield C. and Dobson C. M. (1988) Sequential H t NMR resonance and secondary structure of hen egg white lysozyme in solution. Biochemistry 27, 122-136. Rhodes M. B., Bennett N. and Fceney R. (1959) The flavoprotein-apoprotein system of egg white. J. biol. Chem. 234, 2054-2060.

Robinson D. S. (1987) The chemical basis of albumen quality. In Egg Quality---Current Problems and Recent Advances (Edited by Wells R. G. and Belyavin C. G.), pp. 179-191. Butterworths, London. Robinson D. S. and Monsey J. B. (1971) Studies of the composition of egg-white mucin. Biochem. d. 121, 537-547. Robinson D. S. and Mousey J. B. (1975) The composition and proposed subunit structure of egg-white ovomucin: the isolation of an unreduced soluble ovomucin. Biochem. J. 147, 55-62. Royal A., Garapin A., Cami B., Perrin F., Mandel J. L., LeMenr M., Bregere F., Gannon F., Lepennec J. P., Chambon J. and Kourilsky P. (1979) The ovalbumin region: common features in the organization of three genes expressed in chicken oviduct under hormonal control. Nature 279, 125-132. Ruben C. G., Harris E. D. and Nagase H. (1988) Electron microscopic studies of free and proteinase-bound duck ovostatins (Ovomacroglobulins). J. biol. Chem. 263, 2861-2869. Scott M. J., Huckaby C. S., Kato I., Kohr W. J., Laskowski M., Tsai M.-J. and O'Malley B. W. (1987) Ovoinhibitor introns specify functional domains as in the related and linked ovomucoid gene. J. biol. Chem. 262, 5899-5907. Schwabe C., Anastasi A., Crow H., McDonald J. K. and Barrett A. J. (1984) Cystatin. Amino acid sequence and possible secondary structure. Biochem. J. 217, 813-817. Stein J. P., Catterall J. F., Kristo P., Means A. R. and O'Malley B. W. (1980) Molecular cloning of the ovomucoid gene sequences from partially purified messenger RNA. Cell 21, 681-687. Stein P. E., Tewkesbury D. A. and Carrell R. W. (1989) Ovalbumin and angiotensinogen lack serpin S-R conformational change. Biochem. J. 262, 103-107. Stevens L. and Duncan D. (1988) Peptide mapping of ovoglobulins G2A and G2B in the domestic fowl. Br. Poultry Sci. 29, 665-669. Sugimoto Y., Hanada S., Koga K. and Sakaguchi B. (1984) Egg-yolk trypsin inhibitor identical to albumen ovomucoid. Biochim. biophys. Acta 788, 117-123. Sugimoto Y., Saito A., Kusakabe T., Hod K. and Koga K. (1989) Flow of egg white ovalbumin into the yolk sac during embryogenesis. Biochim. biophys. Acta 992, 400-403. Tewkesbury D. A. and Carrell R. W. (1989) Ovalbumin and angiotensinogen lack serpin S-R conformational change. Biochem. J. 262, 103-107. Tsai S. Y., Wang H., Wang L.-H., Sagami I., Bagchi M., Tsai M.-J. and O'Malley B. W. (1988) A novel transcription factor which binds to the chicken ovalbumin upstream promoter sequence. In Mechanisms o f Control o f Gene Expression, pp. 137-153. Alan R. Liss, Inc. Turk V., Brzin J., Longer M., Ritonia A. and Eropkin M. (1983) Protein inhibitors of eysteine proteinases. III. Amino acid sequence of cystatin from chicken egg white. Hoppe-Seyler's Z. physiol. Chemic 364, 1487-1496. Walker M., Stevens L., Duncan D., Price N. C. and Kelly S. M. (1991) A comparative study of the structure of egg-white riboflavin binding protein from the domestic fowl and Japanese quail. Comp. Biochem. Physiol. (submitted). Weaver L. H., Grutter M. G., Remington S. J., Gray T. M., Isaacs N. W. and Matthews B. W. (1985) Comparison of goose-type, chicken-type, and phage-type lysozymes illustrates the changes that occur in both amino acid sequence and thrce-dimensional structure during evolution. Y. molec. EvoL 21, 97-111. White H. B. (1987) Vitamin-binding proteins in the nutrition of the avian embryo. J. exp. Zool. (suppl.) 1, 53-63.

Egg white protzins White H. B. and Merrill A. H. (1988) Riboflavin.binding proteins. A. Rev. Nutr. 8, 279-299. Wilchek M. and Bayer E. A. (1990) Avidin-biotin technology. Meth. Enzymol. Vol 184. Williams J. (1968) A comparison of glycopeptides from the ovotransferdn and serum transferdn of the hen. Biochem. J. 108, 57-67. Williams J. (1982) The evolution of transferrin. Trends biochem. Sci. 7, 394-397. Williams J. and Moreton K. (1988) The dimerization of half-molecule fragments of transferrin. Biochem. J. 251, 849--855.

Williams J., Moreton K. and Goodeafl A. D. J. (1985) Selective reduction of a disulphide bridge in hen ovotransferrin. B~ochem. J. 7,28, 661-665. Winter W. P., Buss E. G., Clagett C. O. and Boucher R. V. (1967) The nature of the biochemical lesion in avian renal riboflavinuria--II. The inherited change of a riboflavin-binding protein from blood and eggs. Comp. Biochem. Physiol. 22, 897-906. Zahnley J. C. (1974) Evidence that the two binding sites for trypsin on chicken ovoinhibitor are not equivalent. J. biol. Chem. 249, 4282-4285.