Pergamon Comp. Biochem. PhysioL Vol. 107B, No. 4, pp. 59%604, 1994 Elsevier Science Ltd Printed in Great Britain 0305-0491/94 $6.00 + 0.00 Purification and characterization of the riboflavin- binding protein from goose (Anser anser) egg yolk Lewis Stevens, Kay Nicol, Sharon M. Kelly, Catherine Scott, J. S. Grant Reid and Nicholas C. Price Department of Biological and Molecular Sciences, University of Stirling, Stirling FK9 4LA, Scotland, U.K. The riboflavin-binding protein, purified from goose egg yolk, is significantly larger than those previously isolated from other avian species. This is, in part, due to a larger polypeptide chain, but mainly to a much higher carbohydrate content, principally glucose and sialic acid. Approximately one-third of the glucose residues can be released by incubation with a mixture of exo-l,4-0c- and exo-l,4-p-D-glucosidases. The amino acid composition is similar to that of other riboflavin-binding proteins, but the peptide maps are distinct from those of domestic fowl. The goose egg yolk protein has a much higher proportion of p-sheet than either domestic fowl or quail riboflavin-binding protein. Key words: Riboflavin-binding protein; .4nser anser. Comp. Biochem. Physiol. I07B, 597-604, 1994. Introduction Riboflavin-binding protein (RfBP) is present in both avian egg yolk and egg white, in contrast to reptilian eggs where it is only present in the yolk (White and Merrill, 1988). It has an im- portant role in the uptake of riboflavin to the developing oocyte. In mutants of the domestic fowl lacking RfBP, the developing embryos die of riboflavin deficiency at around 13 days of incubation (Winter et al., 1967). It is at this stage that a rapid increase occurs in flavin kinase activity, required for the synthesis of FMN and FAD (Zak and McCormick, 1982). RfBPs from domestic fowl have been investi- gated most thoroughly. Those from egg white, egg yolk and plasma have been purified and sequenced (Norioka et al., 1985). All three proteins are the product of the same gene although plasma and egg-yolk RfBP are syn- thesized in the liver, and egg-white RfBP in the Correspondence to: L. Stevens, Department of Biological and Molecular Sciences, University of Stirling, Stirling FK9 4LA, Scotland, U.K. Tel: 44 786-467783; Fax: 44 786-464994. Abbreviations---CD, circular dichroism; RfBP, riboflavin- binding protein. Received 19 July 1993; accepted 22 September 1993. oviduct. All three show polymorphism at pos- ition 14. Egg-yolk RfBP differs from the other two in having 11-13 fewer amino acids residues. When the plasma RfBP is taken up into the egg yolk the C-terminal peptide (11/13 residues) is cleaved. There are also some differences in the glycosylation pattern, although in all three the oligosaccharides are linked by asparagines 36 and 147 (Miller et al., 1982). Little detailed characterization has been car- ried out on any RfBPs from other avian sources. Walker et al. (1991) have shown that RfBP from quail egg white shows a distinct peptide map from that of domestic fowl after cleavage with thermolysin, chymotrypsin and V8 protease, and also has differences in the tertiary structure as revealed by the near UV CD spectra, although the far UV CD spectra are similar, indicative of similar secondary structures. RfBP has been purified from duck egg white, and it is reported to have a quite distinct amino acid composition from that of the domestic fowl (Muniyappa and Adiga, 1980). Duck egg-white RfBP is reported to lack proline, has a much lower proportion of methionine and arginine residues, but has higher proportions of valine, 597
Purification and characterization of the riboflavin- binding protein from goose (Anser anser) egg yolk
Lewis Stevens, Kay Nicol, Sharon M. Kelly, Catherine Scott, J. S. Grant Reid and Nicholas C. Price Department of Biological and Molecular Sciences, University of Stirling, Stirling FK9 4LA, Scotland, U.K.
The riboflavin-binding protein, purified from goose egg yolk, is significantly larger than those previously isolated from other avian species. This is, in part, due to a larger polypeptide chain, but mainly to a much higher carbohydrate content, principally glucose and sialic acid. Approximately one-third of the glucose residues can be released by incubation with a mixture of exo-l,4-0c- and exo-l,4-p-D-glucosidases. The amino acid composition is similar to that of other riboflavin-binding proteins, but the peptide maps are distinct from those of domestic fowl. The goose egg yolk protein has a much higher proportion of p-sheet than either domestic fowl or quail riboflavin-binding protein.
Introduction Riboflavin-binding protein (RfBP) is present in both avian egg yolk and egg white, in contrast to reptilian eggs where it is only present in the yolk (White and Merrill, 1988). It has an im- portant role in the uptake of riboflavin to the developing oocyte. In mutants of the domestic fowl lacking RfBP, the developing embryos die of riboflavin deficiency at around 13 days of incubation (Winter et al., 1967). It is at this stage that a rapid increase occurs in flavin kinase activity, required for the synthesis of FMN and FAD (Zak and McCormick, 1982). RfBPs from domestic fowl have been investi- gated most thoroughly. Those from egg white, egg yolk and plasma have been purified and sequenced (Norioka et al., 1985). All three proteins are the product of the same gene although plasma and egg-yolk RfBP are syn- thesized in the liver, and egg-white RfBP in the
Correspondence to: L. Stevens, Department of Biological and Molecular Sciences, University of Stirling, Stirling FK9 4LA, Scotland, U.K. Tel: 44 786-467783; Fax: 44 786-464994.
Received 19 July 1993; accepted 22 September 1993.
oviduct. All three show polymorphism at pos- ition 14. Egg-yolk RfBP differs from the other two in having 11-13 fewer amino acids residues. When the plasma RfBP is taken up into the egg yolk the C-terminal peptide (11/13 residues) is cleaved. There are also some differences in the glycosylation pattern, although in all three the oligosaccharides are linked by asparagines 36 and 147 (Miller et al., 1982).
Little detailed characterization has been car- ried out on any RfBPs from other avian sources. Walker et al. (1991) have shown that RfBP from quail egg white shows a distinct peptide map from that of domestic fowl after cleavage with thermolysin, chymotrypsin and V8 protease, and also has differences in the tertiary structure as revealed by the near UV CD spectra, although the far UV CD spectra are similar, indicative of similar secondary structures. RfBP has been purified from duck egg white, and it is reported to have a quite distinct amino acid composition from that of the domestic fowl (Muniyappa and Adiga, 1980). Duck egg-white RfBP is reported to lack proline, has a much lower proportion of methionine and arginine residues, but has higher proportions of valine,
597
598 Lewis Stevens et al.
phenylalanine and histidine (see Table 2). Riboflavin is present in the egg white and egg yolk from domestic fowl and related species, but this appears unusual; in many birds the binding protein is present in both yolk and white, but the egg white is practically devoid of riboflavin (White, 1990).
Because of the differences in amino acid composition of egg-white RfBP reported for duck and domestic fowl, a wider range of species needs to be investigated in order to establish the common features and differences. In a preliminary investigation in which we used polyacrylamide gel electrophoresis to separate egg-white RfBPs from a number of species, there appeared to be a higher concentration of RfBP in the order Galliformes than in Anseri- formes. A partial purification of RfBP from goose egg white was carried out but its concen- tration in egg white was less than 15 mg/100 ml compared with about 100 mg/100 ml for dom- estic fowl egg white. The goose egg yolk, on the other hand, appeared to be a much richer source, and we therefore undertook the purifi- cation and characterization of RfBP from this source. It appears to have a significantly higher Mr than other RfBPs, and an unusual carbo- hydrate composition.
Materials and Methods Enzymes
TLCK-treated chymotrypsin, thermolysin (protease Type X), V8 protease (protease Type XVII), amyloglucosidase (exo-l,4-ct-glucosi- dase) and fl-D-glucosidase were purchased from Sigma Chemical Co. (St Louis, MO).
Purification of riboflavin-binding protein from goose (Anser anser) egg yolk
Eggs from domestic geese (Anser anser) were obtained from a local source. The whites and yolks were separated and either used immedi- ately or stored at -12°C. RfBP was purified from goose egg yolk using a modification of the procedure described for egg yolk (Miller and White, 1986). Goose egg yolks (700ml) were mixed with an equal volume of 0.1 M sodium acetate buffer, pH 4.3, and then centrifuged at 12,000g for 1 hr. All subsequent procedures were performed at 4°C. The supernatant was dialysed against three changes of distilled water and then against 0.1 M acetate buffer, pH 4.3. The sample was mixed with 4 g swollen Sepha- dex A-50 for 2 hr. The mixture was then poured into a column, washed with 0.05 M sodium acetate buffer, pH 5.5, and then eluted with the same buffer containing 0.5 M NaCI. The peak fractions were collected, dialysed against 0.05 M
sodium acetate buffer, pH 5.5, and then passed down a second Sephadex A-50 column. The second column was eluted using the same buffer containing increasing concentrations of NaC1 (0.1, 0.15 and 0.5M). RfBP eluted with the 0.5 M NaC1. The peak fractions contained a contaminating protein of higher MT. This was separated from RfBP by passing the peak frac- tions down a Sephadex G-100 column (2.5 cm diameter × 70 cm).
Monitoring of the purification and analyses of the final preparation for homogeneity were performed by polyacrylamide gel electrophor- esis (Laemmli, 1970) both in the presence and absence of SDS. In both, 10% polyacrylamide was used, and in the latter Tris-glycine buffer, pH 8.3, was used (Stevens and Duncan, 1988). When electrophoresis under non-denaturing conditions is performed at pH 8.3, the RfBP moves ahead of all other proteins because of its low pI, and so is easily resolved (Stevens and Duncan, 1988).
For the experiments in which apo-RfBP was required, it was separated from riboflavin on CM-Sephadex C-50 equilibrated at pH 3.8 (Miller and White, 1986).
Circular dichroism and fluorescence spectra
Circular dichroism spectra were recorded at 20°C in a JASCO J-600 spectropolarimeter. Far UV (190-260 nm) spectra were recorded at pro- tein concentrations of about 0.5 mg/ml using a cell of path-length 0.02 cm. Molar ellipticity values were calculated using a value of 112.9 for the mean residue weight of the polypeptide component, derived from the amino acid com- position. The secondary structure content was determined by using the CONTIN procedure over the range 190-240 nm (Provencher and G16ckner, 1981).
The quenching of fluorescence of riboflavin by the apo-riboflavin-binding protein was measured as emission at 520 nm after excitation at 370 nm (Murthy et al., 1976).
Phosphate, carbohydrate and protein estimation
Phosphate was determined by the method of Eng and Noble (1968) after hydrolysis with 70% (v/v) perchloric acid at 180°C for 3 hr.
RfBP was analysed for carbohydrates after hydrolysis using trifluoroacetic acid for 1 hr at 80°C for sialic acid and 1-4hr at 100°C for monosaccharides. The hydrolysed samples were then freeze-dried and redissolved in 0.1 ml dis- tilled water. Monosaccharides and sialic acid were separated by high-performance ion- exchange chromatography (Dionex BioLC with CarboPac-PA1 column, pulsed amperometric detector and post-column base addition). The column (250 × 4 mm; flow rate: 1 ml/min) was
Goose egg-yolk riboflavin-binding protein 599
eluted with 20 mM NaOH for 2 min, with water for 27 min and with a linear gradient of NaOH (0-100 mM) over 20 min. The monosaccharides were eluted with the water, and the sialic acid in the NaOH gradient. The relative proportions of the carbohydrates were calculated assuming identical molar response factors. The identity of glucose was confirmed by assaying the trifluo- racetate hydrolysate using glucose oxidase (Bergmeyer, 1974). Polypeptide determination was by the Biuret method (Layne, 1957) using bovine serum albumin as standard.
Amino acid analysis
Amino acid analysis was performed on RfBP by the Welmet Edinburgh Protein Characteris- ation Facility (Department of Biochemistry, University of Edinburgh, Edinburgh, U.K.). Half-cysteine residues were determined after reduction and pyridinylethylation.
Determination of the tyrosine and trypto- phan content of the protein was performed using the method of Edelhoch (1967) which is based on measurement of A280 and A288 in the presence of 6 M guanidine hydrochloride (Ultrapure grade from Gibco-BRL, Paisley, Scotland, U.K.), and also on the AA295 after the addition of KOH. The concentrations of guanidine hydrochloride solutions were checked by measurements of refractive index (Nozaki, 1972).
Peptide mapping
Purified RfBP (approximately 30#g) from goose egg yolk and domestic fowl egg white were digested separately with TLCK-treated chymotrypsin (5/~g), thermolysin (5/~g) and protease from Staphylococcus aureus strain V8 (10/~g) in 0.125 M Tris-HCl buffer 0.5% SDS, but in the absence of reducing agent, for 60 min at 37°C, and then separated on 12% polyacryl- amide gels as described by Cleveland et al. (1977).
Results Purification and Mr determination
The purified RfBP from goose egg yolk ran as a single band both by SDS-polyacrylamide gel electrophoresis, and by polyacrylamide gel elec- trophoresis at pH 8.3 under non-denaturing conditions in the absence or SDS. Under the latter conditions RfBPs run well ahead of all other proteins initially present in the egg yolk.
Electrophoretic mobilities of purified goose egg-yolk RfBP, domestic fowl egg-white RfBP, and a partially purified goose egg-white RfBP were compared with those of dalton markers. Their mobilities corresponded with the follow- ing Mr values: goose egg-yolk RfBP 37,000,
goose egg-white 36,500, domestic fowl egg-white 33,500. Although there is a slight difference between the mobilities of goose egg-yolk and egg-white RfBP, it is within the limits of resol- ution of the method. However, the difference between these and that of domestic fowl egg- white RfBP is clearly significant. It is known that in the case of domestic fowl egg-white RfBP there is a significant difference between the apparent Mr of 34,000 determined by SDS- polyacrylamide electrophoresis and the value of 29,200, calculated from the amino acid sequence and the carbohydrate and phosphate compo- sition (Hamazume et al., 1984) and this has been attributed to the presence of carbohydrate. For this reason other methods of Mr determination of the goose egg-yolk RfBP were performed.
Riboflavin binds strongly to apo-RfBP (e.g. for the domestic fowl RfBP, Ka = 5.0 x 108 M-I; Nishikimi and Kyogoku, 1973) and the binding is accompanied by a quenching of the riboflavin fluorescence (excitation 370 nm, emission 520 nm), with 1:1 stoichiometry (Becvar and Palmer, 1982). This was used to titrate goose egg-yolk apo-RfBP against known concen- trations of riboflavin. From the equivalence point the Mr of the apo-RfBP corresponded to 43,500. The apoprotein concentration used for this calculation was that determined by the Biuret method using bovine serum albumin as standard. It is assumed to be the M, of the polypeptide alone. A third method was used to estimate Mr using a Sephadex G-100 column (2.5cm diameter, 75cm length) equilibrated with 0.1 M KCI and calibrated with Mr markers. In this case the goose egg-yolk RfBP eluted ahead of bovine serum albumin, corre- sponding with an apparent Mr ~ 74,000. For comparison, domestic fowl egg-white RfBP, which has reported mobilities equivalent to Mr values between 30,000-36,000 on SDS- polyacrylamide gel electrophoresis (see White and Merrill, 1988), eluted from G-100 in a position corresponding to 41,000. Because of the apparent discrepancies between Mr values obtained by different methods it was important to determine the carbohydrate and phosphate contents of goose RfBP.
Carbohydrate and phosphate analysis
The goose egg-yolk RfBP was hydrolysed using trifluoracetic acid for different times and temperatures in order to optimize the yield of monosaccharides and sialic acid. For monosac- charides, 4 hr at 100°C gave the optimum yield, and for sialic acid determinations, 1 hr at 80°C was used. There were two main peaks eluting from the high-performance anion-exchange column with retention times corresponding to glucose and sialic acid. Sialic acid elutes well
600 Lewis Stevens et al.
separated from other carbohydrates, and the retention time corresponded well with that of the standard, and so there was little doubt about its identity.
The presence of glucose as the other main component was surprising, since it is not usually found in glycoproteins and has not been found in any other RfBP to date. Several other mono- saccharides elute from the high-performance anion-exchange column close to glucose, and so further substantiating evidence was important. When an internal glucose standard was mixed with the hydrolysate and eluted from the column, it co-eluted with the purported glucose peak with no evidence of any shoulder. To further confirm the peak as glucose, glucose estimation using glucose oxidase was carried out on the hydrolysate. Not only was the presence of glucose detected, but the amount present corresponded with that estimated by high- performance anion-exchange chromatography. A third, much smaller, peak eluting from the column had a retention time which corre- sponded with either galactose or glucosamine, but no further work has been performed to differentiate between these.
The carbohydrate content of goose egg-yolk RfBP based on the sum of the identifiable carbohydrates is 38% (Table 1). The carbo- hydrate contents of domestic fowl egg-yolk and domestic fowl egg-white RfBP are estimated to be 13.7-15% and 10.7-13%, respectively (Miller et al., 1982; Hamazume et al., 1984), the principal components being mannose, galac- tose, N-acetylglycosamine and sialic acid. When we analysed domestic fowl egg-white RfBP using high-performance anion-exchange chro- matography, as described in the Materials and Methods section, the sum of the glucosamine, galactose, mannose and sialic acid was 8.5%, slightly lower than the 10.7% obtained by Hamazume et al. (1984).
The phosphate moiety, determined after RfBP hydrolysis with perchloric acid, was 2.3% of the polypeptide content. The results of the carbo- hydrate and phosphate analyses are summarized on Table 1, together with the calculated number of moles of each residue based on a polypeptide
Mr = 43,500. The amount of carbohydrate is surprisingly large and, when added together with the phosphate residues, gives an aggregate Mr which corresponds quite closely to that obtained from the G-100 gel-filtration column.
The carbohydrate content is quite different from that present in other RfBPs, both because of the large amount of it and the presence of glucose. In domestic fowl egg-white, egg-yolk and plasma RfBP there are two oligosaccharides linked to asparagines 36 and 147. In order to try to obtain more information as to how the glucose residues are linked together, a prelimi- nary experiment was carried out in which 0.87mg goose egg-yolk RfBP was incubated with 510 units of amyloglucosidase (exo-l,4-~- glucosidase) and 56 units of fl-glucosidase in 0.05 M acetate buffer, pH 5.0, at 18°C for 2 days in a dialysis bag. After the incubation, the RfBP was run on SDS-polyacrylamide gel electro- phoresis together with an untreated RfBP sample. The mobility of the treated RfBP in- creased by 6% compared with the unincubated RfBP. The glucose content of the diffusate corresponded with the release of 35-40% of the glucose residues present in RfBP. A control incubation mixture of amyloglucosidase and fl-glucosidase but lacking RfBP was carried out to ensure that all the glucose in the diffusate originated from RfBP.
Amino acid analysis and peptide mapping
It was important to carry out an amino acid analysis of goose egg-yolk RfBP, particularly in view of the reported differences in composition of domestic fowl egg-white RfBP and duck egg-white RfBP (Muniyappa and Adiga, 1980; Hamazume et al., 1984). The results of the amino acid analysis of goose egg-yolk RfBP are given in Table 2, together with those already determined for domestic fowl (Hamazume et al., 1984) and duck (Muniyappa and Adiga, 1980). The tyrosine and tryptophan contents were ob- tained by spectrophotometric analysis. The re- suits are expressed both as mole %, and as residue number based on polypeptide Mr. The mole % enables comparison of the amino acid composition between the three species, which is
Table 1. Overall composition of goose egg-yolk riboflavin-binding protein Component % Composition Number of residues* Contribution to M r Amino acid residues 60 385 43,500 Glucose residues 19 88 14,256 Sialic acid residues 16 42 12,222 Glucosamine/galactose residues 3 14 2254 Phosphate residues 1.4 13 1001
Total 73,233 *Based on the M r of the polypeptide component = 43,500 together with its overall %
composition.
Goose egg-yolk riboflavin-binding protein 601
Table 2. Comparison of amino acid composition of riboflavin-binding proteins from goose egg yolk, hen egg yolk* and duck egg white1"
*Data from Hamazume et al. (1984); *data from Muniyappa and Adiga (1980); J;normalized to Mr = 43,500; §normalized to Mr = 34,000 (Hamazume et al., 1984); Ilnormalized to Mr = 36,000. For details of amino acid residue determination in goose egg-yolk RiBP, see Materials and Methods section.
m o r e diff icult w i th the res idue n u m b e r since all th ree are n o r m a l i z e d on d i f ferent Mr values .
T h e m o l e % c o m p o s i t i o n o f goose egg y o l k a n d d o m e s t i c fowl a re qu i t e s imi lar ; the la rges t d i f fe rence is in the r a t ios o f G l u / A s p , a l t h o u g h
the to ta l c o n t e n t s o f ac id ic a m i n o ac ids are s imilar . T h e r e a p p e a r to be s igni f icant differ- ences b e t w e e n the goose a n d d u c k pa r t i cu l a r ly in the p ro l ine , a rg in ine , m e t h i o n i n e , pheny l - a l an ine a n d h is t id ine con ten t s .
M r
66000
45000
36000
29000
240O0
20100
14200
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Fig. I. Peptide mapping of riboflavin binding protein from domestic fowl (DRfBP) and goose (GRfBP) using chymotrypsin, thermolysin and V8 protease. Separation of the digests was carried out on 12% polyacrylamide gels containing SDS. Lanes l, 7, 13 contain dalton marker proteins and their M r are shown in the margin. The contents of the other lanes were as follows: 2, chymotrypsin; 3, DRfBP; 4, DRfBP+chymotrypsin 5, GRfBP; 6, GRfBP+chymotrypsin; 8, thermolysin; 9, DRfBP; 10, DRfBP+thermolysin; II, GRfBP; 12, GRfBP+thermolysin; 14, V8 protease; 15, DRfBP; 16,
Fig. 2. CD spectra of riboflavin proteins from domestic fowl (---) and goose (--). Spectra were recorded as described in
the text.
Peptide mapping was carried out using three different enzymes, chymotrypsin, thermolysin and V8 protease (Fig. 1). These proved to be the most useful in a previous comparative study between domestic fowl and quail RfBP (Walker et al., 1991). In the present study a different pattern of peptides was obtained from domestic fowl egg-white and goose egg-yolk RfBP with each enzyme. Trypsin digested only a small proportion of domestic fowl RfBP, just de- tectable on the leading edge of the unchanged RfBP (lane 4). By contrast, goose RfBP pro- duced a major band of apparent Mr 32,000 (lane 6). With thermolysin, which has a broader specificity, most of the RfBP is digested in both cases giving one band common to both (appar- ent Mr 30,000) and also three distinct bands (apparent Mr 27,000 and 19,000 in domestic fowl, lane 10, and apparent Mr 22,000 in goose, lane 12). V8 protease also gave some common bands and some distinct bands (lanes 16 and 18).
Circular dichroism spectra
The far UV CD spectra of the holoforms of RfBPs from goose egg yolk and domestic fowl egg white are compared in Fig. 2. There is a significant difference between them indicative of differences in secondary structures. Analysis of the spectra by the method of Provencher and G16ckner (1981) gives the following structural contents: domestic fowl 24% ~-helix, 39% fl- sheet, 37% remainder; goose 8% ~-helix, 6 4 o fl-sheet, 28% remainder.
D i s c u s s i o n
The goose egg-yolk RfBP has a number of properties in common with those previously characterized (Hamazume et al., 1984; White and Merrill, 1988; Muniyappa and Adiga, 1980; Walker et al., 1991) as would be expected of
homologous proteins. It has similar amino acid composition, it is multiply phosphorylated, and binds riboflavin with high affinity. By compar- ing the amino acid compositions the difference index (DI) can be calculated to obtain an indi- cation of relatedness (Cornish-Bowden, 1983). When applied to data in Table 2 it gives the following values: goose and domestic fowl 7.30, goose and duck 19.1, and domestic fowl and duck 16.8. For proteins with a polypeptide Mr between 30,000 and 40,000, values of ~< 7.5-8.5 are indicative of their being strongly related, values of i> 11-13 are indicative of their being weakly related (Cornish-Bowden, 1983). The goose and domestic fowl RfBP thus fall just within the "strongly related" criterion as far as the polypeptide component is concerned. How- ever, even though the amino acid compositions are similar, there are undoubtedly sequence differences which are revealed by the peptide mapping.
The most significant differences between goose egg-yolk RfBP and other RfBPs so far characterized are its apparently higher Mr, higher carbohydrate content and, in particular, the presence of glucose residues. The proposed Mr in the region of 73,000 is based on a number of assumptions. The first of these is the assump- tion that goose egg-yolk RfBP has a single binding site for riboflavin. An alternative possi- bility would be that RfBP was a dimer with two riboflavin-binding sites, and that it dissociated in SDS. This might explain the difference be- tween the apparent Mr determined by SDS- polyacrylamide gel electrophoresis and gel filtration. This seems unlikely for two reasons. Firstly, it could not also account for the high carbohydrate content. Secondly, all egg proteins which have so far been characterized are monomeric, and many, including RfBP, are rich in disulphide bridges, characteristic of many extracellular proteins, which are rarely oligomeric. There is a precedent for a larger egg protein in the goose, namely lysozyme which has 185 amino acid residues compared with 129 in the domestic fowl egg-white lysosome (Weaver et al., 1985). There is little sequence identity between the two lysozymes.
If the Mr of goose RfBP, determined by fluorescence quenching is correct, then the atyp- ical mobility on SDS-polyacrylamide gel elec- trophoresis remains to be explained.
Determination of Mr by gel filtration and SDS-polyacrylamide gel electrophoresis are both comparative methods which depend for their validity on the "normal" behaviour of the protein concerned. Glycoproteins are known to give anomalous results for both. The gel filtration depends on average volume occupied by the protein in solution (Stokes radius), and
Goose egg-yolk riboflavin-binding protein 603
this parameter is particularly sensitive to the shape and physical properties of the protein. The branching of the chain caused by the oligo- saccharides means that glycoproteins may have an unusual shape. The electrophoretic mobility of a protein in SDS-polyacrylamide gel electro- phoresis depends on its having a uniform coat- ing of SDS, on average one SDS per two amino acid residues. Glycoproteins are unable to bind the normal amounts of SDS because of steric hindrance by the oligosaccharide residues. In a detailed study of the behaviour of glycoproteins in SDS-polyacrylamide electrophoresis, Leach et al. (1980) found that glycoproteins gave abnormally high Mr estimates, and that selective removal of sialic acid residues increased their mobility. They also concluded from the proteins studied that it was not possible to make a reliable correction of the Mr based on its carbo- hydrate content. Since goose egg-yolk RfBP has an unusual carbohydrate composition when compared with typical glycoproteins, and when taken together with the high carbohydrate con- tent, it may behave differently on SDS- polyacrylamide gel electrophoresis. This is suggested by the observation that although its mobility on SDS-polyacrylamide gel electro- phoresis is similar to domestic fowl RfBP (ap- parent Mr 37,000 and 33,500), its elution from Sephadex G-100 is very different (apparent Mr 74,000 and 41,000). In order to resolve this it will be necessary to study the completely degly- cosylated protein. Although both goose and domestic fowl RfBPs have similar amino acid compositions, the sequence differences evident from the peptide maps are more important in accounting for the differences in secondary structure deduced from the CD spectra. It is also possible that the differences in the carbo- hydrate content and composition lead to differ- ences in the secondary structures of the goose and domestic fowl RfBP.
A further apparent difference between the goose and domestic fowl RfBP is the relation- ship between the egg-yolk and egg-white pro- teins. In domestic fowl 11-13 amino acids are cleaved from the plasma RfBP on entering the yolk, lowering its Mr by 1400. In the goose there is no evidence that the egg-white protein is smaller than that of the egg yolk, judged by electrophoretic mobility. Goose egg-yolk RfBP appears to have a larger number of phosphate residues (13 compared to seven or eight) than that reported for domestic fowl egg-yolk protein. It has been suggested that the enzyme catalysing the RfBP phosphoryl- ation recognizes the motif-S-X-E/S(P) (Roach, 1991) which may be more abundant in goose egg-yolk RfBP, and hence lead to more phos- phorylation.
In conclusion, this characterization of goose egg-yolk RfBP has revealed some interesting differences from other RfBPs, particularly in the nature of the carbohydrate moiety. The large amount of carbohydrate present in goose egg- yolk RfBP is in marked contrast to that of duck egg white, where it is reported to be absent (Muniyappa and Adiga, 1980). The function of the carbohydrate moiety, where present, is un- known. Further work on the carbohydrate is important both for the further understanding of the RfBP structure, and the possible influence of the carbohydrate on the polypeptide confor- mation. It may also provide clues as to its function.
Acknowledgements--We thank the Science and Engineering Research Council for provision of the c.d. facility, and Dr S. Provencher for supplying the CONTIN Program.
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