Comp. Biochem. Physiol. Vol. 100B, No. 1, pp. 77-81, 1991 0305-0491/91 $3.00+0.00 Printed in Great Britain Pergamon Press pie

A COMPARATIVE STUDY OF THE STRUCTURE OF EGG-WHITE RIBOFLAVIN BINDING PROTEIN FROM

THE DOMESTIC FOWL AND JAPANESE QUAIL

MAR~ON WALKER, LL~ws ST~WNS,* DOmS DUNCAN, NICHOLAS C. PRICE and SHAnON M. KELLY

Department of Biological and Molecular Sciences, University of Stirring, Stirling FK9 4LA, Scotland, UK (Tel: 0786 73171); (Fax: 44 786 64994)

(Received 6 March 1991)

Abstract--1. The riboflavin binding proteins from domestic fowl and Japanese quail have been isolated and their structures compared by circular dichroism, fluorescence and peptide mapping.

2. The two proteins have similar secondary structures, but differ in their tertiary structures as reflected in the environments of aromatic amino acid side chalus.

3. Differences in amino acid sequence between the proteins are indicated by the digestion patterns obtained with thermolysin, chymotrypsin and V8 proteinase from Staphylococcus aureus. Both proteins are resistant to digestion by trypsin.

INTRODUCTION

A number of comparative studies have been made of the proteins from egg white of different avian species (Feeney and Allison, 1969; Joll~s et al., 1979; Fukamizo et al., 1983; Iwase et al., 1984; Weaver et al., 1985), the most extensive being that for ovomucoid (Laskowski et al., 1987). In the latter, the third domain of ovomucoid was sequenced from I00 different avian species. The physiological roles of several of the egg white proteins, e.g. ovalbumin, ovomucoid, ovoinhibitor, ovostatin, cystatin and ovoglobulins, are unclear. Some may simply act as a nutrient reserve for the developing ~mbryo, in which case their precise structure may not be important. Others are known to be proteinase inhibitors, but the relevance of this is not understood. One group of proteins to which a function can be assigned with reasonable certainty is the group of binding proteins. Of these, conalbumin, which binds Fe 2+ is the most abundant. Three vitamin binding proteins are known: avidin, riboflavin binding protein (RfBP) and thi- amin binding protein. These are believed to be im- portant in maintaining the supply of vitamins to the developing embryo (White, 1987; White and Merrill, 1988).

The essential role of the RfBP has been demon- strated from a study of the homozygous recessive mutant (rdrd) of the domestic fowl (Winter et al., 1967). Developing embryos having this genetic con- stitution die at around 13 days incubation, from riboflavin deficiency. This is at present the only documented example of a lethal mutant involving an egg-white protein.

It is therefore useful to make a comparative study of a protein for which the function is known, and in

*Author to whom correspondence should be addressed. Abbreviations used---c.d., circular dichroism; R.fBP, ribo-

flavin binding protein.

77

which the function must impose evolutionary con- straints on the structure. RfBP from the domestic fowl has been purified (Rhodes et al., 1959) and sequenced (Hamazume et al., 1984). The binding of riboflavin to the apoprotein is accompanied by a quenching of fluorescence of riboflavin, which is most likely to be due to stacking of the ravin with aromatic amino acid residues (Farrell et al., 1969). Although the three-dimensionai structure of the RfBP has not been determined, a preliminary study of its circular dichroism spectrum has been made (Nishikimi and Kyogoku, 1973).

In this work we have purified RfBP from both the domestic fowl and Japanese quail and compared their structures using circular dichroism, fluorescence spec- troscopy, and peptide mapping (Cleveland et al., 1977). In another egg-white protein, lysozyme, there are six amino acid replacements between domestic fowl and quail, but none of these occur in the substrate binding site (Fukamizo et al., 1983).

MATERIALS AND ~ T H O D S

Purification of riboflavin binding proteins from egg white Commercially available domestic fowl eggs were used,

and Japanese quail (Coturnix japonica) eggs were obtained from a local source. RfBPs were prepared by a scaled down modification of the method of Hamazume et al. (1984). Egg white (20 nil) was homogenized and the pH adjusted to 5.5 with 1 M HCI; the precipitated protein was removed by centrifugation. The supernatant was mixed with preswollen DEAE--Sephadex A-50 (0.4 g per 100 mi supernatant). After standing for 60 min at 4°C the suspension was poured into a column (15ram diameter, 120mm length) and washed successively with 20 ml 0.05 M sodium acetate buffer pH 5.5, followed by 20ml 0.05 M sodium acetate buffer pH 5.5 containing 0.15M NaCI, and finally 50ml 0.05M sodium acetate buffer pH 5.5 containing 0.5 M NaCI. Hution of the riboflavin-binding protein was monitored by A~0. The pooled fractions were concentrated by dialysis against 50% w/w polyethylene glycol 6000, and then dialyzed overnight against distilled water. They were then further purified by a

78

Mr 66000

MAR~ON WALKER et al.

45000

36000

29000 24000

20100

14200

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Fig. 1. Peptide mapping of riboflavin binding protein from domestic fowl (DRfBP) and quail (QRfBP) using trypsin, chymotrypsin, thermolysin and V8 proteinase. Separation of the digests was carried out on 12% polyacrylamide gels containing SDS. Lanes 6, 12 and 18 contained Dalton marker proteins and their M, are shown in the margin. The contents of the other lanes was as follows: 1. DRfBP; 2. DRfBP+trypsin; 3. trypsin; 4. QRfBP; 5. QRfBP+trypsin; 7. chymotrypsin; 8. QRfBP; 9. QRfBP + chymotrypsin; 10. DRfBP + chymotrypsin; I 1. QRfBP + chymotrypsin; 13. DRfBP; 14. DRfBP+thermolysin; 15. thermolysin; 16. QRfBP; 17. QRfBP+thermolysin; 19. DRfBP; 20.

DRfBP + V8 proteinase; 21. V8 proteinase; 22. QRfBP; 23. QRfBP + V8 proteinase.

second passage down a DEAE-Sephadex A-50, eluting as before.

To prepare the apoprotein, the peak fraction was dialyzed against 0.1 M sodium acetate buffer pH4.0 to release ri- boflavin (Murthy et al., 1976), and then adjusted to pH 7.0 by dialysis against 0.1 M sodium phosphate buffer pH 7.0.

Peptide mapping

Purified RfBP (approximately 30 #g) from domestic fowl and quail were digested separately with TPCK-treated trypsin (1 #g). TLCK-treated chymotrypsin (5 #g), ther- molysin (5 #g) and proteinase from Staphylococcus aureus strain V8 (10 #g) in 0.125 M Tris-HC1 buffer 0.5% SDS, but in the absence of reducing agent, for 60 min at 37°C, and then separated on 12% w/v polyacrylamide gels as described by Cleveland et al. (1977).

Circular dichroism, fluorescence and absorption spectra C.d. spectra were recorded at 20°C in a JASCO J-600

spectropolarimeter, Far u.v. (190-260nm) and near u.v. (260-320 nm) spectra were recorded at protein concen- trations of about 0.5 mg/ml using cells of pathlength 0.02 cm and 1 cm respectively. Molar ellipticity values were calcu- lated using a value of 133.3 for the mean residue weight, a value which takes into account the carbohydrate and phos- phoryl group content of the protein (Hamazume et al., 1984). The secondary structure content was determined by using the CONTIN procedure over the range 190-240 nm (Provencher and Glfckner, 1981). The ~t-helical content was also determined by the method of Siegel et al. (1980).

The concentration of domestic fowl RfBP was deter- mined speetrophotometrically using the published value of A2~ 2 = 1.68 for a 1 mg/ml solution (path-length = 1 cm) (Nishikimi and Kyogoku, 1973). The concentration of the quail RfBP was determined relative to that of the domestic fowl using the method of Waddell (1956), based on measure- ments of A2, 5 and A225, which directly measures the concen- tration of peptide bonds. Using this procedure, the A2a 2 for

the quail protein was found to be the same as that of the domestic fowl protein within 2%. Determinations of the concentration of the quail protein by the Coomassie Blue binding method (Sedmak and Grossberg, 1977) using bovine serum albumin as a standard gave values within 10% of that obtained by measurements of A282.

Fluorescence spectra were recorded at 20°C in a Perkin Elmer MPF 3A fluorimeter with excitation at 280 nm. The protein concentration was 0.055 mg/ml. The quenching of fluorescence of riboflavin by the aporiboflavin-binding pro- tein was measured as emission at 520 nm after excitation at 370 nm (Murthy et al., 1976).

Determination of the tyrosine and tryptophan content of the proteins were performed using the method of Edelhoch (1967) which is based on measurement of A280 and A28 s in the presence of 6 M guanidine hydrochloride (Ultrapure grade from Gibeo-BRL). The concentrations of guanidine hydrochloride solutions were checked by measurements of refractive index measurements (Nozaki, 1972).

RESULTS

Purification o f riboflavin binding proteins from domestic fowl and quail

Riboflavin binding proteins from egg white of both domestic fowl and quail were purified by ion exchange chromatography using DEAE-Sephadex A-50. The method described by Hamazume et al. (1984) uses ion exchange chromatography followed by gel filtration on Sephadex G-100. It was found in this study that a better purification was obtained using two successive ion exchange chromatography steps. Riboflavin binding protein constitutes less than 1% of egg-white protein (Osuga and Feeney, 1977), and after the two stage purification there is less than 5% contamination by other proteins as can be seen

Riboflavin binding proteins 79

by SDS-polyacrylamide gel electrophoresis (Fig. 1, 20000 lanes 1 and 4). Using the purified apo-protein it was found by fluorescence quenching (Choi and ~-6 McCormick, 1980) that the number of binding sites ~ for riboflavin was close to 1.0 for both domestic fowl and quail. Spectrophotometric measurements of A2~0/ co. A,~5 led to the conclusion that the apo-proteins g~ contained <0.2 mol fiboflavin/mol protein. ~

Peptide mapping

In order to compare the structures of the domestic fowl and quail R/'BP, peptide mapping was carried out using trypsin, chymotrypsin, V8 proteinase, and thermolysin. The undigested RfBP has a mobility corresponding to a Mr of 35,000. Since RfBP is a glycoprotein it can be expected to give a diffuse band, which does not correspond exactly to the true M,. This will also apply to any fragments that also contain carbohydrate moieties. Neither domestic fowl nor quail RfBP was appreciably degraded by trypsin (Fig. 1, lanes 2 and 5) under the conditions used. When higher concentrations of trypsin were used there was still no appreciable digestion. Each of the other three enzymes catalyzed appreciable digestion, with thermolysin (Fig. 1, lanes 14 and 17) and V8 proteinase (Fig. 1, lanes 20 and 23) leaving no intact RfBP. The patterns obtained in each case differed between domestic fowl and quail. With chymotrypsin the domestic fowl RfBP was degraded to a diffuse band corresponding to M, 28,000, and with little other staining material visible (Fig. 1, lane 10). The quail RfBP, on the other hand gave three much sharper bands corresponding to M, values of 17,000, 21,000 and 22,000 (Fig. 1, lanes 9 and 11). With both domestic fowl and quail, RfBP V8 proteinase pro- duced a diffuse band corresponding to M, 33,000 and three much sharper bands corresponding to M, values of 13,000, 14,000 and 17,000. Two prominent bands in the quail digest corresponded to Mr values of 23,000 and 26,000 and there were no comparable bands in the domestic fowl (Fig. 1, lanes 20 and 23). Thermolysin caused the most extensive degradation and produced the most contrasting patterns, with three diffuse bands at M, values of 15,000, 18,000 and 25,000 in the domestic fowl, and six much sharper bands corresponding to M, values of 13,800, 14,500, 14,800, 15,700, 19,500 and 20,200 in the quail (Fig. 1, lanes 14 and 17).

Tyrosine and tryptophan contents

Using the procedure of Edelhoch (1967), the tryptophan and tyrosine contents of the protein from the domestic fowl were determined to be 6.7 and 9.9 per mole, respectively. These values compare well with those found by amino acid sequencing (6 and 9 for tryptophan and tyrosine respectively, Hamazume et al., 1984). The values found for the quail protein were 7.1 (tryptophan) and 8.7 (tyrosine). Given the relatively large errors in determination of tyrosine by this method (arising from the small contribution of tyrosine to the overall absorbance), it can be con- eluded that the content of these aromatic amino acids in the two proteins are similar. This conclusion will require confirmation by direct sequencing.

-20000

0

-~-200

I I

190 210 230 250

Wavelength (rim)

(b) ~ ._.~/~'-o"" -"--Y

- 3 0 0 ' ' 260 280 300 320

Wavelength (nm)

Fig. 2. C.d. spectra of riboflavin binding proteins from domestic fowl ( - - - - ) and quail ( ). Spectra were recorded as described in the text, (a) far u.v. spectra, and (b)

near u.v. spectra.

Circular dichroism spectra

Far u.v. The far u.v.c.d, spectra of the apo- forms of the riboflavin binding protein from domestic fowl and quail are shown in Fig. 2(a). The spectrum of the domestic fowl protein is similar to that reported previously (Nishikimi and Kyogoku, 1973; Kumosin- ski et al., 1982) with a sharp minimum at 206 um and a shoulder at about 225 nm. The spectrum for the quail protein is broadly similar with slightly greater (negative) ellipticities at these wavelengths. Analysis of the spectra by the method of Provencher and G16ckner (1981) gives the following structural con- tents: domestic fowl 24% ~-helix, 39% t-sheet, 37% remainder; quail protein 31% ~-helix, 31% t-sheet, 38% remainder. Using the method of Siegel et al. (1980) which examines the spectra over a more restricted wavelength range (210-240 urn), the ,,-helix contents of the domestic fowl and quail proteins were determined as 25% and 29%, respectively.

Addition of riboflavin to the apoproteins led to little change in the far u.v.c.d, spectra, consistent with earlier findings for the domestic fowl protein (Nishikimi and Kyogoku, 1973).

Near u.v. The near u.v.c.d, spectra of the apo- proteins from the domestic fowl and quail are shown in Fig. 2(b). The spectrum for the domestic fowl protein is similar to that reported previously, as are the changes brought about by addition of riboflavin (Nishikimi and Kyogoku, 1973), i.e. a shift of the band in the 290-295 nm region by about 1.5 nm to the red. The near u.v.c.d, spectrum of the quail protein shows significant differences from that of the domestic fowl protein. In particular, there is a shift in the peak in the 290-295 nm region (characteristic

80 MAsaot~ WALK~g et al.

I

.~ 6o

..~_

"~ ~o

~ -

~ 20 - ~ ,-r

0 i

300 330 360 390

Wavelength (nm) Fig. 3. Fluorescence spectra of riboflavin binding proteins. The excitation wavelength was 280 nm and the concen- tration of protein 0.055 mg/ml; (a) RfBP from domestic

fowl, (b) RfBP from quail.

of tryptophan side-chains). Although these changes are difficult to interpret in detail, they are character- istic of differences in the tertiary structure between the proteins. On addition of riboflavin to the quail protein, a shift is observed of similar magnitude to that noted with the domestic fowl protein.

Fluorescence spectra

The fluoresence spectra of the apoproteins from domestic fowl and quail are shown in Fig. 3. The emission maximum for the domestic fowl protein is at 340 nm, characteristic of tryptophan side chains with a fairly high average degree of exposure to the solvent (Teipel and Koshland, 1971). In the quail protein, the emission maximum is shifted to 337 nm and the fluorescence enhanced by about 50%. These data are characteristic of an altered tertiary structure with the tryptophan side chains in the quail protein being, on average, somewhat less exposed to solvent (Teipel and Koshland, 1971).

DISCUSSION

Riboflavin binding proteins have been studied from a limited number of species of birds, but the only detailed structural information at present is for that of the domestic fowl, for which the complete sequence is known ('Norioka et al., 1985). The amino acid compositions for the RfBPs from the domestic duck Anas platyrhynckos (Muniyappa and Adiga, 1980) and for the Muscovy duck, Cairina moschata (Abrams et aL, 1988) have been measured. These reveal substantial differences, particularly in the num- ber of proline, methionine, histidine and arginine residues compared with those of the domestic fowl. The Japanese quail is much more closely related to the domestic fowl, both belonging to the family Phasianidae, which is believed to have split less than 31 million years ago (Helm-Bychowski and Wilson, 1986). This closer relationship is also borne out by the differences in sequences of other egg-white proteins such as ovomucoid (Laskowski et al., 1987) and lysozyme (Joll~s et aL, 1979). In the latter the differ- ence between the Japanese quail and domestic fowl

corresponds to a minimal mutation distance of 5, whereas that between the domestic fowl and the duck corresponds to 15.5.

In this paper, we have shown that there are some small, but significant differences between the riboflavin binding proteins from quail and domestic fowl. Although the overall secondary structures of the two proteins are very similar, there are differences in the environments of aromatic amino acid side chains as revealed by the near u.v.c.d, and fluorescence properties. These differences in tertiary structures must reflect differences in amino acid sequence. Although a detailed sequence analysis of the quail protein has not been undertaken, the Cleveland peptide maps show that sequence differ- ences do occur between the two proteins. It is inter- esting to note that both proteins are resistant to digestion by trypsin under the conditions employed, despite the fact that the protein from domestic fowl is known to contain 15 lysine and five arginine residues (Norioka et aL, 1985). Presumably the high number (nine) of disulphide bonds in this protein restricts access of trypsin to potential scissile bonds under the conditions used for digestion. It has been previously reported that trypsin can be used to digest the RfBP from domestic fowl after prior cleavage of the protein by cyanogen bromide (Hamazume et al., 1987).

Acknowledgements--The Science and Engineering Research Council for provision of the c.d. facility, and to Dr. S. Provencher for supplying and CON-TIN Program.

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