143Biochem. J. (1983) 209, 143-150Printed in Great Britain

Hydrophobic chromatography of proteins in urea solutions

The separation of apoproteins from a lipoprotein of avian egg yolk

Ralph W. BURLEY and Robert W. SLEIGHC.S.LR.O. Division ofFood Research, North Ryde, N.S. W. 2113, A ustralia

(Received 22 June 1982/Accepted 13 September 1982)

A method is described for the chromatographic separation of mixtures of egg-yolkproteins of low solubility, by using a hydrophobic column (phenyl-Sepharose) andeluting with increasing concentrations of aqueous urea at low pH. The resolving powerof the method was established by tests on proteins and protein fragments of knownsequence. The theoretical basis for the method remains, however, unclear. Factors suchas the aggregation of the protein often appeared to be more important than itshydrophobicity in determining the urea concentration needed for elution. The methodwas applied to the mixture of apoproteins from the low-density lipoprotein (densityabout 0.95 g/ml) of avian egg yolk. For the previously studied apoproteins from eggyolk of the hen (Gallus domesticus), hydrophobic chromatography provided a new andconvenient method for isolating the main apoproteins (hen apovitellenins I-VI). For thehitherto unexplored apoproteins from egg yolk of the duck (A nas platyrhynchos) themethod has now been used to isolate three new proteins, two of which were not readilyseparated by methods based on molecular size. The elution pattern obtained with duckegg-yolk apoproteins is not the same as that of the hen egg-yolk apoproteins, althoughwe suggest a relationship for the three new apoproteins based on their amino acidcompositions and other properties. Possible roles for the apoproteins in avian egg yolkare described.

Hydrophobic chromatography, in which thestationary medium contains hydrophobic groups(Er-el et al., 1972), has been successfully applied tothe separation of soluble proteins (see, e.g., thereview by Shaltiel, 1976). Theoretical aspects havebeen considered in detail by Srinivasan & Ruckens-tein (1980). Usually the proteins are applied to thehydrophobic column in water or dilute aqueoussolution, and eluted with salt solutions, detergents ormixed solvents. It has more recently been found thatthe range of hydrophobic chromatography can beextended to include highly insoluble proteins, includ-ing those associated with lipids, by using increasingconcentrations of urea as eluent and thus avoid-ing the use of detergents and salts (Creamer &Matheson, 1981; Burley & Sleigh, 1981).We have applied this method to the problem of the

resolution of the complicated mixture of apo-proteins isolated from the major, i.e. low-density,lipoprotein fraction (density about 0.95 g/ml) ofavian egg yolk. The number, nature and purpose ofthese proteins is a problem in avian biochemistry. It

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has been shown that one of them ('hen apovitelleninI') is transferred unchanged in sequence from theblood very-low-density lipoprotein to egg-yolk lipo-protein (Dugaiczyk et al., 1981), where it possiblyhas a structural role (Burley, 1973). None of theother apoproteins has been studied in such detail.Their study has been complicated by the problem ofisolation, as most of them are insoluble in aqueousbuffers. Methods used for their isolation have so fardepended on molecular size. These methods includegel-filtration chromatography in various media suchas detergents, dilute HCI and urea (Burley, 1975;Raju & Mahadevan, 1976; Bengtsson et al., 1977;Burley & Sleigh, 1980), and also gel electrophoresisin detergent plus urea (Burley & Sleigh. 1980).Hydrophobic chromatography in urea solutionsprovides a useful alternative to these methods. andshould enable large amounts of the yolk apoproteinsto be isolated for further study. In the present paperwe give examples of its use on known and unknownapoprotein mixtures. A brief account of some of ourresults has been reported (Burley & Sleigh, 1981).

0306-3275/83/010143-08$02.00 © 1983 The Biochemical Society

R. W. Burley and R. W. Sleigh

Materials and methodsUrea solutions

Standard solutions of urea of analytical gradewere passed through a mixed-bed ion-exchange resincolumn and a micropore filter (pore size 0.45,pm).Small volumes of concentrated acid, or occasionallybuffer salt, were then added. Solutions that con-tained concentrations of urea higher than 3 M wereacidified with HCl (final concn. 20mM, pH 3.0-3.5).For lower concentrations of urea, HCl was replacedby formic acid or acetic acid to prevent the pH fromfalling below about 3, because of possible damage tothe chromatographic gels.

Chromatographic materialsSephadex, Sepharose and the hydrophobic

materials phenyl- and octyl-Sepharose CL-4B werefrom Pharmacia, Sydney, N.S.W., Australia. For theresults reported in the present paper one batch ofphenyl-Sepharose (lot no. FM 19078) was used. Nochange in its properties was observed over 10months, during which it was in contact with acidicurea at 21 0 C. A batch used for experiments reportedpreviously (lot no. 9705; Burley & Sleigh, 1981)gave slightly different results in that the resolution ofthe less-retarded proteins was improved and theywere eluted at lower urea concentrations. The orderof elution was, however, unchanged. Treating thisbatch of Sepharose with the proteolytic enzymePronase (Kaken Chemical Co., Tokyo, Japan) incase its properties had been altered by the permanentadsorption of a small amount of protein did notaffect its behaviour, which probably depended on theoriginal agarose.A 2,4-dinitrophenyl derivative of Sepharose CL-

4B, referred to as 'dinitrophenyl-Sepharose' wasprepared by coupling bis-(2,4-dinitrophenyl)lysine toSepharose CL-4B by the procedure of Axen et al.(1967). The coupling was performed in a tetrahydro-furan at room temperature by using [1-cyclohexyl-3-(2-morpholinoethyl)carbodi-imide methotoluene-p-sulphonatel (from Aldrich Chemical Co., Mil-waukee, WI, U.S.A.).

ProteinsEgg-yolk apoproteins. These were from the

low-density lipoprotein of fresh egg yolk fromAustralorp hens (Gallus domesticus) or Pekin ducks(Anas platyrhynchos). The lipoproteins were ob-tained by centrifuging granules-free yolk in 2 M-NaClas described previously (Burley, 1978). Apoproteinswere isolated by removing the lipid with chloro-form/methanol (1: 1, v/v) at neutral pH (Burley,1975) and dissolved in 3M-urea (pH3) to give aprotein concentration of about 0.5%.

The low-molecular-weight apoprotein, duck's apo-vitellenin I, and its CNBr-cleavage fragments wereisolated as described by Inglis & Burley (1977).

Hen's apovitellenin I was isolated by gel-filtrationchromatography on Sephadex G-75 (Burley, 1975).The monomeric, i.e. reduced, form was obtained bytreating a solution (0.4%) in 6 M-urea, pH 7, with2-mercaptoethanol (final concn. 0.1 M) for about 4 hat 21 IC and then dialysing it against acidic ureasolution.

Other proteins. A mixture of a-, /,-, and K-caseinswas obtained by acid precipitation from fresh bovinemilk (Manson & Annan, 1971). Phosphate groupswere removed with potato acid phosphatase(Bingham et al., 1976).

Hydrophobic chromatographyA column (50cm x 2.2 cm) containing 175 ml of

phenyl-Sepharose CL-4B was used at 210C withupward flow at a rate of 16-18 ml/h, during whichthree fractions were collected. Linear urea gradientswere prepared by means of a gradient mixer (modelGM- I from Pharmacia). Usually acidic 3 M- and9 M-urea in equal volumes of 200-280 ml weremixed. Proteins in the fractions were detected at280nm in 1 cm cells in a Unicam SP. 3000 spectro-photometer (Pye-Unicam Instruments, Cambridge,U.K.). Proteins or fragments with low absorbance at280nm were detected by applying spots (25,p1) tofilter paper and staining with Coomassie Blue (0.1%)in acetic acid/ethanol/water (1:4:5, by vol.).

Hydrophobicity indexAs a measure of the hydrophobicity of proteins

and peptides, the hydrophobicity index (1) ofBigelow (1967) was used. This index represents theaverage value of the free energy of transfer of theiramino acid residues from ethanol to water, expressedas AFTkJ/mol of amino acid. The corrected valuesgiven by Nozaki & Tanford (1971) were taken intoaccount.

ElectrophoresisYolk proteins and polypeptides were identified on

tubes (8 cm x 0.7 cm) or slabs (8 cm x 8 cm x 0.2 cm)of polyacrylamide gel in minor modifications (Burley& Sleigh, 1980) of the procedure of Weber &Osborn (1969). Better resolution was obtained withtubes, but slabs were more convenient for com-parisons and for photoelectric scanning. For the gelpatterns given in the present paper, slabs containing6 M-urea /65 mM-Tris /borate buffer/i mM-EDTA,pH 8.6, were used. Gels used for proteins (Figs. 3and 5) contained 7.5% acrylamide. Those used forpeptides (Fig. 6) contained 14% acrylamide. Thegels were stained with a mixture of Coomassie Blueplus Amido Black [each 0.05% in acetic acid/water/ethanol (1:4:5, by vol.)] and scanned on aShimadzu photoelectric densitometer (SchimadzuCorp., Kyoto, Japan). Finally, the scans were traced.Molecular weights were estimated by using a series

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of standard proteins, as described previously (Burley& Sleigh, 1980).

Milk caseins were identified by electrophoresis inurea without detergent.

A nalyticalproceduresProtein concentrations were- determined gravi-

metrically after the solution had been dialysed intowater. Dialysis tubing with high retention(Spectrapor; Spectrum Medical Industries, LosAngeles, CA, U.S.A) was used.Amino acid compositions of proteins were deter-

mined on HCl hydrolysates, by using a Beckmanmodel 120C analyser (Beckman Industries, PaloAlto, CA, U.S.A.).

Tryptophan was determined by the spectrophoto-metric method of Edelhoch (1967).

Results

Hydrophobic chromatography of egg-yolk apo-vitellenin I and otherproteins of known sequence

Fig. 1 shows the separation of a synthetic mixtureof two similar proteins of low molecular weight fromegg yolk of different avian species, duck's apo-vitellenin I and the reduced form of hen's apo-vitellenin I. Table 1 gives the positions of elution andother data for these proteins, and shows that theywere eluted in the order expected from theirhydrophobicities, i.e. the more hydrophobic proteinwas eluted later. Subsequent tests suggested, how-ever, that hydrophobicity was not always dominant.Thus the oxidized, dimeric, form of hen's apo-vitellenin I was eluted much later (Table 1) and wascompletely separated from the monomer; and testson other mixtures of known proteins showed that,although separation was often complete, there wasno consistent relationship between elution pattern

and hydrophobicity. For example, Table 1 also givesdata for the elution of two polypeptide series: (i)three CNBr-cleavage fragments of duck's apo-vitellenin I, and (ii) three caseins from bovine milkbefore and after removal of phosphate groups. Foreach series the constituents were separated at leastpartly. Resolution could be improved by using ashallower gradient, but neither the composition (e.g.number of aromatic residues) nor the sequence (e.g.

0.8

0.6

0oo

C 0.4

0.2

O La20 40

Fraction no.

iCZ

Fig. 1. Hydrophobic chromatography ofapovitellenin IA column of phenyl-Sepharose was used (see theMaterials and methods section), and samples (each10 mg) of duck apovitellenin I (peak A) and thereduced form of hen apovitellenin I (peak B)were applied in 8.Oml of acidic 2M-urea. Thefractions were each of 5.3 ml, and the points refer tooptical absorbance at 280 nm. The straight linerefers to the urea concentration gradient. Peaks wereidentified by the amino acid composition of theisolated proteins.

Table 1. Hydrophobic chromatography ofproteins andpolypeptidesPositions of elution of proteins and protein fragments during chromatography as in Fig. 1 are given, plus other data.For experimental details see the text.

ProteinDuck apovitellenin IHen apovitellenin I (reduced)Hen apovitellenin I (oxidized)Duck apovitellenin ICNBr fragment 1CNBr fragment 2CNBr fragment 3as1-CaseinfJ-CaseinK-Casein

Elution position Hydrophobicity indext([urea], M*) (kJ/mol)6.85 +0.10 5.156.64 + 0.11 4.698.0 + 0.2 4.69

5.66.76.1

7.0 (7.4t)4.9 (5.0)4.5 (4.7t)

5.524.186.364.486.824.69

No. ofresidues

8282164

293914

199209169

Aromaticresidues

97

14

423

191313

* Urea concentration at centre of peak. Standard errors are given where three or more values were available.t See the Materials and methods section.t Values in parentheses refer to the dephosphorylated protein.

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145

R. W. Burley and R. W. Sleigh

0.3 G 6 j-

0.24

0.1 2

0 020 40 60 80 100 120

Fraction no.

Fig. 2. Hydrophobic chromatography ofproteins from hen's-egg yolkA mixture (172mg) of the apoproteins from hen's-egg-yolk lipoprotein was applied within 3 h of dissolving in acidic3 M-urea (45 ml); other conditions were as given in Fig. 1 legend, although the fractions were each 5.4 ml. The letters(A-H) denote the principal peaks and the horizontal bars the proteins identified by electrophoresis (Fig. 3), which areindicated by roman numerals. The fractions used for electrophoresis in Fig. 3 are indicated by arrow heads. Thestraight lines refer to the urea concentration gradient.

distribution of hydrophobic residues) was helpful inpredicting the order of elution.

Before using phenyl-Sepharose for the tests inTable 1, we tried other materials. Octyl-Sepharoseretained the yolk apoproteins more firmly than didphenyl-Sepharose, although the order of elution wasthe same. We also observed some difference betweendifferent batches of phenyl-Sepharose (see theMaterials and methods section). A new material,dinitrophenyl-Sepharose, did not retain any of theyolk apoproteins.

Separation of the apoprotein mixture from hen's-egg-yolk lipoprotein

Table 1 suggested that chromatography onphenyl-Sepharose in acidic urea solution couldprofitably be applied to mixtures of similar hydro-

I ~~~~~~vVI

A

C

DLJ/\t

E

F|I

AG A-

50 10010-3 x Mol.wt.

Fig. 3. Photometric scans of gel-electrophoretic patternsofproteins from hen's-egg yolk

Polyacrylamide slabs containing detergent wereused, as described in the Materials and methodssection. Gel T, total apoprotein mixture from thelipoprotein of hen's egg yolk; roman numerals referto apovitellenins identified previously. Gels A-G,selected chromatographic fractions denoted byarrow heads in Fig. 2. The approximate molec-ular-weight scale derived from standards is indicatedat the bottom of the Figure.

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Hydrophobic chromatography of yolk apoproteins

phobic proteins. Fig. 2 refers to the separation of afreshly prepared mixture of apoproteins from hen'segg-yolk lipoproteins. Seven peaks (A-G) werepartly or completely resolved. The proteins in thepeaks were identified by gel electrophoresis indetergent by comparison with the original mixture(Fig. 3, gel T), which had been elucidated previously(Yamauchi et al., 1976; Burley & Sleigh, 1980).Electrophoretic patterns of fractions within thepeaks A-G are shown in Fig. 3. Table 2 summarizes

data from Figs. 2 and 3 and gives the hydro-phobicities of the principal apoproteins. Table 2 alsoindicates how these proteins could be resolved orpurified further by gel-filtration chromatography, sothat it was possible to isolate the seven previouslydescribed apoproteins, apovitellenins I-VI (Burley,1975; Burley & Sleigh, 1980), starting with hydro-phobic chromatography.From a comparison of Table 2 and Table 1 it is

apparent that apovitellenin I emerged at a slightly

Table 2. Hydrophobic chromatography ofa protein mixturefrom hen's-egg yolkData for the separation of the mixture of lipoprotein apoproteins were as given in Fig. 2 legend. Identification of theprincipal proteins was by electrophoresis (Fig. 3) and gel-filtration chromatography (Burley & Sleigh, 1980). Forexperimental details see the text.

[Ureal at centreof peak (M)

34.75

Principal proteins(apovitellenins)*

III (4.52), V (4.27)II (3.64), VI (4.14)

5.3 III (4.52)

6.2 Ia (4.69)6.8 VI (4.10)7.6 1 (4.69)

9 V (4.27)

Minor proteins(apovitellenins)

IV, VIIII

Ia, IV

IV, IIIVa and others unidentifiedVa, VI

III, IV, VI

Isolation ofprincipal proteinst

Not attemptedII and IV separated on

CL-6B or G-100Ia removed on G-75 and

IV on CL-6BIa separated on G-75VI isolated on CL-6BI isolated on G-100

or G-75V isolated on CL-6B

* The hydrophobicity index (kJ/mol) is given in parentheses.tGel-filtration chromatography was used with columns (80cmx2cm) of Sepharose CL-6B, Sephadex G-100, or

Sephadex G-75 (Burley, 1975; Burley & Sleigh, 1980) in acid 6M-urea.

80Fraction no.

10

-

It

Fig. 4. Hydrophobic chromatography of a protein mixturefrom duck's-egg yolkA sample (136mg in 25 ml of acidic 3M-urea) was applied to the same column of phenyl-Sepharose used for Fig. 2and eluted in the same way. The letters a, b and c refer to proteins that were isolated and subsequently purified. Theirelectrophoretic patterns are given in Fig. 5. I refers to duck apovitellenin I, the principal constituent. This peak wascut off because of its size. The straight lines refer to the urea concentration gradient.

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Peak(Fig. 2)AB

C

DEF

G

147

0OC,1

R. W. Burley and R. W. Sleigh

Table 3. Amino acid compositions and other datafor duck's-egg-yolk apoproteinsThe apoproteins a, b, and c are those indicated in Fig. 4. For comparison, compositions of the corresponding proteinsfrom the hen are also given, i.e. hen's-egg-yolk apovitellenins IV, III and VI (Burley & Sleigh, 1980). The standarderrors (S.E.M.) refer to three or more estimations. For experimental details see the text.

Amino acidAspThrSerGluProGlyAlaCySValMetIleLeuTyrPheTryLysHisArg10-4 X Mol.wt.*

Protein a

Mean S.E.M.

11.62 0.148.42 0.1811.90 0.069.86 0.064.19 0.136.98 0.076.56 0.110.26 0.065.05 0.062.09 0.015.10 0.189.93 0.133.13 0.255.25 0.203.06 0.257.41 0.411.51 0.062.87 0.147.54.446.1

Henapovitellenin

IV17.166.759.0710.073.856.345.510.315.351.795.35

10.073.175.341.057.172.173.847.54.14

Protein b

Mean S.E.M.12.81 0.327.90 0.368.48 0.12

13.28 0.313.97 0.206.30 0.095.86 0.1006.37 0.181.84 0.064.44 0.109.43 0.293.45 0.265.97 0.173.26 0.257.12 0.272.42 0.09.4.40 0.256.54.4811.8

Henapovitellenin

III12.275.706.2712.342.864.647.260.315.462.178.579.933.044.771.049.361.264.286.54.52

* Estimated by gel electrophoresis in detergent; see the Materials and methods section.t Hydrophobicity index (kJ/mol); see the Materials and methods section.t Amino acid difference index, referred to the neighbouring hen's-egg-yolk apoprotein.

Protein c

Mean S.E.M.9.42 0.186.98 0.018.27 0.1114.19 0.394.53 0.105.77 0.196.78 0.260.93 0.035.86 0.192.45 0.044.69 0.0410.09 0.183.49 0.104.08 0.072.79 0.088.80 0.691.71 0.043.76 0.06

12.04.486.7

lower urea concentration when part of the wholemixture. Moreover, the chromatographic pattern inFig. 2 would not be expected from the hydro-phobicities. Peak A, which was not retarded andwas also eluted with 2 M-urea, was clearlyanomalous. It contained two proteins (apo-vitellenins III and V) that were also present in twoother peaks (peaks C and G). It is possible that peakA contained a complex of these proteins, because wehave previously found evidence (Burley & Sleigh,1980) that they are present as an unstable complexwhen the apoprotein mixture is first dissolved inurea. Peak A decreased in size after the mixture hadbeen left at 20C in acidic urea solution, as expectedfor an unstable complex. We also observed thatapovitellenin III (peak C) changed on standing inurea solution after separation, so that on

rechromatography it was eluted in the position ofpeak B, possibly a result of the formation of an

aggregate. A more definite example of the effect ofaggregation of a single protein was provided byapovitellenin VI (peak E), which is known toaggregate slowly in urea (Burley & Sleigh, 1980). Inold solutions this protein emerged earlier, betweenpositions D and E in Fig. 2.

The hen's egg-yolk apoproteins were more

difficult to elute from phenyl-Sepharose if the pH ofthe urea solution was 4 or 7 instead of 3.

Separation of the apoproteins from the lipoproteinofduck's-egg yolk

Fig. 4 shows the hydrophobic chromatography ofthe total apoprotein mixture. The correspondingelectrophoretic pattern is shown in Fig. 5 (gel T).Four of the proteins were isolated. The others havenot been studied. As expected from its large size,peak I contained duck's egg-yolk apovitellenin 1,identified by its amino acid composition (Inglis &Burley, 1977). Peaks a, b, and c contained singleproteins with some overlapping, according to gelelectrophoresis (Fig. 5). They were purified byrechromatography on Sepharose CL-6B, althoughthe proteins in peaks a and b could not be separatedfrom each other in this way because their molecularweights were too close: 65 000 and 75 000 accordingto gel electrophoresis in detergent (Table 3). Hen's-egg-yolk apoproteins with the same molecularweights as these were separated by gel-filtrationchromatography because they behaved abnormallyon Sepharose CL-6B (Burley & Sleigh, 1980). Theproteins in peaks a and b were well separated on

1983

Henapovitellenin

VI10.746.859.4310.503.745.716.790.716.312.044.9310.423.284.300.487.851.724.2017.04.10

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Hydrophobic chromatography of yolk apoproteins

T

a J

10 50 100

10-3 X Mol.wt.Fig. 5. Photometric scans of gel-electrophoretic patterns

ofproteins from duck's-egg yolkPolyacrylamide slabs were used as indicated in Fig.3 legend. Gel T represents the total apoproteinmixture. Most of the proteins have not beenidentified. The large peak corresponded to duckapovitellenin I. Gels a, b and c refer to proteinsisolated from three of the peaks in Fig. 4.

phenyl-Sepharose by using a shallower gradient thanthat shown in Fig. 4.Amino acid compositions and other data for the

purified proteins from peaks a, b and c (Fig. 4) aregiven in Table 3. This Table also gives data for thehen's-egg-yolk apoproteins that were judged to beequivalent by the amino acid 'difference index'(Metzger et al., 1968). According to this testproteins a and c were closest to hen's-egg-yolkapovitellenins IV and VI respectively. Protein bresembled apovitellenin III, although the differencewas larger than for the others. As already noted,proteins a and b corresponded to apovitellenin IVand III in molecular weight and in chromatographicbehaviour on phenyl-Sepharose, but protein cdiffered by about 30% in molecular weight fromapovitellenin VI and behaved differently on phenyl-Sepharose. CNBr digests of the proteins of lowermolecular weight (i.e. proteins a and b) gavedifferent electrophoretic patterns (Fig. 6). Neither

20 10 5i0-3 x MoI.wt.

Fig. 6. Photometric scans of gel-electrophoretic patternsof CNBr digests of duck's-egg-yolk proteins

Duck's-egg-yolk apoproteins a and b (see Fig. 4)were digested as described in the Materials andmethods section and applied to a polyacrylamide-gelslab with a high proportion of acrylamide (14%).Other conditions were as indicated in Fig. 5 legend.

agreed completely with the closest hen's egg-yolkproteins (apovitellenins IV and III; Burley & Sleigh,1980), but in general they are similar. From all thesedata it seems appropriate to name proteins a, b and cduck's-egg-yolk apovitellenins IV, III and VIrespectively.

Discussion

Our results show that elution of proteins from ahydrophobic column is sensitive to small changes inurea concentration. It is likely that proteins areattached to such a column by hydrophobic groups,i.e. phenyl groups on phenyl-Sepharose, and not byother groups, because the hen's-egg-yolk apo-proteins were not retained on a column of dinitro-phenyl-Sepharose. Urea interacts with protein sidechains, especially with aromatic groups (Nozaki &Tanford, 1963; Prakash et al., 1981), so it would beexpected to weaken the binding of proteins tophenyl-Sepharose. Only for very similar proteins,however, did the elution position depend on thehydrophobicity alone (Fig. 1). Co-operative bindingwould explain the large difference between themonomeric (reduced) and dimeric forms of hen's-egg-yolk apovitellenin I (Table 1) and possibly someof the difference between duck's-egg-yolk apo-vitellenin I and its CNBr-cleavage peptides (Table1). We suggest that an important additional factor isthe effect of urea on the protein structure. In general,proteins that are unfolded in 3 M-urea would beexpected to expose more binding sites and so bemore firmly attached to the column; thus compactaggregates should be more easily eluted than

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150 R. W. Burley and R. W. Sleigh

extended monomers. Data on the shapes in 3 M-ureaof the proteins we have tested and on the effect ofincreasing concentrations of urea are not yetavailable, so that the validity of this suggestioncannot be assessed. It is not, however, surprisingthat there is no consistent relationship betweenelution pattern and hydrophobicity, as also noted byCreamer & Matheson (1981) for elution of milkproteins by a combination of aqueous buffers and agradient of neutral urea.

Although the theoretical basis for hydrophobicchromatography in urea solutions is not clear, wehave found it useful for isolating the apoproteinsfrom avian lipoproteins, especially in conjunctionwith other methods. As a result, four proteins arenow available for comparison between two species,and it is already apparent that there are surprisinglylarge differences (Table 3) that will have to be takeninto account in explaining the function of theseproteins. As mentioned above, it is likely thatapovitellenin I, the main constituent that has alsobeen isolated from a blood lipoprotein (Dugaiczyk etal., 1981), is a determinant of lipoprotein structure.For the other apoproteins there are several possi-bilities. They may also be structural proteins withoutstringent requirements as to composition or size.They could be highly specific and essential for thefunctions of the lipoprotein as it exists in yolk. Theyneed not be related to the lipoprotein, but could bedriven into the lipoprotein fraction during isolation;a related example of such behaviour has beendescribed (Burley, 1978). Information that woulddistinguish among these possibilities is not available.As a guide to further experiments we emphasize thatin yolk, with 50% solids, the environment of thelipoprotein is different from that in blood, with about7% solids, so that the requirements for lipid-proteininteractions may be different. Thus the differencesthat we have observed may be an indication ofimportant functional differences between the yolksof different species. Such functions might include thestabilization of the lipoprotein before incubation andcontrol of its accessibility during incubation. Minorapoproteins that are not present in sufficientamounts to take part in all the lipoprotein particlespossibly represent other lipid-protein structures inyolk.

We thank Dr. M. Howden for a supply of duck's eggs.

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1983