Separation of branched sialylated oligosaccharides using high-pH anion-exchange chromatography with pulsed amperometric detection

182,1-8

ANALYTICALBIOCHEMISTRY

(1989)

Separation of Branched Sialylated Oligosaccharides Using High-pH Anion-Exchange Chromatography with Pulsed Amperometric Detection R. Reid Townsend,*,’ *Department TDepartments $Department

Received

Mark

R. Hardy,*j2

Dale A. Cumming,tT3

January

correspondence of Pharmaceutical 513 Parnassus, address: Dionex

address: Genetics Institute, MA 02140. address: The Biomembrane 305, Seattle, WA 98119.

0003-2697/89

$3.00

0 1989 by Academic of reproduction

and Brad Bendiak$p4

Baltimore, Maryland 21218; Canada M5S lA8; and Ontario, Canada M5G 1X8

16,1989

’ To whom Department San Francisco, ’ Present 94086. 3 Present Cambridge, 4 Present West, Suite

All rights

P. Carver,?

of Biology and the McCollum-Pratt Institute, The Johns Hopkins University, of Medical Genetics and Medical Biophysics, University of Toronto, Ontario, of Biochemistry, Hospital for Sick Children, 555 University Avenue, Toronto,

Ten characterized sialylated oligosaccharides from bovine fetuin (B. Bendiak, M. Harris-Brandts, S. W. Michnick, J. P. Carver, and D. A. Cumming, Biochemistry, in press; and D. A. Cumming, C. G. Hellerqvist, M. Harris-Brandts, S. W. Michnick, J. P. Carver, and B. Bendiak, Biochemistry, in press) were chromatographed using high-performance anion-exchange chromatography with pulsed amperometric detection. At near neutral pH values, oligosaccharides were separated according to their number of formal negative charges from sialic acid; however, at alkaline pH, the neutral portion of the oligosaccharides enhanced resolution due to oxyanion formation. Specifically, trisialylated triantennary oligosaccharides containing a GalB(1,3)GlcNAc sequence were more retained and could be completely separated from those having only Gal/3( 1,4)GlcNAc units. Oligosaccharides containing the same number of sialic acids were separated according to the combination of a(2,6)and a(2,3)-linked sialic acids (a(2,6)-linked sialic acid reduced retention time). The relative molar electrochemical responses for di-, tri-, tetra-, and pentasialylated oligosaccharides were found to be similar (4.8 +- 14% relative to glucose). Coelution studies were performed with each of the characterized oligosaccharides and the mixture of oligosaccharides which were released from fetuin with N-

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Jeremy

should be addressed. Present address: Chemistry, University of California, San Francisco, CA 94143. Corp., 1228 Titan Way, Sunnyvale, CA

Press, Inc.

in any form

reserved.

87 Cambridge Institute,

Park

201 Elliott

glycanase. of sialylated ta-) varied sources.

The relative proportion of the major classes oligosaccharides (bi-, tri-, tetra-, and pensignificantly in bovine fetuin from different 0 1989 Academic

Press, Inc.

It has recently been shown that HPAE-PAD5 can separate a diverse array of branch and linkage isomers of neutral oligosaccharides (l-6). Recently, we have shown that anionic oligosaccharides (sialylated and phosphorylated) can be separated not only on the basis of their formal negative charge, but also according to the “neutral” oligosaccharide portion using HPAE (2). In these studies (2), linear, monosialylated di- and tetrasaccharides were resolved according to (i) sialic acid linkage ((w(2,3) or a(2,6)), (ii) which residue the sialic acid is linked, and (iii) the structure of the neutral portion of the chain. In the present study, we applied HPAE-PAD to the separation and detection of multisialylated branched oligosaccharides purified from bovine fetuin (degree of polymerization = 14-16) which have been characterized using one- and two-dimensional 500 mHz ‘H NMR and methylation analysis (7,8). We found that chromatography under alkaline conditions resolved these larger branched oligosaccharides not only by sialic acid content, but also according to the combination of (~(2,3)- and a(2,6)-linked sialic acids within each charge class (i.e., di-, tri- and tetrasialylated oligosaccharides). Oligosaccharides with a greater number of cx(2,6)- versus a(2,3)-linked sialic acids were the least retained. Separation was also achieved on the basis

Drive, Avenue

’ Abbreviations used: HPAE-PAD, matography with pulsed amperometric oligosaccharide; M, Man; SA, NeuAc;

high-pH anion-exchange chrodetection; TRI, triantennary G, Gal; GN, GlcNAc. 1

2

TOWNSEND

ET

AL.

of the neutral carbohydrate structure. Of the oligosaccharides studied, those containing a GalP( 1,3)GlcNAc sequence were more retained than those with a Gal@(1,4)GlcNAc unit. The electrochemical response relative to Glc was found to be similar for di-, tri-, tetra-, and pentasialylated oligosaccharides which enabled estimation of the relative amounts of various species in N-glycanase-released oligosaccharide mixtures from bovine fetuin. Using HPAE-PAD, we found significant variations in the relative amounts of sialylated oligosaccharides in fetuins from different sources.

sodium acetate gradient: Gradient A: isocratic at 50:33: 17 (Eluant 1:Eluant 2:Eluant 3, respectively) for 5 min, then a linear gradient to 50:17:33 at 60 min, to 50:0:50 at 80 min, followed by return to initial conditions at 90 min. Sialylated oligosaccharides were eluted at near neutral pH with Gradient B: Eluant 1 was 10 mM acetic acid and Eluant 2 was 500 mM acetic acid both with pH adjusted to 6.0 using a 50% NaOH solution. A linear gradient up to 100% Eluant 2 in 60 min was generated after 10 min of isocratic elution with Eluant 1. The column was reequilibrated for at least 15 min prior to the next analysis.

MATERIALS

Determination of electrochemical response factors. The sialylated oligosaccharides were quantified by glucosamine composition essentially as previously described (10). An aliquot of the stock solution of oligosaccharides in water was added to a 1.5-ml screw-capped polypropylene tube (Sarstedt, Princeton, NJ) to a final volume of 200 ~1. An equal volume of 12.1 N HCl solution was added and hydrolysis was carried out at 100-103°C for 4 h. The samples were dried in a Speed-Vat, resuspended in 200 ~1 of water, transferred to a 250 ~1 autosampler vial, and analyzed for glucosamine without further processing. The relative response of the oligosaccharides was determined as the peak area of the sample per picomoles of sample divided by the peak area of Glc per known picomoles of Glc.

AND METHODS

Materials. Fifty percent (w/w) NaOH solution (low in carbonate) was purchased from Fisher Scientific (Rockville, MD). Sodium acetate was reagent grade from J. T. Baker (Phillipsburg, NJ). Bio-Gel P-6 was purchased from Bio-Rad (Richmond, CA). N-Glycanase was supplied by Genzyme (Boston, MA). Fetuin was purchased from Sigma Chemical Co. (Type III and IV), (St. Louis, MO), Calbiochem (LaJolla, CA), and GIBCO (purified by the method of Spiro (9), Grand Island, NY). Chromatography. The system used for HPAE-PAD of sialylated oligosaccharides and monosaccharides consisted of a Dionex Bio-LC gradient pump, CarboPac PA1 column (4.9 X 250 mm) and a Model PAD 2 detector. The following pulse potentials and durations were used for detection: El = 0.01 V (tl = 300 ms); E2 = 0.70 V (tz = 120 ms); E3 = -0.50 V (t3 = 300 ms). The response time of the PAD 2 was set to 3 s. A Dionex DQP-1 single piston pump was used to add 0.3 M NaOH to the column eluant through a tee at 1 ml/min before the PAD cell to minimize baseline drift during gradient analyses. The eluants were prepared by suitable dilution of 50% NaOH solution with 18 Mohm, deionized water (Hydro Services Corp., Raleigh NC). Eluants containing sodium acetate were filtered through O.&pm nylon filters prior to use. Samples were injected with a Spectra-Physics autosampler via a Rheodyne 7010 valve equipped with a 200-~1 sample loop and a Tefzel rotor seal to withstand the alkalinity of the eluants. The resulting chromatographic data were integrated and plotted using either a Spectra-Physics Model SP4270 integrator or a Waters Model 840 (DEC Professional 350-based) data station. The capacity of the gradient pump module to handle up to four different eluants was used to afford maximum flexibility for chromatographic methods development. Eluant 1 was installed as 200 mM NaOH, Eluant 2 as water, and Eluant 3 as a 600 mM sodium acetate solution. In this manner, both pH and acetate concentration were varied during scout analyses to optimize elution conditions. The Dionex Eluant Degas Module was employed to saturate the eluants with helium to degas and to minimize absorption of COZ. Sialylated oligosaccharides were separated at alkaline pH and retention times determined (Table 1) at alkaline pH using the following

Preparation of oligosaccharides using N-glycanase. Oligosaccharides were prepared from either bovine fetuin (Methods A) or from tryptic glycopeptides prepared as previously described (Method B) (11). For Method A, fetuin (10 mg) was dissolved in 2 ml of 0.25 M phosphate buffer, pH 8.5. Two units of N-glycanase (2 X 10m3 IU) was added followed by incubation at 37°C for 3 days. The sample was then applied to a Bio-Gel P-6 column (70 X 1.5 cm) equilibrated in 50 mM ammonium acetate buffer, pH 6.9. The column eluant was monitored at 280 nm. The fractions (2 ml) between the 280-nm absorbing peak and the total bed volume were assayed for sialic acid on the Dionex carbohydrate analyzer after hydrolysis with 0.1 N HCl at 80°C. The sialic acid containing fractions were pooled and lyophilized, and the total was assayed for sialic acid. The yield was 28% of the total oligosaccharides from 10 mg of fetuin (192 nmol) which was based on an average of 12 mol of sialic acid per mole of glycoprotein. More complete release of oligosaccharides (93%) was obtained by incubating 100 nmol of fetuin tryptic glycopeptides with 5 munits of N-glycanase in 40 ~1 of 0.2 M sodium phosphate buffer, pH 8.2, for 48 h at 37°C (Method B). The oligosaccharides were separated from the peptides and unreacted glycopeptides using an octadecyl-silica column equilibrated in 50 mM ammonium acetate, pH, 6.9. The column effluent was monitored at 215 nm. The oligosaccharides (measured by GlcNAc content after hydrolysis) were slightly retained while the

CHROMATOGRAPHY

OF

SIALYLATED

peptide containing compounds were eluted with 50% acetonitrile in 50 mM ammonium acetate. By comparing the GlcNAc content of the oligosaccharide and peptide containing fractions, it was determined that 50% of the oligosaccharide was released. The recovered glycopeptides were then redigested with 10 units of enzyme in 40 ~1 of the same buffer for 5 days at 37°C and separated by HPLC as describe above. After the second incubation with N-glycanase, 93% of GlcNAc applied to the column was recovered in the oligosaccharide fractions. Since some of the O-linked oligosaccharides also contain GlcNAc (la), 93% is a minimal estimate of the amount of oligosaccharides released. Oligosaccharide fractions from both N-glycanase digestions were pooled, lyophilized, and analyzed using HPAE-PAD. RESULTS

HPAE-PAD of Characterized Sialylated Oligosaccharides from Bovine Fetuin Table 1 shows the structures and retention times of 10 oligosaccharides which have been purified from bovine fetuin and characterized using 500 mHz ‘H NMR and methylation analysis (7,8). In compounds 1 and 2 all of the Gals are linked p(1,4). These two structures differ only in the linkage of the sialic acid on the Man cu(1,6) branch and are separated by HPAE by about 1 min. The remaining 8 structures contain a Galfi(l,S)GlcNAc sequence-linked p(1,4) to the ol(l,3)-linked Man. This sequence (Galfi(l,3)GlcNAc-) signals sialylation at the g-position of the GlcNAc (13) resulting in tetra- and pentasialylated triantennary structures (oligosaccharides 8-10). Also, this sequence is related to striking sialylation heterogeneity of trisialylated triantennary structures (oligosaccharides 3-7), which are retained 410 min longer than compounds 1 and 2. Compound 3 has all three sialic acids in a(2,6) linkage and of the Galfl(1,3) TRIs elutes first. Oligosaccharides 4 and 5 have two cY(2,6)-linked and one cu(2,3)-linked sialic acids. They differ only in the branch location of one a(2,6)-linked sialic acid (branch positional isomers) and coeluted under these gradient conditions. Compounds 6 and 7 have two (u(2,3)- and one a(2,6)-linked sialic acids and were the most retained of the characterized Galfi(1,3) TRIs. In compound 6, the a(2,6) sialic acid is linked to a Gal whereas in compound 7 sialic acid is attached to the GlcNAc. Thus, these two compounds (6 and 7) differ not only in the branch location of (r(2,6)linked sialic acid, but also in the residues to which this sialic acid is linked. The difference in both branch and residue location of sialic acid (Gal versus GlcNAc) may account for the partial separation of these two isomers. The Galfi(1,3) TRI tetrasialylated relatives of compounds 1 and 2 are oligosaccharides 8 and 9 which also differ only in the linkage of the sialic acid on the a(1,6) branch. The same effect of sialic linkages (sum of (r(2,3)and cu(2,6)-linked sialic acids) was evident in the reten-

3

OLIGOSACCHARIDES

tion times of these two tetrasialylated oligosaccharides. Compound 8 has three a(2,6)- and one a(2,3)-linked sialit acids and eluted before oligosaccharide 9 with two a(2,6)- and two a(2,3)-linked sialic acids. Compound 10 possesses a Galp(l,S)GlcNAc sequence on the (~(1,6) branch and is sialylated on the GlcNAc of this branch resulting in a pentasialylated triantennary oligosaccharide. It eluted 13 min after the tetrasialylated oligosaccharides. HPAE-PAD of the Sialylated Bovine Fetuin

Oliosaccharides

from

Figure 1A shows the separation of N-glycanase released oligosaccharides from fetuin (GIBCO) on the pellicular anion-exchange column at pH 6.0. As previously shown using DEAE chromatography (7), there are five classes of oligosaccharides in fetuin corresponding to 0 (n), 1 (i), 2 (ii), 3 (iii), 4 (iv), and 5 (v) sialic acids. The major species are di-, tri-, and tetrasialylated oligosaccharides. Chromatography at alkaline pH using the same column resulted in the resolution of bovine fetuin oligosaccharides into approximately 20 peaks (Figs. 1B and 1C). Coelution studies with the characterized oligosaccharides given in Table 1 and the mixture of fetuin oligosaccharides were performed by adding sufficient amounts of each standard oligosaccharide to the N-glycanase-released oligosaccharide mixture to increase the height of the coeluting peak by 50-100%. The standard oligosaccharides coeluted with the numbered peaks (Figs. 1B and 1C) in the total mixture without detectable distortion of peak symmetry. The starred peaks which trail major peaks by ~2 min are likely due to epimerization of the reducing terminal (GlcNAc + ManNAc) from chromatography under alkaline conditions. These later eluting peaks disappear upon sodium borohydride reduction of the oligosaccharide mixture. These oligosaccharides were detected using pulsed amperometry which we have used in the chromatography of smaller anionic oligosaccharides (2). In this study (2), the PAD response among isomers of sialylated tetrasaccharides varied by as much as twofold. However, in these larger sialylated oligosaccharides, we found that the molar response factor was 4.8 f 14% (3.9-6.1) relative to an external glucose standard (Table 1). The relative electrochemical response for a disialylated biantennary oligosaccharide was found to be 5.2 (unpublished data). The degree of similarity in electrochemical response enabled estimation of these sialylated oligosaccharides directly from the electrochemical current. Duplicate acid hydrolyses were performed on compounds 3 and 8-10 for GN determination. As shown in Table 1, the difference in response factors (from two different hydrolyses) for the same compound was smaller than the range of relative responses among the compounds. The two major peaks released from fetuin were TRIs which coeluted with compounds 1 and 2 and contained

TOWNSEND

ET AL.

TABLE Retention

Times

and Response

Factors

of Sialylated

Fetuin

Structure

Compound number (Tri-i

1 Oligosaccharides

using HPAE-PAD

Retention time”

PAD response *

MBW)-R

30.0

4.2

W(W)-R

31.1

5.4

MB(U)-R

35.2

4.8,4.9d

W(W)-R

38.6

5.7

MPMbR

38.6

4.9

MP(W-R

40.2

3.9

4B)’ SAa(2,6)Gfl(l,4)GN/3(1,2)Ma(l,6) L SAa(2,6)G@(l,4)GNfl(1,2)Mao/ SAn(2,3)G~(l,4)GN/3(1,4)’ 2

(Tri-S 2A) SAa(2,3)GP(1,4)GNP(1,2)Ma(1,6) L /= SAa(2,6)G~(l,4)GNb(l,2)Mcu(1,3) SAa(2,3)Gfl(1,4)GNP(l,4)’ 3 (Tri-S 6B) SAa(2,6)GP(1,4)GNfl(1,2)Ma(l,6) I 7 SAa(2,6)G~(l,4)GN/3(1,2)Ma(l,3) WLW’JPW~~ SAn(2,6; 4 (Tri-S 5D) Gfl(1,4)GNp(1,2)Ma(l,6) L 7 SAa(2,6)G/3(1,4)GNfl(1,2)Ma(l,3) SAa(2,3)GP(l,3)GN/3(1,4)’ SAa(2,6; 5 (Tri-S 5C) SAcx(2,6)G~(1,4)GN~(l,2)Ma(l,6) L /* GP(1,4)GNP(1,2)Ma(l,3) SAa(2,3)GP(1,3)GNfl(1,4J7 SAcr(2,6; 6 (Tri-S 2B) SAa(2,3)G~(1,4)GN~(1,2)Ma(l,6) I /* SAa(2,6)GP(1,4)GNP(1,2)Ma(1,3) /1 SACU(~,~)GP(~,~)GN~(~,~)

CHROMATOGRAPHY

OF TABLE

Comnound

SIALYLATED

l-Continued Retention

Structure

number

5

OLIGOSACCHARIDES

time”

PAD

response

*

7 (Tri-S

3D) SAol(2,3)Gp(l,4)GNP(1,2)Mo1(1,6) L 41.0

6.

MP(1,4)-R

51.2

5.1,5.2

MP(lA-R

58.2

4.4,4.2

MP(W-R

71.8

4.1, 3.8

MN

1,4)-R

/1 GP(1,4)GNP(1,2)Mol(l,3) 7 SAol(2,3)G/%l,3)GNP(L4) /1 SAo1(2,6)

8 (Tetra-S

5) SAol(2,6)GP(1,4)GNP(1,2)Mo(l,6) I 7 SAol(2,6)Gp(l,4)GN/3(1,2)M~~(l,3) SA~(2,3)G/3(1,3)GN@(l,4)’ SAn(2.6)’ 9

(Tetra-S

3)

/’ SA~~(2,6)GP(1,4)GN/3(1,2)Mo(l,3) SAol(2,3)G@(l,3)GNfl(l,4;^ SAo1(2,6) 10 (Penta-S

/*

6) SAa(2,6) SAIY(2,3)Gfi(l,3);N~(1,2)Mn(1,6) L 7 SAcu(2,6)G@(l,4)GNP(1,2)Ma(1,3) SA~u(2,3)GP(1,3)GN@(1,4j SAo((2,6)’ R = GNP(1,4)GNa,P

’ Retention times observed using Gradient A described under Materials and Methods. * The PAD response is given relative to Glc which was 4128 rVs/pmol at 300 nA full scale. ’ The designations in parentheses correspond to nomenclature given these compounds in Refs. d The duplicate values are from two separate acid hydrolyses.

all Gals linked p(1,4). These two peaks constituted about 61% of the total PAD response. The remainder of the trisialylated TRIs (3-7), which contained the Gal/3(1,3)GlcNAc sequence, coeluted between these two major peaks and the tetrasialylated fractions. The peaks in this region of the chromatogram comprised 10% of the total PAD response. In this fetuin (GIBCO, Spiro method (9)), the di-, tetra-, and pentasialylated oligosac-

(7) and (8).

charides contributed the remainder with an estimated 10, 12, and 1% of the total PAD response, respectively (Table 2). Since incomplete enzymatic release of oligosaccharides from glycoproteins may introduce bias into the structures detected, we compared the profile of oligosaccharides obtained after treatment of the glycoprotein (28% of the oligosaccharides released) (Fig. 2A) or tryp-

6

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ET

AL. TABLE

Distribution

2

of Sialylated Oligosaccharides in Bovine Fetuins Sialylated Asialo

Source

* 4 3

GIBCO Sigma III Sigma IV Calbiochem a Percent

a

9

4.5

67

10 &

FIG. 1. HPAE-PAD of N-glycanase released oligosaccharides from bovine fetuin. Five nanomoles of oligosaccharides, enzymatically released from bovine fetuin (GIBCO, Spiro method (9)) was injected into the Dionex carbohydrate analyzer equipped as described under Materials and Methods. The PAD sensitivity was set at 300 nA full scale. (A) The column was equilibrated in 10 mM acetic acid with pH adjusted to 6.0 using 50% NaOH. The oligosaccharides were separated using Gradient A as described under Materials and Methods. In A, n, i, ii, iii, iv, and v denote the elution position of neutral, mono-, di-, tri-, tetra- and pentasialylated oligosaccharides, respectively. (B) The column was equilibrated in 0.1 M NaOH and 0.1 M sodium acetate. Gradient A, described under Materials and Methods, was developed as indicated by the dashed line. (C) A 3.5-fold expansion of the electrochemical response. The numbers indicate the elution position of the respective oligosaccharides shown in Table 1. The starred peaks likely represent epimers of the reducing terminal as discussed under Results.

tic glycopeptides (>93% released) (Fig. 2B). Qualitatively, the peak profiles in the tri- and tetraregions of the chromatograms were similar. Interestingly, the oligosaccharides representing at least 93% of those on fetuin showed proportionally increased amounts of oligosaccharide(s) coeluting with tri- and tetraantennary structures having an cY(2,3)-linked sialic acid on the Mana(l,G) arm (compounds 2 and 9). The asialo-, mono-, and disialylated regions of the chromatogram were also different between the two sets of oligosaccharides. With the above exceptions noted, the overall profiles were similar. We next examined the oligosaccharides released from bovine fetuin which had been purified using different methods and were from different commercial sources. The relative proportions of the major species are given in Table 2. Figure 3A is the oligosaccharide profile obtained from fetuin purified by an ammonium sulfate precipita-

of total

+ mono-

species

Di-

Tri-


10 26 14

71 66 68

6

50

43

PAD

Tetra-

Penta-

12 5 14

1 0.2 2 10.1

0.6

response.

tion method (Sigma, Type III) (14) and Fig. 3B shows oligosaccharides derived from the same type of fetuin which was further purified by Sephadex G-75 purification (Sigma, Type IV) to remove the high molecular weight contaminants (15). The oligosaccharide profile, shown in Fig. 2C, was from fetuin (Calbiochem) purified by the same ammonium precipitation method (14). The fetuin oligosaccharides shown in Figs. 1 and 2 was purified by the method of Spiro (9). Fetuins purified either by the method of Pederson (14) or by the method of Spiro (9) gave similar oligosaccharide profiles (Fig. 1 and Fig. 3A). Further purification by gel filtration approximately tripled the amount of tetrasialylated oligosaccharides (Fig. 3B) (Table 2). However, another fetuin, also purified by the method of Pederson, gave a very different profile. Two to five times more disialylated oli-

0

10

20

30

50

II

60

70

80

Tirne4~rnin)

FIG. 2. HPAE-PAD of fetuin oligosaccharides released to different extents by N-glycanase. Fetuin (GIBCO, Spiro method (9)) was incubated with N-glycanase and the oligosaccharides were separated from the glycoprotein as described for Method A under Materials and Methods. (A) The resulting chromatogram using Gradient A. The chromatogram of oligosaccharide released from tryptic glycopeptides (Method B) is shown in (B). The PAD sensitivity was set at 300 nA full scale.

CHROMATOGRAPHY

OF

SIALYLATED

1

2 -1 1

FIG. 3. HPAE-PAD of oligosaccharides released from bovine fetuins from different commercial sources. Oligosaccharides were released from Sigma Type III (A), Sigma Type IV (B), and Calbiochem (C) bovine fetuin and chromatographed using Gradient A. The PAD sensitivity was set at 300 nA full scale.

gosaccharides were found from this fetuin and a sharp reduction in tetra- and pentasialylated forms was observed (Fig. 3C). DISCUSSION

The chromatography of these branched tri- and tetrasialylated oligosaccharides showed features similar to our previously described separations of smaller sialylated oligosaccharides (2). We found that linear oligosaccharides containing sialic acids linked (r(2,6) were less retained than those having sialic acids linked a(2,3) (2). In the present study of multisialylated oligosaccharides, containing -both a(2,6)- and a(2,3)-linked sialic acids in a single structure, the order of elution was related to the combination of these two types of sialic acid linkages. We found that, within each charge class, oligosaccharides with a greater proportion of cu(2,6)-linked sialit acids were less retained. Apparently, some degree of oxyanion formation at the 6-OH of Gal or GlcNAc contributes to the greater retention time of a(2,3)-substituted oligosaccharides and, therefore, substitution instead at the ~-OHS negates this effect. In amine-bonded HPLC of sialylated oligosaccharides at near neutral pH (7) and at pH 4.0 (16), the reverse elution order has been observed in that cy(2,6)-linked sialic acid tends to increase retention times.

OLIGOSACCHARIDES

7

A unique advantage of HPAE chromatography of sialylated oligosaccharides is the distinct contribution of the neutral portion of the molecule (2). In this previous study, linear di-, tetra-, and monosialylated oligosaccharides containing a Gal/3(1,3)GlcNAc sequence were retained 5 to 10 min longer than structures in the same charge class containing only Gal@(l,4)GlcNAc units. In the present study, this same trend was observed in larger branched oligosaccharides containing three and four sialit acids. Thus, this retention time rule has been found to hold for both neutral (1) and sialylated oligosaccharides as well as glycopeptides with one to three amino acids (1). The sialylated oligosaccharides were detected using pulsed amperometry. We previously found that neutral oligosaccharides larger than pentasaccharides produced a response threefold greater than Glc (3). Small sialylated oligosaccharides (2-6 units) gave a response range of 1.4-2.9 (2). These branched anionic oligosaccharides (14-16 monosaccharide units) gave a response of 3.9 to 6.1(4.8 -t 14%). From these data, we conclude that there are significant differences in the electrochemical response among these branched oligosaccharides, but to a proportionately lesser extent than we found for the smaller oligosaccharides. Therefore, within this variation, the PAD response can be used to quantify branched di-, tri-, and tetra-, and pentasialylated oligosaccharides. The carbohydrate of bovine fetuin has been a paradigm for structural studies since the initial report by Spiro (17). Only recently, are the structural aspects of the sialylated structures being defined (7,8,16). In addition to the previously discussed separation and analytical difficulties (7,8,16), we found significant differences in the proportion of oligosaccharides from fetuins which apparently depended on the source and not necessarily the method or extent of purification. Fetuin was originally purified from fetal serum as a 45% ammonium sulfate fraction (14). Higher molecular weight contaminants (>48,000), that are usually found in this fraction, can be removed by gel filtration (15) or alternate precipitation strategies (9). We found that the two fetuins which had been purified using the same method had very different oligosaccharide profiles when analyzed by HPAE-PAD. These differences in oligosaccharide composition of fetuins from different sources may explain, for example, the reported range (8.5-40% of the total) (16,18,19) of the amount of Galp(l,3)GlcNAc TRIs found in different studies. The source of these variations is currently unknown. The differences may be related to the procurement of fetal calf serum or subtle alterations of the standard purification protocols. Alternatively, these differences in the proportion of sialylated species may reflect biological variation related to the age of the donor population, since the plasma concentration of fetuin changes rapidly perinatally (20). The availability of this method (HPAEPAD) for resolving and sensitively detecting compli-

8

TOWNSEND

ET

cated mixtures of oligosaccharides in a single chromatographic step should facilitate biological correlations with the oligosaccharides of fetuin and other glycoproteins. ACKNOWLEDGMENTS

1. Townsend, R. R., and USA 85,3289-3293.

Hardy,

M. R. (1988)

2. Townsend, R. R., Hardy, M. R., Hindsgaul, (1988) Anal. Biochem. 174,459-470. 3. Townsend, R. R., Hardy, in Enzymology, 179,65-76. R.,

7.

W-T., and Zopf, D. (1988) Carbohydr. L-M., Yet, M-G., and Shao, M.-C. 2819-2824. Bendiak, B., Harris-Brandts, M., Michnick, and Cumming, D. A., Biochemistry, in press.

Res., in press. (1988) FASEB S. W.,

Carver,

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R.R.T. was supported by the National Institutes of Health Grant DK31376. M.R.H. was the recipient of a Postdoctoral Carbohydrate Research Fellowship from Dionex Corporation (Sunnyvale, CA). The authors thank the MRC of Canada and the Terry Fox Special Initiatives Program for support. B.B. thanks Harry Schachter for MRC support. The Dionex carbohydrate analyzer was purchased with an instrumentation supplement grant to National Institutes of Health Research Grant DK-09970 to Dr. Yuan Chuan Lee. This paper is contribution number 1483 from the McCollum-Pratt Institute, The Johns Hopkins University. The authors thank Dr. Yuan Chuan Lee for the use of his laboratory for the chromatography studies.

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