Microanalysis of enzyme digests of hyaluronan and chondroitin/dermatan sulfate by fluorophore-assisted carbohydrate electrophoresis (FACE)

Anthony Calabro1, Maria Benavides, Markku Tammi2, Vincent C. Hascall and Ronald J. Midura

Department of Biomedical Engineering/ND20, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA and 2Department of Anatomy, University of Kuopio, P.O. Box 1627, SF-70211, Kuopio, Finland

Received on July 14, 1999; revised on September 3, 1999; accepted on September 9, 1999.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Hyaluronan and chondroitin/dermatan sulfate are glycosaminoglycans that play major roles in the biomechanical properties of a wide variety of tissues, including cartilage. A chondroitin/dermatan sulfate chain can be divided into three regions: (1) a single linkage region oligosaccharide, through which the chain is attached to its proteoglycan core protein, (2) numerous internal repeat disaccharides, which comprise the bulk of the chain, and (3) a single nonreducing terminal saccharide structure. Each of these regions of a chondroitin/dermatan sulfate chain has its own level of microheterogeneity of structure, which varies with proteoglycan class, tissue source, species, and pathology. We have developed rapid, simple, and sensitive protocols for detection, characterization and quantitation of the saccharide structures from the internal disaccharide and nonreducing terminal regions of hyaluronan and chondroitin/dermatan sulfate chains. These protocols rely on the generation of saccharide structures with free reducing groups by specific enzymatic treatments (hyaluronidase/chondroitinase) which are then quantitatively tagged though their free reducing groups with the fluorescent reporter, 2-aminoacridone. These saccharide structures are further characterized by modification through additional enzymatic (sulfatase) or chemical (mercuric ion) treatments. After separation by fluorophore-assisted carbohydrate electrophoresis, the relative fluorescence in each band is quantitated with a cooled, charge-coupled device camera for analysis. Specifically, the digestion products identified are (1) unsaturated internal {Delta}disaccharides including {Delta}DiHA, {Delta}Di0S, {Delta}Di2S, {Delta}Di4S, {Delta}Di6S, {Delta}Di2,4S, {Delta}Di2,6S, {Delta}Di4,6S, and {Delta}Di2,4,6S; (2) saturated nonreducing terminal disaccharides including DiHA, Di0S, Di4S and Di6S; and (3) nonreducing terminal hexosamines including glcNAc, galNAc, 4S-galNAc, 6S-galNAc, and 4,6S-galNAc.

Key words: chondroitin sulfate/dermatan sulfate/fine structure/hyaluronan/microanalysis


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Sulfated glycosaminoglycans such as chondroitin sulfate or dermatan sulfate are composed of three regions, a linkage oligosaccharide, connecting the chain to the core protein, a variably sulfated disaccharide repeat structure within the chain and a non-reducing terminus. These regions are of current interest since they are suggested to confer biologic functions on particular chain populations (see Introduction to Calabro et al., 2000). Many microanalytical techniques are now available to separate and quantitate nanogram amounts of the nonreducing termini (Otsu et al., 1985Go; Hascall et al., 1995Go; Midura et al., 1995Go; Plaas et al., 1996Go, 1997), unsaturated disaccharides (Carney and Osborne, 1991Go; Karamanos et al., 1994Go; Midura et al., 1994Go; Deutsch et al., 1995Go; Kitagawa et al., 1995Go) and linkage oligosaccharides (Sugahara et al., 1988Go; Shibata et al., 1992Go) produced by chondroitinase digestion of chondroitin/dermatan sulfate chains. The analyses include capillary zone electrophoresis (Carney and Osborne, 1991Go; Deutsch et al., 1995Go; Kitagawa et al., 1995Go) and high-performance liquid chromatography (Otsu et al., 1985Go; Shibata et al., 1992Go; Karamanos et al., 1994Go; Midura et al., 1994Go, 1995; Hascall et al., 1995Go; Plaas et al., 1996Go, 1997). However, these existing analytical methods have distinct disadvantages. They normally require long, labor intensive preparation times which include: (1) isolation of the chondroitin sulfate chains from tissues or cells, (2) purification of the chondroitin sulfate chains from other macromolecules, (3) purification of the chondroitinase digestion products from the enzyme after digestion, (4) purification of the chondroitinase digestion products from reducing agents used to stabilize the digestion products during analysis, and/or (5) purification of derivatized chondroitinase digestion products from unreacted fluorescent tag used as a reporter of mass. In addition, they normally allow for analysis of only one sample at a time with each analysis taking on the order of hours. 1H-NMR and mass spectroscopy have the additional disadvantage of requiring large quantities of starting material (Sugahara et al., 1988Go). The present work was therefore undertaken to provide a new methodological approach, which would allow for rapid, simple, and sensitive detection and quantitation of internal disaccharide and nonreducing terminal structures of chondroitin/dermatan sulfate and hyaluronan without the disadvantages of previous methods.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Fluorescent derivatization and polyacrylamide gel electrophoresis of the internal {Delta}disaccharide structures derived from hyaluronan and chondroitin/dermatan sulfate chains
The internal disaccharides of hyaluronan and chondroitin/dermatan sulfate chains yield unsaturated {Delta}disaccharides after chondroitinase digestion (Figure 1). Standard hyaluronan and chondroitin/dermatan sulfate {Delta}disaccharides were derivatized with 2-aminoacridone (AMAC) as described in Materials and methods (Figure 1). Figure 2A shows the positions of the two unsulfated and three monosulfated AMAC-derivatized {Delta}disaccharides in the FACE analysis: {Delta}DiHA from hyaluronan (lane 1), and {Delta}Di0S, {Delta}Di2S, {Delta}Di4S, and {Delta}Di6S from chondroitin/dermatan sulfate (lanes 2–5, respectively). Figure 2B shows the positions of the three disulfated and one trisulfated AMAC-derivatized {Delta}disaccharides from chondroitin/dermatan sulfate: {Delta}Di2,4S (or {Delta}DiB), {Delta}Di2,6S (or {Delta}DiD), {Delta}Di4,6S (or {Delta}DiE), and {Delta}Di2,4,6S (or {Delta}DiTriS) (lanes 6–9, respectively). With the exception of the {Delta}Di2,4S and {Delta}Di2,4,6S, which run at the same position in the electrophoresis front, all of these {Delta}disaccharides are clearly resolved. Figure 3 shows standard curves for four {Delta}disaccharides, {Delta}DiHA, {Delta}Di0S, {Delta}Di4S, and {Delta}Di6S. The four curves superimpose indicating that the AMAC fluorotag gives the same molar fluorescence value for each derivative with a free reducing terminus. The curves show linearity on a log-log plot over the range from 6.25 to 100 pmol, (~2–50 ng for these {Delta}disaccharides) as measured by hexuronic acid assay.



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Fig. 1. Schematic showing the structure of a 4-sulfated tetrasaccharide (Di4SDi4S), and its two chondroitinase digestion products (reaction 1). The left column shows the products expected following subsequent mercuric ion treatment (reaction 2), and AMAC derivatization (reaction 3) of the saturated disaccharide product (Di4S), which is representative of other nonreducing terminal disaccharide structures of chondroitin sulfate chains. The right column shows the products expected following similar treatments of the unsaturated {Delta}disaccharide product ({Delta}Di4S), which is representative of other internal disaccharide structures of chondroitin sulfate chains. The structure of the fluorotag, 2-aminoacridone (AMAC), is shown at the bottom left.

 


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Fig. 2. FACE analyses of hyaluronan and chondroitin sulfate {Delta}disaccharides derivatized with AMAC as described in Materials and methods. Each lane contains the following AMAC-derivatized {Delta}disaccharide standard: {Delta}DiHA from hyaluronan (lane 1), and {Delta}Di0S, {Delta}Di2S, {Delta}Di4S, {Delta}Di6S, {Delta}Di2,4S, {Delta}Di2,6S, {Delta}Di4,6S, and {Delta}Di2,4,6S from chondroitin sulfate (lanes 2–9, respectively). The standard lanes (S7) contain a mixture of seven AMAC-derivatized {Delta}disaccharides from top to bottom: {Delta}DiHA, {Delta}Di0S, {Delta}Di6S, {Delta}Di4S, {Delta}Di2S, {Delta}Di4,6S, and {Delta}Di2,4,6S.

 


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Fig. 3. Quantitation of AMAC-derivatized hyaluronan and chondroitin sulfate {Delta}disaccharides after separation by FACE. Mixtures containing from 6.25 to 100 pmol each of the indicated AMAC derivatives were separated by FACE. The gel (inset) was then imaged using a Quantix cooled-CCD camera, and the images analyzed using the Gel-Pro Analyzer program as described in Materials and methods. The relative fluorescence for each band is plotted versus pmoles of {Delta}disaccharides as determined by hexuronic acid analysis (R = 0.997, P = 0.00022).

 
Fluorescent derivatization and polyacrylamide gel electrophoresis of hyaluronan ladders
Figure 4 shows FACE analyses of AMAC-derivatized products from partial digestion of a constant amount of hyaluronan for a constant time with serial dilutions of testicular hyaluronidase. This enzyme is a hydrolase with endo-hexosaminidase specificity, and gives a ladder of oligomer digestion products that differ by one disaccharide repeat. The partial digests at the lower enzyme concentrations (lanes 4 and 5) reveal a ladder extending above 50 disaccharides. As the enzyme concentration increases, the primary end products HA4 and HA6 increase. They no longer migrate on the basis of size and show an inversion in mobility, with the HA6 overlapping the HA8 and the HA4 migrating at the level of HA14. The digests at the higher enzyme concentrations (lanes 2 and 3) also show a small amount of the HA2 (DiHA) disaccharide, a minor product of this enzyme, which comigrates with {Delta}DiHA (data not shown). This shift in mobility away from one based solely on size is presumably the result of the smaller oligosaccharides interacting with borate in the electrophoresis buffer. The presence of borate in the Glyko gel running buffer was inferred from experiments in which a gel running buffer from Novex (#LC6675) containing 89 mM Tris, 89 mM boric acid, and 2 mM EDTA, pH 8.3 similar to that described in Jackson et al. (1991, 1994) was substituted, and produced identical results (data not shown). The interaction with borate makes possible the separation of AMAC-derivatized oligosaccharides with similar molecular weights, but different chemistries as seen in Figures 2, 5, 6, and 7. A standard mixture of three purified, AMAC-derivatized hyaluronan oligomers, HA10, HA14 and HA18, is shown in lane 1 to index the ladder.



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Fig. 4. FACE analyses of AMAC-derivatized products from partial digestion of a constant amount of hyaluronan (100 µg) for 4 h at 37°C with 1:3 serial dilutions of testicular hyaluronidase starting at 1000 U/ml (lanes 2–5). The relative positions of the saturated hyaluronan oligomers containing 1 (HA2), 2 (HA4), 3 (HA6), 4 (HA8), 5 (HA10), 10 (HA20), 15 (HA30), 20 (HA40), and 25 (HA50) disaccharides are indicated. Lane 1 contains a standard mixture of three purified, AMAC-derivatized hyaluronan oligomers (HA10, HA14, and HA18) used to index the ladder.

 


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Fig. 5. FACE analyses of hyaluronan and chondroitin sulfate {Delta}disaccharides treated with mercuric ion, and then AMAC derivatized. A shift in mobility of a band from that of the original derivatized {Delta}disaccharide structure to that of its derivatized product(s) after mercuric ion treatment is indicated by arrows. Equal signs indicate that co-migrating bands are the same structure. The standard lanes labeled S5 contain the following mixture of five AMAC-derivatized hexosamines listed as they appear from top to bottom: galNAc, glcNAc, 6S-galNAc, 4S-galNAc, and 4,6S-galNAc.

 


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Fig. 6. FACE analyses of a Di4SDi4S tetrasaccharide prepared from testicular hyaluronidase digests of chondroitin sulfate chains isolated from rat chondrosarcoma aggrecan as described in Materials and methods. Samples were derivatized directly with AMAC (lanes 1), chondroitinase ABC digested then AMAC derivatized (lanes 2), or chondroitinase ABC digested followed by mercuric ion treatment then AMAC derivatized (lanes 3). Analyses of the major tetrasaccharide (Di4SDi4S) and minor tetrasaccharides (Di0SDi4S and Di4SDi0S) are highlighted in (A) and (B), respectively. The positions of nonreducing terminal structures are indicated (x). The standard lanes labeled S8 contain a mixture of eight AMAC-derivatized {Delta}disaccharides from top to bottom: {Delta}DiHA, {Delta}Di0S, {Delta}Di6S, {Delta}Di4S, {Delta}Di2S, {Delta}Di4,6S, {Delta}Di2,6S, and {Delta}Di2,4,6S.

 


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Fig. 7. FACE analyses of a Di6SDi6S tetrasaccharide prepared from testicular hyaluronidase digests of chondroitin sulfate chains isolated from human adult cartilage aggrecan as described in Materials and methods. Samples were derivatized directly with AMAC (lanes 1), chondroitinase ABC digested then AMAC derivatized (lanes 2), or chondroitinase ABC digested followed by mercuric ion treatment then AMAC derivatized (lanes 3). Analyses of the major tetrasaccharide (Di6SDi6S) and minor tetrasaccharides (Di6SDi4S and Di4SDi6S) are highlighted in (A) and (B), respectively. The positions of nonreducing terminal structures are indicated (x).

 
Fluorescent derivatization and polyacrylamide gel electrophoresis of the nonreducing terminal monosaccharide structures derived from chondroitin/dermatan sulfate chains
The nonreducing ends of some chondroitin/dermatan sulfate chains yield substituted galNAc after chondroitinase digestion. Mercuric ion treatment quantitatively removes the {Delta}hexuronic acid from the {Delta}disaccharides, releasing the hexosamine portion (Ludwigs et al., 1987Go), Figure 1. This chemistry was used to assign elution positions for all the potential galNAc non-reducing termini. Figure 5 shows FACE analyses of the one hyaluronan and eight chondroitin/dermatan sulfate derived {Delta}disaccharides analyzed in Figure 2 after treatment with, and subsequent removal of mercuric ion, followed by derivatization with AMAC. {Delta}DiHA, {Delta}Di0S, {Delta}Di4S, {Delta}Di6S, and {Delta}Di4,6S yield exclusively AMAC-derivatized glcNAc, galNAc, 4S-galNAc, 6S-galNAc, and 4,6S-galNAc, respectively, each of which migrates to a unique location relative to their original derivatized {Delta}disaccharide.

{Delta}Di2S, {Delta}Di2,4S, and {Delta}Di2,4,6S yielded quantitatively derivatized galNAc, 4S-galNAc, and 4,6S-galNAc, respectively, as expected. However, in each case they also yielded a major unknown band (X1), which migrates at a position behind derivatized 4S-galNAc, as well as two minor, more rapidly migrating unknown bands (X2 and X3). The sum of the intensities of these unexpected bands in each case is nearly equal to the intensity of the respective derivatized galNAc product. Thus, they appear to be products from the 2S-substituted {Delta}hexuronic acid moiety that are stable to the mercuric ion treatment, and that contain a free reducing group that reacts with the fluorotag. In the absence of a 2S-substituted {Delta}hexuronic acid only minor amounts of the X1 product are detected (see lanes 1, 2, and 8). As indicated in the analysis of the products from {Delta}Di2,6S (lane 7), this major new band (X1) comigrates with derivatized 6S-galNAc.

Fluorescent derivatization and polyacrylamide gel electrophoresis of the nonreducing terminal saturated disaccharide structures derived from chondroitin sulfate chains
The nonreducing ends of some chondroitin sulfate chains yield saturated disaccharides after chondroitinase digestion, which are not altered by mercuric ion treatment, Figure 1. Thus, FACE analyses were used to identify the locations for derivatized Di0S, Di4S, and Di6S. Tetrasaccharides were prepared from testicular hyaluronidase digests of chondroitin sulfate isolated from Swarm rat chondrosarcoma aggrecan (almost exclusively 4-sulfated) and from human adult cartilage aggrecan (almost exclusively 6-sulfated) as described in Materials and methods. Since chondroitinase is an eliminase, digestion of these tetrasaccharides with this enzyme gives equal quantities of both an unsaturated and a saturated disaccharide, Figure 1. FACE analyses are shown in Figures 6 and 7 for the 4-sulfated tetrasaccharide, Di4SDi4S, and the 6-sulfated tetrasaccharide, Di6SDi6S, respectively. Analyses were made on intact derivatized tetrasaccharides (lanes 1), and for the derivatized products of chondroitinase digests of the tetrasaccharides either directly (lanes 2) or after treatment with mercuric ion (lanes 3).

In Figure 6, the undigested 4-sulfated tetrasaccharide sample shows a single dominant band representative of derivatized Di4SDi4S (panel A, lane 1). After chondroitinase digestion, the 4-sulfated tetrasaccharide gives a broad band composed of two major, overlapping bands, one of which is the previously identified {Delta}Di4S, and the other of which is Di4S, which migrates slightly ahead (panel A, lane 2). The identities of these two bands were confirmed after mercuric ion treatment (panel A, lane 3). The saturated Di4S was resistant to the mercuric ion treatment and therefore did not change mobility. However, the mercuric ion treatment removed the {Delta}hexuronic acid from the {Delta}Di4S shifting its elution position to that of 4S-galNAc.

In Figure 6, a minor tetrasaccharide band migrates slower than the Di4SDi4S band. This band contains 2 tetrasaccharides, Di4SDi0S and Di0SDi4S (panel B, lane 1). The presence of the Di4SDi0S is indicated by the production of identifiable {Delta}Di0S after chondroitinase digestion (panel B, lane 2) that gives rise to galNAc after mercuric ion treatment (panel B, lane 3). The presence of the Di0SDi4S is indicated by the production of Di0S (panel B, lane 2) which is not altered by the mercuric ion treatment (panel B, lane 3). A small amount of {Delta}DiHA is also present, indicating some contamination of the original chondroitin sulfate sample with hyaluronan (panel B, lane 2). In Figure 6, several additional minor peaks are also apparent, indicating some heterogeneity in the size and in the sulfation patterns of the undigested oligosaccharides. The presence of 4S-galNAc in the fluorotagged chondroitinase products (panel A, lane 2) indicates the presence of oligosaccharides with nonreducing terminal 4S-galNAc in the preparation. This indicates that some of the minor bands in the preparation (panel A, lane 1) are trisaccharides and/or pentasaccharides.

In Figure 7, the analysis of the 6-sulfated tetrasaccharide reveals similar information. The undigested 6-sulfated tetra­saccharide sample shows a single dominant band representative of derivatized Di6SDi6S (panel A, lane 1). The {Delta}Di6S and Di6S bands in the chondroitinase digest overlap, with the latter migrating slightly ahead (panel A, lane 2). The former gives rise to 6S-galNAc, while the latter is unaffected by the mercuric ion treatment (panel A, lane 3). A minor tetrasaccharide band migrates just ahead of the Di6SDi6S band. This band contains 2 tetrasaccharides, Di6SDi4S and Di4SDi6S (panel B, lane 1). The presence of the Di6SDi4S is indicated by the production of identifiable {Delta}Di4S after chondroitinase digestion (panel B, lane 2) that gives rise to 4S-galNAc after mercuric ion treatment (panel B, lane 3). The presence of the Di4SDi6S is indicated by the production of Di4S (panel B, lane 2) which is not altered by the mercuric ion treatment (panel B, lane 3). As with the Di4SDi4S tetrasaccharide in Figure 6, several additional minor peaks are also apparent in the Di6SDi6S preparation, indicating some heterogeneity in the size and in the sulfation patterns of the undigested oligo­saccharides. Further, the presence of 6S-galNAc in the fluorotagged chondroitinase products (panel A, lane 2) indicates that some of the minor bands are trisaccharides and/or pentasaccharides with nonreducing terminal 6S-galNAc. As predicted from the chemistry (Figure 1), the intensities of the bands for both derivatized tetrasaccharides, and their respective derivatized saturated and unsaturated disaccharides should be equivalent, and all are within 5% or less of each other.

Specificity of chondro-4-sulfatase and chondro-6-sulfatase for sulfated {Delta}disaccharides and sulfated galNAc structures
The suitability of the sulfated {Delta}disaccharides, and the sulfated galNAc structures to serve as substrates for chondro-4-sulfatase and chondro-6-sulfatase was tested. In all cases, both the chondro-4-sulfatase and chondro-6-sulfatase showed only specificity for sulfate at the 4- and 6-positions of the hexosamine, respectively, with neither enzyme showing specificity for sulfate at the 2-position of the hexuronic acid. Importantly, 4S-galNAc, 6S-galNAc, and 4,6S-galNAc were not substrates for either of the sulfatases (data not shown). The results for only those {Delta}disaccharides that proved to be substrates for the two sulfatases are shown in Figure 8. Digestion with the chondro-6-sulfatase alone specifically and quantitatively removed sulfate from the 6-position of {Delta}Di6S, {Delta}Di2,6S, and {Delta}Di4,6S yielding {Delta}Di0S, {Delta}Di2S, and {Delta}Di4S, respectively (lanes 4 – 6). Digestion with the chondro-4-sulfatase alone specifically and quantitatively removed sulfate from the 4-position of only {Delta}Di4S, yielding {Delta}Di0S (lane 1). The chondro-4-sulfatase only partially removed the sulfates from the 4-position of {Delta}Di2,4S and {Delta}Di4,6S, yielding minor amounts of {Delta}Di2S, and {Delta}Di6S, respectively (lanes 2 and 3). However, when the chondro-4-sulfatase and chondro-6-sulfatase were used together, the sulfates from both the 4- and 6-positions were quantitatively removed from {Delta}Di4,6S, yielding {Delta}Di0S (lane 7).



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Fig. 8. FACE analyses of sulfated chondroitin sulfate {Delta}disaccharides digested with or without chondro-4-sulfatase (4Sase) and/or chondro-6-sulfatase (6Sase) prior to AMAC derivatization (lanes 1–8) or with mercuric ion treatment (Hg2+) after sulfatase digestion, but prior to AMAC derivatization (lanes 9–12). The standard lanes (S11) contain a mixture of eleven AMAC-derivatized saccharides from top to bottom: galNAc, glucose, {Delta}DiHA, {Delta}Di0S, 6S-galNAc, 4S-galNAc, {Delta}Di6S, {Delta}Di4S, {Delta}Di2S, {Delta}Di4,6S, and {Delta}Di2,4,6S.

 
Detection of the expected products after sulfatase digestion of {Delta}Di2,4,6S required mercuric ion treatment prior to derivatization with AMAC, since after derivatization the expected products, {Delta}Di2,4S and {Delta}Di2,6S, migrate at or near the electrophoresis front. The migration of derivatized {Delta}Di2,4,6S at the electrophoresis front is shown in lane 8. The expected 4,6S-galNAc, X1, X2, and X3 products are seen after mercuric ion treatment of {Delta}Di2,4,6S (lane 9). Digestion with the chondro-6-sulfatase alone specifically and quantitatively removed sulfate from the 6-position of {Delta}Di2,4,6S yielding {Delta}Di2,4S which, after mercuric ion treatment, yields 4S-galNAc along with the X1, X2, and X3 bands (lane 10). However, the chondro-4-sulfatase was unable to remove the sulfate from the 4-position of {Delta}Di2,4,6S as indicated by the unchanged, derivatized 4,6S-galNAc band (lane 11). Any minor amounts of the 6S-galNAc resulting from chondro-4-sulfatase digestion of {Delta}Di2,4,6S to yield {Delta}Di2,6S is undetectable since, after mercuric ion treatment, 6S-galNAc comigrates with the X1 band. When both the chondro-4-sulfatase and chondro-6-sulfatase were used, the sulfate from the 6-position was quantitatively removed from {Delta}Di2,4,6S, but the sulfate from the 4-position was only partially removed yielding a mixture of {Delta}Di2S and {Delta}Di2,4S which, after mercuric ion treatment, yields galNAc and 4S-galNAc, respectively, along with the X1, X2, and X3 bands (lane 12). These results confirm that sulfation at either the 6-position of the hexosamine or the 2-position of the hexuronic acid inhibits the chondro-4-sulfatase activity. Higher enzyme concentrations or longer incubation times did not change these results (data not shown). The incomplete nature of the mercuric ion treatment in lanes 9–12 is discussed below in Important considerations.

Important considerations
This section describes important considerations for FACE analysis for which the data are not shown. A major consideration throughout these analyses was pH. The chondroitinase enzyme showed incomplete digestion of hyaluronan and unsulfated chondroitin sulfate when the pH was above 7. For this reason 0.0005% phenol red was added to the sodium acetate or ammonium acetate buffers at pH 7 to insure that the pH of resuspended samples was at or below 7. This was done visually based on the color of the phenol red indicator dye, which appears yellow at a pH of 6.8 and red at a pH of 8.2. Resuspended samples which appeared pink to red were titrated back to a pH of 6.8 to 7.0 by adding 1 µl aliquots (total of 10 maximum) of either a 0.1, 1, or 10 M acetic acid solution as appropriate until the dye first turned yellow. The phenol red was reduced to a colorless compound in the presence of cyanoborohydride, and therefore was not detected in the FACE gels. The amount of 0.0005% phenol red in 100 µl of solution is ~1 nmol, which is minor compared to the 50,000 nmol of cyanoborohydride present in the derivatization reaction. Maintaining an acidic pH was also necessary because the unsaturated disaccharides produced by chondroitinase are unstable at alkaline pH prior to derivatization. Also for this reason, samples were always frozen on dry ice and then lyophilized, rather than concentrated, to avoid a potential rise in pH as a result of the acetate ion evaporating faster than the ammonium ion. The major danger of low pH is desulfation, but even in the presence of acetic acid at a pH of 3.5 (see below) there was little evidence of desulfation using our protocols.

The pH in the final derivatization reaction was approximately 6.8 due to the buffer formed from the acetic acid and cyanoborohydride. If while maintaining the acetic acid concentration, the cyanoborohydride concentration was reduced to 125 mM, a 10-fold molar excess of cyanoborohidride above that of the AMAC, then the pH of the final derivatization solution was ~3.5. Under these reaction conditions the derivatization proceeded to completion. However, if the acetic acid was removed and the cyanoborohydride concentration was left at 1.25 M, then the resulting pH was ~10.5 and no derivatized products were detected. This was independent of the nature of the oligosaccharide to be derivatized and not the result of alkaline degradation of unsaturated disaccharide structures, since glucose showed similar results.

Previous methods have used derivatization reaction volumes of as little as 10 µl (Jackson, 1991Go, 1994). This allows for a greater percentage of the total sample to be loaded on the FACE gel. However, we found a high degree of variability in samples as a direct result of the difficulty in complete resuspension of lyophilized oligosaccharides in such small volumes. Therefore, we use a derivatization reaction volume of 80 µl, which was determined to be the minimum volume required to access easily the entire inner surface of a 1.5 ml microcentrifuge tube by simple vortexing, and thus confidently resuspend all of a lyophilized sample. If lower reaction volumes are required, then the concentration of AMAC should be increased proportionally to maintain a 10-fold molar excess above that of the oligo­saccharides to be derivatized. We routinely load only 5 µl or 1/20th of the 100 µl of the final derivatization reaction. At the salt concentrations we use, higher loading volumes caused the faster moving bands to narrow as they entered the gel. Larger loading volumes of from 10 to 20 µl were possible, but require that the salt concentration be proportionally lowered. This can be accomplished without decreasing the reaction volume by decreasing both the cyanoborohydride and acetic acid concentrations to the same extent so as to maintain the proper pH. The cyanoborohydride can be decreased to 125 mM while still maintaining a 10-fold molar excess above that of the AMAC.

Sodium acetate can be used in place of ammonium acetate for the chondroitinase or sulfatase digestion steps and, although it is not volatile, its presence after lyophilization does not affect the derivatization reaction (see Figure 8, lanes 1–8). However, it is important to use a volatile buffer such as ammonium acetate for those samples that are to be treated with mercuric ion after chondroitinase digestion. This is because the nonvolatile sodium acetate buffer, pH 7, remains behind after lyophilization and thus increases the pH of the mercuric acetate solution above the normal pH of 5 used for the mercuric ion treatment. This results in incomplete removal of the {Delta}hexuronic acid from {Delta}disaccharides and production of unidentified products (see Figure 8, lanes 9–12). The mercuric ion treatment is done prior to derivatization, and then the mercuric ion is removed by treatment with Dowex H+ resin, because the AMAC-derivatized {Delta}disaccharides are unstable to mercuric ion. When 50 nmol aliquots of AMAC-derivatized {Delta}disaccharides were treated with mercuric ion at concentrations of from 0.15 to 35 mM for 30 min at room temperature only the 4 mM mercuric ion concentration produced the expected AMAC-derivatized hexosamine products. At lower mercuric ion concentrations only the original AMAC-derivatized {Delta}disaccharides were seen indicating insufficient reagent, while at higher mercuric ion concentrations no AMAC-derivatized material was detected indicating either loss of the AMAC and/or destruction of the {Delta}disaccharide. This is in contrast to mercuric ion treatment prior to derivatization, which is quantitative over a wide concentration range.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We have developed fast, simple and sensitive protocols for determining the fine structure of hyaluronan and chondroitin/dermatan sulfate chains. These protocols take advantage of bacterial eliminases, such as chondroitinase ABC or hyaluronidase SD, which are specific for chondroitin/dermatan sulfate and/or hyaluronan. These enzymes generate specific oligosaccharide products with free reducing aldehydes of known chemistry that were previously blocked in the intact chains. These newly generated reducing aldehydes are then quantitatively derivatized with the fluor, 2-aminoacridone (AMAC), by a Schiff’s base reaction which is stabilized by reduction with cyanoborohydride. This provides for the same molar fluorescence per reducing aldehyde for each derivatized saccharide independent of its chemistry, and for detection in the 1 pmol or less range when imaged with our cooled CCD camera system after separation by fluorophore-assisted carbohydrate electrophoresis (FACE).

Two additional chemistries are used in our protocols. Since the bacterial enzymes are eliminases, they release internal disaccharides with unsaturated hexuronic acid residues, while any nonreducing terminal disaccharides contain saturated hexuronic acid residues (see Figure 1). This provides a means for distinguishing these two structures, since only the internal disaccharides with their unsaturated hexuronic acid are sensitive to mercuric acid treatment. In addition, chondro-4-sulfatase and chondro-6-sulfatase, enzymes which selectively remove sulfate esters from chondroitin/dermatan sulfate derived disaccharides (see Figure 8), can be used to confirm the sulfation pattern of the digestion products analyzed by FACE.

Our standard protocols for FACE analysis of an unknown sample involves a minimum of four steps. Step 1 involves direct AMAC derivatization of the starting material. This identifies any structures with reducing groups that are present in the starting material, whether expected, such as in the tetrasaccharide preparations shown in Figures 6 and 7, or unexpected, such as glucose, which is often seen in samples obtained directly from tissue extracts or culture medium (see Calabro et al., 2000Go). Step 2 involves AMAC derivatization of the chondroitinase digestion products. This establishes the fine structure of the mass of the chondroitin sulfate chain (i.e., internal {Delta}disaccharide composition), and some nonreducing termini that are clearly resolved (i.e., galNAc, 4S-galNAc, and 6S-galNAc). Step 3 involves AMAC derivatization of the chondroitinase digestion products after treatment with mercuric ion. This unmasks the remaining nonreducing termini including saturated disaccharides and 4,6S-galNAc, and helps confirm the identities of the internal {Delta}disaccharides. Step 4 involves AMAC derivatization of the chondroitinase digestion products after treatment with chondro-4-sulfatase and/or chondro-6-sulfatase. This confirms the identity of any sulfated disaccharides, and allows for resolution of comigrating bands as described below. Together these four protocols allow for complete fine structure analysis of chondroitin/dermatan sulfate chains and for measurement of hyaluronan content. In most cases, they provide for several independent measurements of each saccharide structure. Application of these protocols for the analysis of the hyaluronan and chondroitin sulfate on aggrecan from cartilage is shown in an accompanying article (Calabro et al., 2000Go).

The specific chondroitinase digestion products which were identified with these protocols are the unsaturated internal {Delta}disaccharides including {Delta}DiHA, {Delta}Di0S, {Delta}Di2S, {Delta}Di4S, {Delta}Di6S, {Delta}Di2,4S, {Delta}Di2,6S, {Delta}Di4,6S, and {Delta}Di2,4,6S (see Figure 2), the saturated nonreducing terminal disaccharides including DiHA (see Figure 4), Di0S, Di4S, and Di6S (see Figures 6 and 7), and the nonreducing terminal hexosamines including glcNAc, galNAc, 4S-galNAc, 6S-galNAc, and 4,6S-galNAc (see Figure 5). In addition, we have identified the relative position of AMAC-derivatized glucose (see Figure 8, lane S11). Of these 19 AMAC-derivatized saccharides, all clearly resolve by FACE with the exception of: (1) {Delta}Di0S from Di0S and {Delta}Di4S from Di4S; (2) {Delta}Di6S from both Di6S and 4,6S-galNAc; (3) {Delta}Di2,4S, {Delta}Di2,6S, and {Delta}Di2,4,6S from each other (The resolution of {Delta}Di2,6S from {Delta}Di2,4S and {Delta}Di2,4,6S at the electrophoresis front was variable depending on the gel lot as seen in the standards (lanes S8) in Figures 6 and 7); (4) {Delta}DiHA from both glcNAc and DiHA; and (5) 6S-galNAc and the X1 product. The oligosaccharide composition of specific samples will determine which of these problems must be addressed as described below.

The lack of resolution of AMAC-derivatized {Delta}Di0S and {Delta}Di4S from AMAC-derivatized Di0S and Di4S, respectively, is easily resolved by mercuric ion treatment (step 3 above). Treatment with mercuric ion converts the typically abundant internal {Delta}disaccharide structures, {Delta}Di0S and {Delta}Di4S, to galNAc and 4S-galNAc, respectively, which both migrate at different and unique positions in the FACE analysis. This permits subsequent quantitation of any nonreducing terminal Di0S and Di4S structures whose chemistries and therefore relative mobilities are unaffected by mercuric ion treatment. Since mercuric ion treatment contributes galNAc and 4S-galNAc from {Delta}Di0S and {Delta}Di4S, respectively, the amounts of galNAc and 4S-galNAc originally present as nonreducing terminal structures are measured in the original chondroitinase digest (step 2 above).

The lack of resolution of AMAC-derivatized {Delta}Di6S from AMAC-derivatized Di6S is complicated by the comigration of AMAC-derivatized 4,6S-galNAc. Treatment with mercuric ion (step 3 above) converts the typically abundant internal {Delta}disaccharide structure, {Delta}Di6S, to 6S-galNAc, which migrates at a different and unique position in the FACE analysis. Since mercuric ion treatment contributes 6S-galNAc from {Delta}Di6S, the amount of 6S-galNAc originally present as a nonreducing terminal structure is measured using the original chondroitinase digest (step 2 above). However, unlike for Di0S and Di4S above, direct quantitation of Di6S in samples treated with mercuric ion is only possible if no detectable 4,6S-galNAc is present in the sample. Samples containing 4,6S-galNAc as either a non-reducing terminal structure or as internal {Delta}Di4,6S, which subsequently appears as 4,6S-galNAc after mercuric ion treatment, require an additional calculation. The amount of 4,6S-galNAc resulting from mercuric ion treatment of {Delta}Di4,6S is easily estimated from the {Delta}Di4,6S band in the chondroitinase digest (step 2 above), and this value subtracted. Generally, since the remaining Di6S and 4,6S-galNAc are both nonreducing terminal structures, their lack of resolution is not a major problem, because the estimation of number averaged chain length for chondroitin sulfate chains is unaffected (Calabro et al., 2000Go). However, if individual quantitation of Di6S and 4,6S-galNAc is desired then the chondroitinase digests (step 2 above) can be digested with both chondro-4-sulfatase and chondro-6-sulfatase prior to derivatization to convert all the saturated and unsaturated disaccharides to Di0S and {Delta}Di0S, respectively. This permits the non-reducing terminal 4,6S-galNAc to be measured directly (step 4 above), and the amount of Di6S is then calculated by subtraction.

The lack of resolution of AMAC-derivatized {Delta}Di2,4S, {Delta}Di2,6S, and {Delta}Di2,4,6S which run at or near the electrophoresis front, is partially resolved by select sulfatase digestion of the chondroitinase digest (step 4 above). For example, the appearance of {Delta}Di2S following chondro-4-sulfatase digestion alone or chondro-6-sulfatase digestion alone indicates the presence of {Delta}Di2,4S, or {Delta}Di2,6S, respectively, in the electrophoresis front (see Figure 8). However, due to the incomplete nature of digestion of {Delta}Di2,4S and {Delta}Di2,4,6S with chondro-4-sulfatase (see Figure 8), quantitation of only the {Delta}Di2,6S may be possible. We are currently investigating different running buffers, gel buffers, gel compositions, and electrophoresis conditions in an effort to resolve our current problems with resolution of these {Delta}disaccharides.

The lack of resolution of AMAC-derivatized {Delta}DiHA from glcNAc and DiHA is primarily in the estimation of number averaged chain length for hyaluronan chains. The internal {Delta}disaccharide from hyaluronan ({Delta}DiHA), unlike those from chondroitin sulfate, migrates close to its mercuric ion treatment product, glcNAc, so that mercuric ion treatment (step 3 above) does not unmask the nonreducing terminal DiHA structure. Fortunately, quantitation of total hyaluronan is unaffected since all three structures contribute to the total mass of hyaluronan, and their comigration means they do not interfere with structures related to chondroitin/dermatan sulfate.

The advantages of the FACE procedure over previous metho­dologies include sensitivity, speed and simplicity. As mentioned above, AMAC gives the same molar fluorescence value for every derivatized saccharide independent of chemistry. Figure 2 shows that the standard curves for four {Delta}disaccharides superimpose. In this case, the curves show linearity on a log-log plot over the range from 6.25–100 pmol. With the new, highly sensitive Quantix CCD camera, the standard curves are linear and readily quantitated at one log lower concentrations, i.e., in the 0.5 pmol range. The FACE analyses in Figures 6 and 7, from digestion of the samples to electrophoresis, image capture and quantitation, can now be completed in 2 days, and require only a few micrograms of starting material. With the exception of the mercuric ion treatment, which requires a Dowex step to remove the reagent, all steps are carried out in a single tube, and enzymes and reagents are not removed before the electrophoresis. In comparison, the previous HPLC based method required a week or more to complete, as derivatized samples need to be purified from enzymes and reagents, and each must be analyzed separately on the Dionex column. Further, the FACE resolves products that are either lost or poorly quantitated in the previous protocol, namely {Delta}Di0S, Di0S, {Delta}DiHA, DiHA, and 6S-galNAc.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
2-Aminoacridone, HCl (AMAC) was purchased from Molecular Probes. Mercuric acetate, glacial acetic acid (99.99+%), dimethylsulfoxide (DMSO, 99.9%), sodium cyanoborohydride (95%), and glycerol (99.5%) were from Aldrich-Sigma. Phenol red (0.5% w/v) was from Gibco. MONO® composition gels (#60100) and MONO® gel running buffer (#70100) were purchased from Glyko. Dowex AG50W-X8 (200–400 mesh) was from Bio-Rad. Chondroitinase ABC, chondro-4-sulfatase, chondro-6-sulfatase, and unsaturated hyaluronan and chondroitin sulfate disaccharide standards were purchased from Seikagaku, America. Bovine testicular hyaluronidase (Type VI-S) was from Aldrich-Sigma. High purity hyaluronan was obtained from Pharmacia (Healon®).

Preparation of {Delta}disaccharide standards for fluorescent derivatization
Standard {Delta}disaccharides (in ultrapure water) from Seikagaku, including {Delta}DiHA from hyaluronan, and {Delta}Di0S, {Delta}Di2S, {Delta}Di4S, {Delta}Di6S, {Delta}Di2,4S, {Delta}Di2,6S, {Delta}Di4,6S, and {Delta}Di2,4,6S from chondroitin/dermatan sulfate, were processed for derivatization as follows. Five identical aliquots of each {Delta}disaccharide were frozen on dry ice, and lyophilized until dry on a vacuum concentrator. Each aliquot contained no more than 50 nmol of disaccharide reducing equivalents based on hexuronic acid (Blumenkrantz and Asboe-Hansen, 1973Go). One aliquot of each {Delta}disaccharide was derivatized directly. The second aliquot of each {Delta}disaccharide was resuspended in 100 µl of 17.5 mM mercuric acetate, 50 mM sodium acetate, pH 5.0, and incubated 30 min at room temperature (Ludwigs et al., 1987Go). The mercuric ion was removed by addition of 30 µl of a 50% slurry of Dowex H+ resin, and the Dowex H+ resin was removed by filtration through a glass wool plugged pipette tip (Plaas et al., 1996Go). One hundred microliters of ultrapure water was used to rinse the reaction tube and glass wool plugged pipette tip. Sample volume trapped in the glass wool plug was recovered by centrifugation at 2000 x g. The mercuric ion treated samples were then frozen on dry ice, and lyophilized until dry on a vacuum concentrator prior to derivatization.

The three remaining aliquots of each {Delta}disaccharide were resuspended in 100 µl of 0.0005% phenol red, 100 mM sodium acetate, pH 7.0. Each aliquot was digested for 1 h at 37°C with chondro-4-sulfatase alone, chondro-6-sulfatase alone or chondro-4-sulfatase and chondro-6-sulfatase together (100 mU of each enzyme/ml). The three sulfatase digests of each {Delta}disaccharide were frozen on dry ice, and lyophilized until dry on a vacuum concentrator. The sulfatase digests of all the {Delta}disaccharides, except {Delta}Di2,4,6S, were derivatized directly. The three sulfatase digests of {Delta}Di2,4,6S were mercuric ion treated as described above prior to derivatization.

Preparation of purified tetrasaccharides for fluorescent derivatization
Tetrasaccharides from 68-year-old human aggrecan (predominantly 6-sulfated, Di6SDi6S) and rat chondrosarcoma aggrecan (predominantly 4-sulfated, Di4SDi4S) were prepared by testicular hyaluronidase digestion, MicroCon 10 filtration and Toyopearl HW40S chromatography as previously described (Plaas et al., 1996Go). The tetrasaccharide preparations were generous gifts of Dr. Anna Plaas (Shriners Hospital for Crippled Children, Tampa, FL). Tetrasaccharide preparations (in ultrapure water) were processed for derivatization as follows. Three identical aliquots from each preparation were frozen on dry ice, and lyophilized until dry on a vacuum concentrator. Each aliquot contained no more than 50 nmol of potential disaccharide reducing equivalents. Each aliquot was resuspended in 100 µl of 0.0005% phenol red, 100 mM ammonium acetate, pH 7.0. One aliquot was immediately frozen on dry ice, and lyophilized until dry on a vacuum concentrator prior to derivatization. The remaining two aliquots were digested with chondroitinase ABC (100 mU/ml) for 3 h at 37°C, and then frozen on dry ice, and lyophilized until dry on a vacuum concentrator. One chondroitinase digest was derivatized directly. The other chondroitinase digest was treated with mercuric ion as described above prior to derivatization.

Preparation of hyaluronan ladders for fluorescent derivatization
Hyaluronan ladders were generated from partial testicular hyaluronidase digests as follows. A 1 mg/ml solution of high purity hyaluronan in 0.0005% phenol red, 100 mM ammonium acetate, pH 7.0 was made from Healon® (10 mg/ml), and the concentration confirmed by hexuronic acid assay (Blumenkrantz and Asboe-Hansen, 1973Go). Four 100 µl aliquots containing 100 µg of hyaluronan were each digested for 4 h at 37°C with 37, 111, 333, or 1000 U/ml of bovine testicular hyaluronidase. The hyaluronidase digests were then immediately frozen on dry ice, and lyophilized until dry on a vacuum concentrator prior to derivatization.

Fluorescent derivatization with 2-aminoacridone, fluorophore-assisted carbohydrate electrophoresis, gel imaging, and data analysis
All samples were derivatized by addition of 40 µl of 12.5 mM AMAC (500 nmol) in 85% DMSO/15% acetic acid followed by incubation for 15 min at room temperature. Then 40 µl of 1.25 M sodium cyanoborohydride (50,000 nmol) in ultrapure water was added followed by incubation for 16 h at 37°C (Jackson, 1991Go, 1994). After derivatization, 20 µl of glycerol (20% final concentration) was added to each sample prior to electrophoresis. If necessary, samples were diluted prior to electrophoresis with a solution containing 0.0005% phenol red, 6% acetic acid, 20% glycerol, 34% DMSO, and 480 mM sodium cyanoborohydride. All derivatized samples were stored in the dark at -70°C. Some samples formed a precipitate during storage, which was dissolved by heating the samples to 60°C for 5–10 min.

MONO® composition gels were stored at 4°C. MONO® gel running buffer was initially dissolved in ultrapure water at room temperature for 1 h, and then stored at 4°C overnight with mixing just prior to use. The wells of each gel were rinsed extensively with running buffer at 4°C, and the glass plates of each gel were thoroughly cleaned with ultrapure water just prior to use. The assembled electrophoresis apparatus from Glyko containing the electrophoresis buffer and one or two gels was placed in a large tank, and packed in ice to equilibrate the buffer to 4°C or less at the start of electrophoresis. All eight lanes of a gel were loaded simultaneously with 5 µl aliquots of sample using a Hamilton 8-channel glass syringe. The samples were electrophoresed for 80 min at a constant 500 V with a starting current of ~25 mA/gel, and a final current of ~10 mA/gel. The final temperature never exceeded 10°C. Prior to loading samples, the gels were briefly electrophoresed to ensure the correct starting current. After electrophoresis, one gel at a time was removed from the apparatus for imaging with the second gel (if any) left in the cool tank buffer to minimize diffusion of bands. The glass plates of each gel were thoroughly cleaned with ultrapure water, and the area around the wells covered with dark tape to mask the intense fluorescence from the unreacted AMAC, which remains in the wells during electrophoresis. The gels were imaged while still in their glass support plates.

The gels were illuminated with UV light (365 nm) from an Ultra Lum Transilluminator, and imaged with a Quantix cooled CCD camera from Roper Scientific/Photometrics. Camera specifications included a research grade Kodak CCD chip, a 5 mega pixel/s transfer rate, a 1317x1035 pixel resolution, and 6.8 µm2 pixels at a 12 bit depth. The camera was fitted with an 11.5–69 mm f1.4 manual zoom lens, a 4x diopter, and an ethidium bromide orange barrier filter. The images were analyzed using the Gel-Pro Analyzer® program version 3.0 from Media Cybernetics. The digital images shown in the Results section depict over saturated pixel intensity for the major derivatized structures in order to allow visualization of less abundant derivatized structures. Quantitation was done with images having all pixels within a linear 12-bit depth range.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank John Coletta, Dr. Jane Grande, John Mako and Stacy Stephenson (Department of Biomedical Engineering), Dr. Preenie Senanayake (Eye Institute), and Carol del la Motte (Colorectal Surgery), all from the Cleveland Clinic Foundation, Cleveland, OH, for their contributions to this work. This work was supported in part by a Mizutani Foundation for Glycoscience grant, NIH Grant HD34831, and funds from the Lerner Research Institute, Cleveland Clinic Foundation.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
glcA, glucuronic acid; glcNAc, N-acetylglucosamine; galNAc, N-acetylgalactosamine; 4S-galNAc, N-acetylgalactosamine-4-sulfate; 6S-galNAc, N-acetylgalactosamine-6-sulfate; 4,6S-galNAc, N-acetylgalactosamine-4,6-di-sulfate; DiHA or glcA-ß1,3-glcNAc, 2-acetamido-2-deoxy-3-O-(ß-D-glucopyrano­syluronic acid)-D-glucose; Di0S or glcA-ß1,3-galNAc, 2-acetamido-2-deoxy-3-O-(ß-D-glucopyranosyluronic acid)-D-galactose; Di4S or glcA-ß1,3–4S-galNAc, 2-acetamido-2-deoxy-3-O-(ß-D-glucopyranosyluronic acid)-4-O-sulfo-D-galactose; Di6S or glcA-ß1,3–6S-galNAc, 2-acetamido-2-deoxy-3-O-(ß-D-glucopyranosyluronic acid)-6-O-sulfo-D-galactose; {Delta}DiHA or {Delta}glcA-ß1,3-glcNAc, 2-acetamido-2-deoxy-3-O-(ß-D-gluco-4-enepyranosyluronic acid)-D-glucose; {Delta}Di0S or {Delta}glcA-ß1,3-galNAc, 2-acetamido-2-deoxy-3-O-(ß-D-gluco-4-enepyranosyluronic acid)-D-galactose; {Delta}Di2S or 2S-{Delta}glcA-ß1,3-galNAc, 2-acetamido-2-deoxy-3-O-(2-O-sulfo-ß-D-gluco-4-enepyranosyluronic acid)-D-galactose; {Delta}Di4S or {Delta}glcA-ß1,3–4S-galNAc, 2-acetamido-2-deoxy-3-O-(ß-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose; {Delta}Di6S or {Delta}glcA-ß1,3–6S-galNAc, 2-acetamido-2-deoxy-3-O-(ß-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose; {Delta}Di2,4S or 2S-{Delta}glcA-ß1,3–4S-galNAc or {Delta}DiB, 2-acetamido-2-deoxy-3-O-(2-O-sulfo-ß-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose; {Delta}Di2,6S or 2S-{Delta}glcA-ß1,3–6S-galNAc or {Delta}DiD, 2-acetamido-2-deoxy-3-O-(2-O-sulfo-ß-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose; {Delta}Di4,6S or {Delta}glcA-ß1,3–4,6S-galNAc or {Delta}DiE, 2-acetamido-2-deoxy-3-O-(ß-D-gluco-4-enepyrano­syluronic acid)-4,6-di-O-sulfo-D-galactose; {Delta}Di2,4,6S or 2S-{Delta}glcA-ß1,3–4,6S-galNAc or {Delta}DiTriS, 2-acetamido-2-deoxy-3-O-(2-O-sulfo-ß-D-gluco-4-enepyranosyluronic acid)-4,6-di-O-sulfo-D-galactose; AMAC, 2-aminoacridone; DMSO, dimethylsulfoxide; FACE, fluorophore-assisted carbohydrate electrophoresis; CCD, charge-coupled device; HA2, DiHA.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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