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.
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Abstract |
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Key words: chondroitin sulfate/dermatan sulfate/fine structure/hyaluronan/microanalysis
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Introduction |
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Results |
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Di2S,
Di2,4S, and
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
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
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
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 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
hexuronic acid from the
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 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
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 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
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 disaccharides and sulfated galNAc structures
The suitability of the sulfated 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
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
Di6S,
Di2,6S, and
Di4,6S yielding
Di0S,
Di2S, and
Di4S, respectively (lanes 4 6). Digestion with the chondro-4-sulfatase alone specifically and quantitatively removed sulfate from the 4-position of only
Di4S, yielding
Di0S (lane 1). The chondro-4-sulfatase only partially removed the sulfates from the 4-position of
Di2,4S and
Di4,6S, yielding minor amounts of
Di2S, and
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
Di4,6S, yielding
Di0S (lane 7).
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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, 1991, 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 18). 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 hexuronic acid from
disaccharides and production of unidentified products (see Figure 8, lanes 912). 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
disaccharides are unstable to mercuric ion. When 50 nmol aliquots of AMAC-derivatized
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
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
disaccharide. This is in contrast to mercuric ion treatment prior to derivatization, which is quantitative over a wide concentration range.
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Discussion |
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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., 2000). 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
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
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., 2000
).
The specific chondroitinase digestion products which were identified with these protocols are the unsaturated internal disaccharides including
DiHA,
Di0S,
Di2S,
Di4S,
Di6S,
Di2,4S,
Di2,6S,
Di4,6S, and
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)
Di0S from Di0S and
Di4S from Di4S; (2)
Di6S from both Di6S and 4,6S-galNAc; (3)
Di2,4S,
Di2,6S, and
Di2,4,6S from each other (The resolution of
Di2,6S from
Di2,4S and
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)
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 Di0S and
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
disaccharide structures,
Di0S and
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
Di0S and
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 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
disaccharide structure,
Di6S, to 6S-galNAc, which migrates at a different and unique position in the FACE analysis. Since mercuric ion treatment contributes 6S-galNAc from
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
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
Di4,6S is easily estimated from the
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., 2000
). 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
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 Di2,4S,
Di2,6S, and
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
Di2S following chondro-4-sulfatase digestion alone or chondro-6-sulfatase digestion alone indicates the presence of
Di2,4S, or
Di2,6S, respectively, in the electrophoresis front (see Figure 8). However, due to the incomplete nature of digestion of
Di2,4S and
Di2,4,6S with chondro-4-sulfatase (see Figure 8), quantitation of only the
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
disaccharides.
The lack of resolution of AMAC-derivatized DiHA from glcNAc and DiHA is primarily in the estimation of number averaged chain length for hyaluronan chains. The internal
disaccharide from hyaluronan (
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 disaccharides superimpose. In this case, the curves show linearity on a log-log plot over the range from 6.25100 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
Di0S, Di0S,
DiHA, DiHA, and 6S-galNAc.
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Materials and methods |
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Preparation of disaccharide standards for fluorescent derivatization
Standard disaccharides (in ultrapure water) from Seikagaku, including
DiHA from hyaluronan, and
Di0S,
Di2S,
Di4S,
Di6S,
Di2,4S,
Di2,6S,
Di4,6S, and
Di2,4,6S from chondroitin/dermatan sulfate, were processed for derivatization as follows. Five identical aliquots of each
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, 1973
). One aliquot of each
disaccharide was derivatized directly. The second aliquot of each
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., 1987
). 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., 1996
). 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 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
disaccharide were frozen on dry ice, and lyophilized until dry on a vacuum concentrator. The sulfatase digests of all the
disaccharides, except
Di2,4,6S, were derivatized directly. The three sulfatase digests of
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., 1996). 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, 1973). 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, 1991, 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 510 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 11.569 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.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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Calabro,A., Hascall,V.C. and Midura,R.J. (2000) Adaptation of FACE methodology for microanalysis of total hyaluronan and chondroitin sulfate composition from cartilage. Glycobiology, 10, 283293.
Carney,S.L. and Osborne,D.J. (1991) The separation of chondroitin sulfate disaccharides and hyaluronan oligosaccharides by capillary zone electrophoresis. Anal. Biochem., 195, 132140.[ISI][Medline]
Deutsch,A.J., Midura,R.J. and Plaas,A.H. (1995) Stucture of chondroitin sulfate on aggrecan isolated from bovine tibial and costochondral growth plates. J. Orthop. Res., 13, 230239.[ISI][Medline]
Hascall,V.C., Midura,R.J., Sorrell,J.M. and Plaas,A.H. (1995) Immunology of chondroitin/dermatan sulfate. Adv. Exp. Med. Biol., 376, 205216.
Jackson,P. (1991) Polyacrylamide gel electrophoresis of reducing saccharides labeled with the fluorophore 2-aminoacridone: subpicomolar detection using an imaging system based on a cooled charge-coupled device. Anal. Biochem., 196, 238244.[ISI][Medline]
Jackson,P. (1994) High-resolution polyacrylamide gel electrophoresis of fluorophore-labeled reducing saccharides. Methods Enzymol., 230, 250265.[ISI][Medline]
Karamanos,N.K., Syrokou,A., Vanky,P., Nurminen,M. and Hjerpe,A. (1994) Determination of 24 variously sulfated galactosaminoglycan- and hyaluronan-derived disaccharides by high-performance liquid chromatography. Anal. Biochem., 221, 189199.[ISI][Medline]
Kitagawa,H., Kinoshita,A. and Sugahara,K. (1995) Microanalysis of glycosaminoglycan-derived disaccharides labeled with the fluorophore 2-aminoacridone by capillary zone electrophoresis and high-performance liquid chromatography. Anal. Biochem., 232, 114121.[ISI][Medline]
Ludwigs,U., Elgavish,A., Esko,J.D., Meezan,E. and Rodén,L. (1987) Reaction of unsaturated uronic acid residues with mercuric salts. Cleavage of the hyaluronic acid disaccharide 2-acetamido-2-deoxy-3-O- (ß-D-gluco-4-enepyranosyluronic acid)-D-glucose. Biochem. J., 45, 795804.
Midura,R.J., Salustri,A., Calabro,A., Yanagishita,M. and Hascall,V.C. (1994) High-resolution separation of disaccharide and oligosaccharide alditols from chondroitin sulphate, dermatan sulphate and hyaluronan using CarboPac PA1 chromatography. Glycobiology, 4, 333342.[Abstract]
Midura,R.J., Calabro,A., Yanagishita,M. and Hascall,V.C. (1995) Nonreducing end structures of chondroitin sulfate chains on aggrecan isolated from Swarm rat chondrosarcoma cultures. J. Biol. Chem., 270, 80098015.
Otsu,K., Inoue,H., Tsuzuki,Y., Yonekura,H., Nakanishi,Y. and Suzuki,S. (1985) A distinct terminal structure in newly synthesized chondroitin sulphate chains. Biochem. J., 227, 3748.[ISI][Medline]
Plaas,A.H., Hascall,V.C. and Midura,R.J. (1996) Ion exchange HPLC microanalysis of chondroitin sulfate: quantitative derivatization of chondroitin lyase digestion products with 2-aminopyridine. Glycobiology, 6, 823829.[Abstract]
Plaas,A.H., Wong-Palms,S., Roughley,P.J., Midura,R.J. and Hascall,V.C. (1997) Chemical and immunological assay of the nonreducing terminal residues of chondroitin sulfate from human aggrecan. J. Biol. Chem., 272, 2060320610.
Shibata,S., Midura,R.J. and Hascall,V.C. (1992) Structural analysis of the linkage region oligosaccharides and unsaturated disaccharides from chondroitin sulfate using CarboPac PA1. J. Biol. Chem., 267, 65486555.
Sugahara,K., Yamashina,I., De Waard,P., Van Halbeek,H. and Vliegenhart,J.F. (1988) Structural studies on sulfated glycopeptides from the carbohydrate-protein linkage region of chondroitin-4-sulfate proteoglycans of Swarm rat chondrosarcoma. Demonstration of the structure gal (4-O-sulfate)ß1,3galß1,4xylß1-O-ser. J. Biol. Chem., 263, 1016810174.