DS is a copolymer GAG constructed by both GlcA- and IdoA-containing repeating disaccharide units. From a biological point of view, it is often important to know whether DS is present in a GAG preparation. Differential digestion with chondroitinase ABC and chondroitinase AC or B may provide useful information on this aspect. When the total amount of A-disaccharides recovered following digestion by chondroitinase AC is less than that obtained by ABC, this is evidence of the presence of DS. Furthermore, comparing the amount of A-disaccharides recovered, using separate digestions with chondroitinase AC and B, we may well determine whether DS is rich in IdoA or GlcA and, at the same time, the sulfation pattern of IdoA- and GlcA-containing disac-charides (see Fig. 4). The protocols are presented below.
1. Dissolve separately the samples containing DS in 10 ^L of digestion buffers I and II (see Note 18).
2. To each one of them, add 40 ^L of DS degradation buffers I and II, respectively, and incubate for 90 min at 37°C.
3. Centrifuge the digestion mixture in a microfuge tube at 11,000g for 5 min.
4. Place aliquots of the supernatant in the electrophoresis vials and keep at 4°C pending use.
5. Start the CE instrument (see Note 11), adjust the detector wavelength to 232 nm, prepare the method, and analyze the samples as described under Subheading 3.1, steps 6-8.
Migration time (min)
Fig. 4. Analysis of DS isolated from porcine skin following separate digestion with chondroitinase ABC (A), AC (B) and B (C). The electropherograms obtained show that DS is rich in IdoA-containing disaccharides (very low susceptibility to chondroitinase AC in contrast to that obtained with chondroitinase B) and that most of the GlcA-containing disaccharides of the DS chain occur in short sequences (extra peaks representing A-oligosaccharides are obtained when DS is treated with chondroitinase B). For identity of peaks (see Figure 3). Reprinted from ref. 1, copyright 1995, Elsevier Science, with permission.
3.3. Disaccharide Composition of heparin/HS
Heparin and HS can be almost quantitatively (>90%) degraded to A-disaccharides by using all three heparin lyases (I, II, and III) in combination (See Note 5). All variously sulfated A-disaccharides and those containing N-acetylated and unsubstituted GlcN at the amino group are completely resolved, using reversed-polarity CZE, according to the following protocol.
Migration time (min)
Fig. 5. CZE profile showing the resolution of all 12 heparin-/HS-derived A-disaccharides. Analysis is performed with 15 mM phosphate buffer, pH 3.50. Peaks: 1 = aAdi-nonSHS; 2 = Adi-nonSHS; 3 = aAdi-mono6SHS; 4 = Adi-mono6SHS; 5 = aAdi-mono2SHS; 6 = Adi-mono2SHS; 7 = Adi-monoNSHS; 8 = aAdi-di(2,6)SHS; 9 = Adi-di(2,6)SHS; 10 = Adi-di(2,N)SHS; 11 = Adi-di(6,N)SHS; 12 = Adi-tri(2,6,N)SHS. Reprinted from ref. 8, with permission of Wiley - VCH, STM Copyright of Licenses. 1. Dissolve the samples containing approximately 0.1-10 ^g of heparin and/or HS in 10 ^L of digestion buffer III.
Add 40 ^L of heparin/HS degradation buffer to the samples and add 1 ^mol calcium acetate from a 2 M stock solution. Digest at 37°C overnight (see Note 8). Centrifuge the digestion mixture in a microfuge tube at 11,000g for 5 min (see Note 9). Place aliquots of the supernatant in the electrophoresis vials and keep at 4°C pending use. Start the CE instrument (see Note 11), adjust the detector wavelength to 232 nm, prepare the method, and analyze the samples as described under Subheading 3.1., steps 6-8 (see Fig. 5).
1. Digestion buffers containing the various lyases and chondrosulfatases should be divided into small portions of 50-200 ^L and kept in sealed plastic tubes at -20°C. Enzyme-containing solutions should not be frozen and used again after thawing. In each protocol, a certain capillary diameter and length are given. When capillaries with different characteristics are available, the resolution may be tested using external standards. Voltage can also be modified (±5-10 kV) to affect the rate of migration and resolution. However, if no complete resolution is obtained, use the proposed capillary and the recommended conditions. Large amounts of injected material may cause peak broadening. Optimal peak shape can be obtained with £100 ng disaccharide injected. Buffers and samples used in capillary electrophoresis should be handled carefully so as to protect the capillary from particles and air bubbles. Therefore, all solutions should be passed through a 0.2-^m membrane filter and degassed in an ultrasonic bath for 5-10 min. Samples dissolved in low volumes are preferably centrifuged at 11,000g for 5 min. A pH of 3.0 in the operating buffer is important for the resolution of the variously sulfated A-disaccharides and should be carefully adjusted. The separation is partly based on ion
suppression (pKa of the carboxyl groups is just above 3 and slightly different in the various disaccharides). Slightly modified pH values (±0.1 pH unit) therefore affect the net charge and the separation of Adi-mono4S and Adi-mono6S and decreased pH may cause close to infinite retardation of nonsulfated A-disaccharides.
5. Due to the different specificities of the three heparin lyases (type of uronic acid and position of GlcN sulfate), it is possible to obtain separate information on HS- and heparin like sequences in the analyte (9). The amounts of enzyme recommended represents an excess to warrant maximal yield of disaccharides, but can be expensive when analyzing large series of samples. Less than 10% of these amounts is sufficient, but one should then be sure that the amounts of GAG is 1 mg or less.
6. The pH of 3.50 in the operating buffer and reversed polarity are critical for the resolution of the heparin- and HS-derived A-disaccharides carrying N-acetylated, N-sulfated, or unsubstituted GlcN. The pH should be checked carefully, since slightly modified pH values during electrophoresis (±0.1 pH) affect the separation (see Note 4). Nonsulfated, nonacetylated A-disaccharide migrates very slowly and can sometimes be difficult to recover at all. In this case, it can be recommended to run one separation in reversed polarity as above, followed by a second separation with normal polarity, moving this A-disaccharide by EOF.
7. Biological samples or tissue extracts taken for analysis of HA and total GalAGs content can be concentrated by precipitation with 4 volumes of 90% (v/v) ethanol, also containing 2.5% (w/ v) sodium acetate. Small amounts of dextran can be used as carrier. Digestion buffer containing the lyases and chondrosulfatases is then added directly to the precipitate. Following heating to stop the enzymic effects, the digests should be centrifuged at 11,000g for 5 min.
8. Incubation times and the units of the enzymes indicated should not be exceeded, since some enzyme preparations may contain contaminating lyase activities. Enzymic digestions should be terminated by boiling the incubation mixtures in a water bath for 1 min.
9. Evaporation during incubation necessitates the use of sealed, capped tubes. Drops on the tube walls are recovered at the same time as particles are removed by centrifugation at 11,000# for 5 min.
10. Removal of chondroitinase-resistant macromolecules is obtained by ultrafiltration in Centricon 3 membrane tubes. However, overly long centrifugation times may cause the membrane to dry and break and therefore should be avoided. In the absence of experience with the handling of these membrane tubes, use them only once.
11. When the capillary is mounted into the capillary cassette, care should be taken to keep the detection window clean. Preferably, use gloves when handling the capillary!
12. Due to some electrolysis during the electrophoretic separation, the operating buffer characteristics (ionic strength and pH) may be affected. Therefore, replace the operating buffers frequently (at least every 5 runs) and run frequent standards (once for each new buffer vial) to ensure uniform performance.
13. When using pressure injection, these protocols give suitable parameters for pressure (mbar) x time (s). When working with low concentrations, it is always possible to increase the injection times without significantly affecting the resolution. However, use the same injection conditions for samples and standards.
14. Electrical current and temperature should be constant during electrophoresis, since changes may give extra peaks or baseline drift. The current should therefore be monitored throughout the run. Temperature also influences the viscosity of the solution and may therefore affect resolution and quantification. Performance can be considerably improved, by creating a "concentration zone" during injection. If the analyte is dissolved in water with as little electrolyte as possible, the conductivity in the zone of injected material will be low, hence increasing the electric field through the injected solvent. This causes the analyte to move rapidly through this zone and it becomes concentrated when entering the running buffer (similar to what happens in TLC when using a concentration zone). To obtain such conditions one can use volatile buffers for digestion (for example, ammonium formate instead of those conventionally recommended), followed by evaporation of the digested material and redissolving it in pure water. The EOF will push the injected low-electrolyte solvent backward out of the capillary, not interfering at the detection window.
15. The amounts of HA and GalAGs are preferably given in terms of uronic acid per milliliter, since the presence of crystal water and the poorly defined weights of the counterions make it difficult to determine the weight of a GAG preparation correctly. Therefore, GAG standards with a well-defined amount of uronic acid (carbazole reaction) can be used. This also monitors the efficiency of the enzymic digestion. Alternatively, the quantity of HA and total GalAGs in samples can be determined, based on standard curves and using commercially available A-disaccharides, as under Subheading 3.2.
16. Disaccharides sulfated in uronic acid migrate earlier than the main nonsulfated peak (mainly in the region of monosulfated disaccharides). However, in most CS preparations from mammalian tissues this peak will be minute and the calculation more readily made when relating to a mammalian CS standard. When analyzing CS/DS preparations with significant amounts of uronic acid sulfation, also include a Adi-di(2,4)S standard (see Subheading 2.2.1) when digesting the standard mixture.
16. LIF detection of AMAC-conjugated A-disaccharides increases the sensitivity at least 100 times more than that obtained by UV detection at 232 nm of underivatized A-disaccharides. However, the AMAC derivatives of the nonsulfated A-disaccharides do not appear on the electropherogram, due to the effect of the fluorochrome. The advantage of this derivatization is its sensitivity, which permits the study of the sulfation pattern with less sample present. The AMAC labeling also allows the non reducing end of the GAG chain to be studied. This fragment does not carry the UV-absorbing A4'5-structure. Recent studies indicate that this part of the chain may be of particular biological importance (10,11).
18. When DS is digested with only one of the chondroitinases AC or B, these digests will also contain A-oligosaccharide fragments. They can be identified in the electropherogram, thereby providing information also regarding longer DS sequences.
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