Hyaluronan can be degraded to smaller fragments by chemical methods (acidic and alkaline conditions), physical stress (high-speed stirring or critical shearing), sonication (38), free-radical-based cleavage (39), or enzymatic methods. Free-radical-based cleavage of hyaluronan in the connective tissue has physiological implications in arthritis and aging. The hydroxyl radical has been shown to be a primary factor in the initiation of hyaluronan degradation by causing non-specific scission of the glycosidic linkage. The higher the concentration of the free radical, greater the decrease in the molecular mass of hyaluronan (40). The use of high-energy ultrasound or sonication is another established method for the cleavage of hyaluronan chains. Preliminary studies have defined the relationship between sonication time, intensity, and reduction in chain length (41,42). After prolonged sonication at a fixed intensity, the molecular size of depolymerized hyaluronan does not change. The fragments produced after this procedure predominantly have N-acetylglucosamine (86%) at their reducing end and glucuronic acid (98%) at the non-reducing end. This suggests that there is some level of specificity during sonication and certain weak linkages related to N-acetylglucosamine are extremely susceptible to sonication (38).
While both the methods described above have been used in various studies for the generation of low molecular weight hyaluronan, the most popular method for the generation of hyaluronan oligomers involves the digestion of hyaluronan by the hyaluronidase enzymes. There are three different types of hyaluronidases known, and they degrade hyaluronan by different mechanisms (5). The first group includes the mammalian-type hyaluronidases (EC 184.108.40.206), which are endo-b-N-acetyl-D-hexosaminidases that degrade hyaluronan to tetrasac-charides and hexasaccharides as the major end products. The testicular hyaluronidase, which belongs to this group, has been shown to have the ability to catalyze transglycosylation reactions also, in addition to its hydrolytic activity (43,44). The second group consists of hyaluronidases (EC 220.127.116.11) that are endo-b-glucuronidases, from leeches and other parasites. Bacterial hyaluronidases (EC 18.104.22.168) form the third group, and they act on hyaluronan via a b-elimination reaction. Based on the bacterial source these enzymes can either yield tetrasaccharides and hexasaccharides, or disaccharides as the final products. The elimination reaction generates a modified uronic acid having a C-4, 5 double bond at the non-reducing end. The formation of this double bond enables detection of the products of digestion by monitoring absorbance at 232 nm (Fig. 2). Therefore, this ultraviolet chromophore represents a good internal 'tag' for following the digestion products during isolation. However, since the conformation adopted by the unsaturated uronic acid is different from the internal glucuronic acid residues it could significantly affect the overall conformation of shorter hyaluronan oligosaccharides. This may eventually affect how they interact with proteins in subsequent studies.
Enzymatic digestion of hyaluronan is usually carried out in sodium acetate buffer adjusted with acetic acid to an acidic pH range (4.8-6.0) where most of these enzymes are active. The temperature for the digestion is usually 37 °C for
Figure 2 The action of bacterial hyaluronidases on hyaluronan (Ac, acetate).
the mammalian enzymes; however, the bacterial enzymes have been shown to be active from room temperature to 60 °C (45). By varying the time of enzymatic digestion the average size of the resulting oligosaccharide pool also varies. Increasing digestion time leads to a larger proportion of low molecular weight species. After the hyaluronan polymer has been treated with the enzyme for the desired time, further reaction is stopped by boiling the digestion mixture for around 5 min. The mixture can then be analyzed and hyaluronan oligomers can be purified using different column chromatography techniques.
B. Separation and Purification of Hyaluronan Oligomers
Various methods have been described for the separation of hyaluronan oligomers. Most widely used among these include size-exclusion chromatography (SEC), ion-exchange chromatography and reversed-phase ion pair (RPIP) highperformance liquid chromatography (HPLC).
SEC separates molecules according to their hydrodynamic volume, i.e., the space a particular polymer molecule takes up when in solution. This results in a separation according to decreasing molecular mass for hyaluronan digestion mixtures (29). This technique is also useful for determining the relative molecular mass of hyaluronan. Based on the enzyme used for digestion, the products eluting off the column can be monitored at either 232 nm (bacterial hyaluronidase) or 206-210 nm (mammalian hyaluronidase). In cases where the buffer contains acetate or citrate, which have strong background UV absorption at these wavelengths (206-210 nm), the uronic acid assay and its modifications are used (46). The smaller size oligomers (4-16mer) are well resolved using this technique and there is also less cross-contamination among peaks (30). However, larger oligosaccharides (>18mer) show clusters of 3-8 sizes within a peak obtained off the column and may need further analysis to determine the individual components (Fig. 3) (29). Therefore, SEC is useful for obtaining pure low molecular weight hyaluronan oligomers and fragments thus obtained are useful in biochemical and crystallography studies.
The HPLC methods for separation and purification of oligosaccharides produced by enzymatic or chemical hydrolysis of hyaluronan include normal phase partition (47), weak anion-exchange (48,49), size-exclusion (50), and RPIP (51). Detection can be based on pulsed amperometric detection (PAD), UV absorbance or fluorescence. The reversed-phase method has been used for the quantification of hyaluronan in biological tissues and samples (51). Hyaluronidase from Streptomyces hyalurolyticus is specific for hyaluronan and quantitatively yields a tetrasaccharide and a hexasaccharide as the final products. These two products were resolved by RPIP HPLC on a C-18 column in the presence of the ion-pairing agent, tetrabutylammonium hydroxide, at pH 7.6 in an acetonitrile gradient. The products were detected and quantified by their absorbance at 232 nm. Based on this quantification, the starting concentration of hyaluronan was estimated to within 93%. A modification of this RPIP method has been applied to study the degradation kinetics of purified hyaluronan oligomers
by bovine testicular hyaluronidase (52). An isocratic elution at pH 9.0 was used and this was consistent with a post-column derivatization procedure using 2-cyanoacetamide. The 2-cyanoacetamide reacted with the reducing end N-acetylglucosamine of hyaluronan oligomers eluting off the column, to yield products that were monitored at 276 nm. This labeling agent offers a variety of detection modes including fluorescence and PAD, and therefore may be compatible with different systems.
Weak-anion exchange HPLC methods utilize an amine-modified stationary phase that becomes protonated under acidic conditions to an extent proportional to the pH of the mobile phase. Modifying the solvent composition and pH of the mobile phase has enabled optimization of the separation of weakly acidic hyaluronan species (49). With most of the earlier HPLC methods the largest hyaluronan fragment that was separated was a dodecasaccharide (52). The development of high-performance anion-exchange chromatography (HPAEC) for the separation of neutral and acidic oligosaccharides (53,54) facilitated the resolution of larger hyaluronan fragments. The initial studies on neutral carbohydrates were performed at high pH to ensure deprotonation of the ring hydroxyls, which could then interact with a pellicular anion-exchange resin. However, since hyaluronan is highly susceptible to degradation at high pH, from 'alkali-peeling' reactions (55), the separation was done in the pH range of 6.3-5.0 by utilizing the carboxylate group. Using this method, hyaluronan oligomers of between 2 and 20 disaccharide units have been resolved (56).
Another approach to address the issue of hyaluronan degradation at the high alkaline pH used in HPAEC, involves the reduction of the hyaluronan oligomers to their alditol forms using borohydride, thereby making them stable to alkali (57). This procedure allows use of the carbohydrate separation abilities of a CarboPac PA1 column when run under alkaline conditions. The chromatographic conditions used afforded a high-resolution and highly sensitive method for the compositional analysis of hyaluronan, and chondroitin sulphates in minute quantities of biological samples. In addition, these conditions were also shown to be ideal for the separation of hyaluronan oligosaccharide alditols in the range of hexasaccharide to dodecasaccharide (12mer). This method has been modified subsequently, by altering the elution conditions, so as to separate hyaluronan
Figure 3 Fractionation of oligosaccharides from a testicular hyaluronidase digest of hyaluronan. A, elution profile of digest. Fractions under the UV absorbing peaks were pooled, and several additional pools were made from fractions 58-77, containing clusters of higher oligomers. V0 and Vt indicate the approximate void and total volumes of the column, respectively. B-D, examples of size distribution of the fluorotagged higher oligomer pools on 20% polyacrylamide minigels. The densitogram in B was material collected under peak 20° in A, while C and D represent material closer to V0, with a nominal size of HA,26 and HA,34, respectively. The number of monosaccharide units in each oligosaccharide peak is indicated above each peak. Reproduced from Ref. 29 with permission from the publisher.
oligosaccharides up to a hexadecasaccharide and chondroitin and dermatan sulfate oligosaccharides up to a hexasaccharide in size (58). A combination of SEC and HPAEC has been used in a recent study for the separation of hyaluronan oligomers ranging from tetrasaccharides to 34mers (59). SEC was primarily used for the separation and purification of low molecular weight hyaluronan oligomers (4-12mer). High molecular weight hyaluronan oligomers were generated by reducing the enzymatic digestion time. An initial gel filtration step was used to select an oligosaccharide pool corresponding to the larger fragments, and these were subsequently purified by anion-exchange HPLC. As noted previously, the larger purified fragments (22-34mer) were mixtures of hyaluronan oligomers of different lengths. It is suggested that these mixtures can be further purified by re-running the samples on the ion-exchange column.
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