Evaluation of high-performance anion-exchange chromatography with pulsed electrochemical and fluorometric detection for extensive application to the analysisof homologous series of oligo- and polysialic acids in bioactive molecules

Shu-Ling Lin, Yasuo Inoue and Sadako Inoue1

Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan, Republic of China

Received on December 15, 1998; revised on February 5, 1999; accepted on February 5, 1999

Our previous studies have shown extensively diverse structures in oligo/polymers of sialic acid (oligo/polySia) that are expressed often in developmentally regulated manner on animal glycoconjugates. The aim of this study was to establish highlysensitive and specific methods that can be used to identify diverse types of oligo/polySia and thus can be applied to studies of biological phenomena associated with the differential expression of oligo/polySia chains with different degree of polymerization (DP). As model compounds, we analyzedfive different homologous series of oligo/polySia, (->8Neu5Ac[alpha]2->)n, (->9Neu 5Ac[alpha]2->)n, (->8Neu5Gc[alpha]2->)n, (->5-Oglycolyl-Neu5Gc[alpha]2->)n, and Neu5Gc9SO4 [alpha]2->(->5-Oglycolyl-Neu5Gc[alpha]2->)n, expressed in various biopolymers. The latter two structures have recently been identified in sea urchin egg receptor for sperm. First we examined application of high-performance anion-exchange chromatography (HPAEC) on a CarboPac PA-100 column with pulsed electrochemical detection (PED) to new types of oligo/polySiacompounds and confirmed that resolution of high polymers (DP > 70) of sialic acids was remarkable as reported previously. However, there are limitations in sensitivity and selectivity in PED that become significant when material is available only in a minute amount or material contained a large proportion of protein. These limitations can be circumvented by fluorometric detection of oligo/polySia tagged with 1,2-diamino-4,5-methyl-enedioxybenzene (DMB) at the reducing terminal residues after separation on a MonoQ HR5/5 column. The latter method can be applied to any type of oligo/polySia we examined if we choose the derivatization conditions and is more sensitive and specific than the method with PED for analysis of oligo/polySia with DP up to 25.

Key words: [alpha]2-9-linked oligo- and polyNeu5Ac/[alpha]2-5-Oglycolyl-linked oligo- and polyNeu5Gc/9-O-sulfated Neu5Gc-capped oligo- and polyNeu5Gc/high performance anion-exchange chromatography/fluorometric detection of oligo- and polysialic acid

Introduction

Oligo/polysialic acid (oligo/polySia) is a collective name for linear oligo/polymers of sialic acid. Oligo/polySia chains constitute a structurally unique group of carbohydrate residues of glyco-conjugates found in living organisms that range evolutionary diversity from bacteria to human. In animal, natural occurrence of oligo/polySia has so far been reported in limited types of cells and is often dependent on onco- and ontodevelopmental stages in sharp contrast to more abundant and widespread distribution of monomeric sialic acid residues that occur on almost all types of cells. Studies have shown that polySia is a biologically important glycotope, being an oncodevelopmental antigen of human and having potential roles in cell growth, differentiation, fertilization, and neuropathogenicity (Troy, 1992).


Figure 1. Separation of [alpha]2-9-linked oligo/polyNeu5Ac on a CarboPac PA-100 column. Elution with 1M NaNO3: 2% for 0-3 min, 10% at 10 min, 16% at 40 min, and 26% at 80 min. (a) A solution of [alpha]2-9-linked polyNeu5Ac (50 µg) in 0.1 M acetic acid (100 µl) was heated for 15 min at 60°C. After hydrolysis pH of the mixture was adjusted to ~13 by adding 50 µl of 0.5 M NaOH and kept at room temperature at least 1 h before injection. A 100 µl portion (equivalent to 33 µg Neu5Ac) was injected. (b) Mild acid hydrolysate of [alpha]2-9-linked polyNeu5Ac (11 µg NeuAc) prepared as (a) was coinjected with a mixture of mono- and [alpha]2-8-linked oligoNeu5Ac (n = 2-7, each equivalent to 100 ng Neu5Ac). Peaks are labeled with DP.

In bacteria linkage isomers of [alpha]2-8- and [alpha]2-9-linked polyNeu5Ac were known in capsules of some strains as homopolymers while in others as copolymers of each linkage type (Troy, 1992). In animal our recent studies on fish eggs and sea urchin eggs and sperm revealed extensively diverse polySia structures: [alpha]2-8-linked oligo/polyNeu5Gc as the first example of polySia glycotope in eukaryote (Inoue and Iwasaki, 1978, 1980); [alpha]2-8-linked copolymers of Neu5Ac and Neu5Gc (Sato et al., 1993); [alpha]2-8-linked oligo/polyKDN (Kanamori et al., 1990); [alpha]2-5-Oglycolyl-linked oligo/polyNeu5Gc (Kitazume et al., 1994a, 1996; Kitazume-Kawaguchi et al., 1997); and capping of oligo/polySia by KDN (Nadano et al., 1986), by 8-O-sulfated Neu5Ac (Ijuin et al., 1996), and by 9-O-sulfated Neu5Gc (Kitazume et al., 1996; Kitazume-Kawaguchi et al., 1997). At present [alpha]2-8-linked polyNeu5Ac is the only chemically established polySia glycotope in mammals, including human, although [alpha]2-9-linked Neu5Ac dimer was reported in human embryonal carcinoma cells (Fukuda et al., 1985). However, wide occurrence of [alpha]2-8-linked oligo/polyKDN was shown in mammalian cells and tissues by immunochemical methods (Kanamori et al., 1994; Qu et al., 1996; Ziak et al., 1996). More recently, occurrence of [alpha]2-8-linked oligoNeu5Gc in pig spleen and Wistar rat tissues was shown by using newly developed monoclonal antibody, and the results were partially supported by chemical analysis (Sato et al., 1998). All these results are indicative of occurrence of diverse polySia structures in mammals including human.

Although the structural diversity of polySia has been identified by chemical methods that include isolation and purification of the compounds and structural studies using instrumental analyses such as 1H-NMR spectroscopy and mass spectrometry, studies of biosynthesis, function, and metabolism of polySia chains depend on immunochemical methods for their detection and identification. However, immunochemical methods need specific antibody for each structural type of polySia group and are not able to detect new type of polySia structure. Moreover, these methods require enzymes specifically reactive to each antigenic glycotope to confirm the specific binding of antibodies to antigens. We have already shown that diversity of polySia is not limited to the structure of building unit sialic acid but is extended to modification of sialic acid residues by substitution of their hydroxyl group(s) with acetyl and sulfate, interresidue linkage types, and the degree of polymerization (DP). Efforts to determine DP-dependent spec-ificities of antibodies against [alpha]2-8-linked oligo/polyNeu5Ac and oligo/polyKDN, and to develop monoclonal antibodies that recognize oligo/polyNeuGc, have recently been made (Sato et al., 1995, 1998). However, at present we have neither antibodies nor enzymes that recognize [alpha]2-5-Oglycolyl-linked oligo/polyNeu5Gc. Another disadvantage of immunochemical methods is they do not give quantitative results that are indispensable to the studies on biosynthesis and metabolism of polySia chains that have often been shown to be developmentally dependent. Thus, in view of both biological significance and structural diversity of oligo/polySia glycotopes, development of highly sensitive and reliable analytical method for detection and identification is urgent. To study (1) biosynthetic mechanism, (2) biological function, and (3) metabolism, the method should provide sufficiently accurate information on the actual chain length of polySia chains because both biological and chemical properties of these unique structural elements are highly dependent on their chain length (Manzi et al., 1994). We have already reported that a capillary electrophoretic method partly fulfills these requirements (Cheng et al., 1998). Recently, two different chromatographic methods were developed, i.e., (1) highly efficient and specific separation of oligo/polymers of diverse types of sialic acid by high performance anion-exchange chromatography (HPAEC) with pulsed amperometric detector (Zhang et al., 1997), and (2) detection and identification of fmol levels of oligosialic acids by fluorometric high performance anion-exchange chromatography (Sato et al., 1998). This paper describes more extensive studies of these chromatographic methods aiming at application to further diverse types of oligo/polySia chains including those with different interresidue linkage types, capped by sulfated Neu5Gc, and expressed on functional glycoproteins that are available only in small amounts. Studies on the function of bioactive molecules, mechanism of biosynthesis, and metabolism of functional polySia chains by using the present techniques will be reported elsewhere.


Figure 2. Separation of [alpha]2-5-Oglycolyl-linked oligo/polyNeu5Gc on a CarboPac PA-100 column. Elution with 1M NaNO3: 2% for 0-3 min, 10% at 10 min, and 25% at 55-60 min. (a) PolySia-gp (Fraction 1sb, 5 µg Neu5Gc) was hydrolyzed in 50 mM sodium acetate buffer (pH 4.8, 200 µl) for 2 h at 80°C, and then the mixture was adjusted to pH ~ 13 with 50 µl of 0.5 M NaOH. A 50 µl portion (equivalent to 1 µg Neu5Gc) was injected. Peaks are labeled with DP and S denotes 9-O-sulfate substitution at the nonreducing terminal Neu5Gc residues. (b) Retention time vs. DP of [alpha]2-5-Oglycolyl-linked oligo/polyNeu5Gc (open circles), and [alpha]2-8-linked oligo/polyNeu5Gc (solid circles).

Results and discussion

Separation and analysis of [alpha]2-8-linked and [alpha]2-9-linked polyNeu5Ac by HPAEC-PED

Resolution of [alpha]2-8-linked polyNeu5Ac on a CarboPac PA-100 column was remarkable as already reported (Zhang et al., 1997). Here we show the separation of [alpha]2-9-linked oligo/polyNeu5Ac generated by mild acid hydrolysis of a highly polymerized sample of Neu5Ac ([alpha]2-9-linked polyNeu5Ac) under similar conditions as described previously (Figure 1a). Figure 1b shows that [alpha]2-9-linked lower oligomers were separated from coinjected (->8Neu5Ac[alpha]2->)n, n = 2-7; thus, the method can be used for identification of each linkage type. The sample of [alpha]2-9-linked Neu5Ac used did not give any peak on the chromatogram without prior hydrolysis, and after hydrolysis in 0.1 M acetic acid for 15 min at 60°C the peaks of oligo/polySia chains up to DP 70 appeared on the chromatogram when 33 µg (as Neu5Ac) was injected. This is in contrast to the commercial product of colominic acid that gave peaks of a series of (->8Neu5Ac[alpha]2->)n, n = 2-90, without prehydrolysis even if we injected newly dissolved solution and the results indicate that the commercial product was a mixture of oligo/polyNeu5Ac having wide range of DP.

   a, b
   c

Figure 3. Separation of [alpha]2-5-Oglycolyl-linked oligo/polyNeu5Gc capped with 9-O-sulfated Neu5Gc on a CarboPac PA-100 column. (a) A mixture of 9-O-sulfated Neu5Gc[alpha]2->(->5-Oglycolyl-Neu5Gc[alpha]2->)n (n = 0-3, each equivalent to 50-250 ng Neu5Gc). Elution with 1 M NaNO3: 2% for 0-3 min, 10% at 10 min, and 15% at 25 min. Peak 1 represents Neu5Gc monomer, externally added, whereas peaks 2-4 represent nonsulfated [alpha]2-5-Oglycolyl-linked (Neu5Gc)n, n = 2-4, originally present in thesolution of 9-O-sulfated Neu5Gc-capped oligo-Neu5Gc. (b) Detection and identification of 9-O-sulfated Neu5Gc[alpha]2->(->5-Oglycolyl-Neu5 Gc[alpha]2->)n in the mild acid hydrolysate of ESP-Sia isolated from sea urchin egg cell surface complex. ESP-Sia (20 µg Neu5Gc) was hydrolyzed in 10 mM trifluoroacetic acid for 15 min at 60°C and then the mixture was adjusted to pH ~ 12 with 0.5 M NaOH and filtered with Centricon-10 (3000 r.p.m. at 4°C). A portion of the filtrate (2 µg Neu5Gc) was injected. Elution with 1 M NaNO3: 2% for 0-3 min, 10% at 10 min, and 25% at 55 min, and 30% at 60 min. (c) Detection and identification of 9-O-sulfated Neu5Gc as a capping residue of polySia-gp isolated from sea urchin jelly. A fraction of polySia-gp (Fraction 2P, 2.7 µg Neu5Gc) was hydrolyzed in 50 mM sodium acetate buffer (pH 4.8) for 2 h at 80°C, and analyzed immediately after neutralization with NaOH to prevent hydrolysis of sulfate ester under alkaline conditions. Injected amount: 1.1 µg Neu5Gc. NaNO3 gradient was same as Figure 2a. Peaks are labeled with DP and S denotes 9-O-sulfate substitution at nonreducing terminal Neu5Gc.

Separation and analysis of oligo/polyNeu5Gc having [alpha]2-5-Oglycolyl linkages by HPAEC-PED

In 1994 we reported identification of a new type of polySia chain unusually linked through the 5-Oglycolyl group of Neu5Gc in sialic acid-rich glycoproteins (polySia-gp) isolated from the jelly coat of sea urchin eggs (Kitazume et al., 1994a). Our earlier study showed that DP of the oligo/polySia chains to be from 4 to more than 40 (average ~20) based on anion exchange chromatography of sialooligosaccharide alditols liberated by alkaline borohydride treatment of the parent glycoprotein although the resolution between each oligomer was insufficient. Result of separation and detection of [alpha]2-5-Oglycolyl-oligo/polyNeu5Gc liberated under very mild acid hydrolysis of a fraction of polySia-gp (1sb) by HPAEC-PED was recorded in Figure 2a. Pretreatment of the glycoprotein sample with 50 mM sodium acetate buffer (pH 4.8) at 80°C for 2 h or 10 mM trifluoro-acetic acid at 60°C for 15 min gave the higher yield of products with high DP than other conditions examined and the polymers up to at least DP = 45 were detectable on the chromatogram. For lower oligomers (DP = 2-10), the retention time differences between [alpha]2-8-linked and [alpha]2-5-oglycolyl-linked isomers were significant (Figure 2b).

Separation and analysis of 9-O-sulfated Neu5Gc-capped oligoNeu5Gc chains by HPAEC-PED

We reported the occurrence of 9-O-sulfated Neu5Gc-capped [alpha]2-5-Oglycolyl-linked oligo/poly-Neu5Gc chains in sea urchin egg cell surface glycoprotein (Kitazume et al., 1996). Our study also showed that sulfated oligoSia chains were on the sea urchin egg receptor for sperm and sulfate residue is important in binding of the receptor to sperm (Kitazume-Kawaguchi et al., 1997). Separation of 9-O-sulfated Neu5Gc and 9-O-sulfatedNeu5Gc-capped [alpha]2-5-Oglycolyl-linked oligo/polyNeu5Gc, i.e., Neu5Gc9SO4[alpha]2->(->5-Oglycolyl-Neu5Gc[alpha]2->)n (n = 1 - 4) was recorded in Figure 3a. Detection and identification of these sulfate-bearing oligoSia chains in ESP-Sia were clear from the chromatogram shown in Figure 3b. Furthermore, we newly identified 9-O-sulfated Neu5Gc in polySia-gp isolated from sea urchin egg jelly (Figure 3c). The ratio of 9-O-sulfated Neu5Gc to total Neu5Gc in this fraction (fraction 2p) estimated from the peak area was 1:8. Almost all separated fractions of polySia-gp from sea urchin jelly were found to contain some proportions of 9-O-sulfated Neu5Gc, but fractions that bound more tightly on anion-exchange gel and were precipitable with saturated ammonium sulfate (e.g., fraction 2p) contained larger proportions of 9-O-sulfated Neu5Gc. For instance, fraction 1sb (cf. Figure 2a) contained the smallest proportion of 9-O-sulfated Neu5Gc and its ratio relative to total Neu5Gc was estimated as 1:90. These values of the relative proportion of 9-O-sulfated Neu5Gc to total Neu5Gc coincided with those estimated by the reverse phase HPLC analysis after complete hydrolysis of interresidue linkages (0.1 M trifluoroacetic acid, 1 h at 80°C; Kitazume-Kawaguchi et al., 1997). The 9-O-sulfated residue may be located at the nonreducing terminal of oligo/polySia chains also in polySia-gp as it was shown for ESP-Sia (Kitazume et al., 1996) because fractions that contained higher proportions of 9-O-sulfated Neu5Gc were more resistant to hydrolysis by bacterial sialidases (results to be published). The chains capped by 9-O-sulfated Neu5Gc may be short and represent a part of oligo/polySia chains present in polySia-gp because a fraction that contained high proportions of 9-O-sulfated Neu5Gc (e.g., fraction 2p) also showed the presence of polySia chains with high DP (cf. Figure 2a , 3c).

Evaluation and limitation of the HPAEC-PED methods in application to biological material

The results of this study, when combined with the previously published one (Zhang et al., 1997), showed that analysis of oligo/polySia chains by HPAEC-PED on a CarboPac PA-100 column was applicable to almost all types of oligo/polySia chains known to occur in living organisms. Compared with the capillary electrophoretic method (Cheng et al., 1998), resolution of polymers of high DP was remarkable with HPAEC-PED. Difference in retention time between the different series of homologous oligo/polySia chains with the same DP was sufficient for identification purpose. This was applied not only to the series with different sialic acid building blocks but also the linkage isomers of the same sialic acid component. Because the resolution of polySia by CarboPac PA-100 column was excellent, we can estimate the real DP of long polySia that is known to be expressed in certain glycoproteins such as embryonic N-CAM and human neuroblastoma cells (Livingston et al., 1988; Troy, 1992).

When HPAEC-PED method is applied to biologically functional oligo/polySia compounds that are available only in minute amount and/or that are difficult to purify, we found that sensitivity of this method is insufficient. For instance, when the same colominic acid sample was analyzed after mild acid-hydrolysis, injection of 180 ng Neu5Ac was sufficient to detect decamer of Neu5Ac. However, for detection of the peaks of polymers with DP 40, 50, and 70, injection amounts of 5, 10, and 30 µg Neu5Ac were necessary. Obviously improvement of prehydrolysis methods (Zhang and Lee, 1999) may partly solve the problem.

Table I. Percentage yielda of parent oligomers of sialic acids after DMB reaction under optimized conditionsb
  n
2 3 4 5 6 7 9 10 11-13
(->8Neu5Ac[alpha]2->)n 95 80 72 67 21 48 - 43c 25d
(->8Neu5Gc[alpha]2->)n 85 74 69 60 - 43 27 24  
(->5-Oglycolyl-Neu5Gc[alpha]2->)n 100 84 61 45 28        
aPercentage of sum of the area for a series of oligomers derived during derivatization.
b20 mM trifluoroacetic acid 2 h at 50°C for [alpha]2-8-linked oligoSia, and 5 mM trifluoroacetic acid 1 h at 50°C for [alpha]2-5-Oglycolyl-linked oligoSia.
cSum of the area for n = 9 and 10.
dSum of the area for n = 11-13.

We examined the possibility that the observed low yields of compounds with high DP might be the result of their low detector response. For this purpose we injected, without hydrolysis, [alpha]2-8-linked oligo/polyNeu5Ac samples separated by preparative anion-exchange chromatography of the partial hydrolysate of colominic acid (Nomoto et al., 1982). The detector response per weight of Neu5Ac (determined by the DMB method after hydrolysis) was practically same for all oligo/polyNeu5Ac in the range DP 3-25 although the value was much lower (about 33%) than the value for the monomer. Based on these results we concluded that decrease in detectability of highly polymerized Neu5Ac and, consequently, difficulty in determining real DP of naturally occurring polySia chains is ascribed solely to the lability of interresidue linkages that apparently increased with DP (Manzi et al., 1994). The DP-dependent self-cleavage of polyNeu5Ac was kinetically studied only for the compounds with lower DP. In the present study we found that for a sample of polyNeu5Ac comprising DP 20-28, the recovery of these polymers was reduced to 89% and 86%, upon the storage of the sample solutions (500 ng of sodium salt was dissolved in 100 µl water) at 23°C for 2 h and 4 h, respectively. The appearance of the peaks of oligoNeu5Ac (DP 2-19) increased during the storage and the sum of the peak area of oligoNeu5Ac was found to compensate the degradation of polyNeu5Ac. The accelerated fragmentation of polysialic acid appeared to be more significant in [alpha]2-8-linked polyNeu5Ac than [alpha]2-8-linked and [alpha]2-5-Oglycolyl-linked polyNeu5Gc though our study was limited to relatively lower oligomers for the latter 2 series of polySia. For instance, in the decamer of [alpha]2-8-linked polyNeu5Ac, the recovery after 2 h at 23°C was 94%, whereas that of [alpha]2-8-linked polyNeu5Gc no sign of degradation during the same storage period as judged from the data based on both the recovery of the decamer and absence of the peaks of lower oligomers. In case of [alpha]2-5-Oglycolyl-linked polyNeu5Gc, contrary to the lability of ketosidic linkages at pH values less than 3.8, the linkage is more stable than the [alpha]2-8 counterpart at pH > 3.8 (Kitazume et al., 1994b). The results of the present study are all in conformity with the previous findings: we used milder conditions of prehydrolysis of the polymer but the oligomers (sodium salts) were stable in water at 23°C.

Another problem is that amperometric response is not highly selective to sialic acid components. When the samples contained a large proportion of peptide components many unidentifiable peaks appear in low salt region and overlap oligoSia peaks, and for these samples purification is necessary before injection.

Evaluation of a HPLC-FD method of oligo/polySia analysis

DMB is now widely used as a highly sensitive fluogenic reagent reactive with sialic acid in identification and differential quantitation of KDN, Neu5Ac and Neu5Gc separated on a reverse phase HPLC column (Hara et al., 1987; Inoue et al., 1996). The detection limit of 100 fmol (30 pg) of sialic acid was attainable in reverse phase chromatography (Hara et al., 1987). Recently, application of DMB-derivatization of oligoSia followed by anion-exchange HPLC was also reported (Sato et al., 1998). The report showed that the reducing terminal sialic acid in oligoSia chain was tagged with DMB under conditions used for derivatization of sialic acid monomers and DMB-oligoSia chains were separated and quantitated by fluorometric anion-exchange HPLC with a detection limit of 13 fmol of Neu5Ac[alpha]2-8Neu5Ac. In the present study, we applied the method for analysis of higher oligomers of sialic acid. The major problem when the DMB-method is applied to oligo/polySia analysis is that derivatization requires acidic medium (pH 2-3) and needs at least 2 h at 50-60°C for the maximum yield. We established conditions that gave most satisfactory results for the analysis of oligo/polySia chains by incubating samples with the DMB reagent without prehydrolysis. Each oligo/polySia samples, (Sia)n, gave ladders of homologous series of DMB-(Sia)n, n = 1, 2, 3, - n, on the chromatogram. The yields of DMB-(Sia)n, i.e., the DMB-tagged parent oligo/polySia were determined for model compounds under optimized conditions and recorded as percentage of total DMB-(Sia)n, n = 2, 3, - n, in Table I. The yield was gradually decreased with increasing DP starting with almost 100% for dimers but still attains about 25% at decamers for [alpha]2-8-linked polySia. For [alpha]2-5-Oglycolyl-linked oligomers of Neu5Gc, the yield dropped down to 28% at hexamer even if we used milder conditions than those used for [alpha]2-8-linked polysialic acids. The elution profiles for biological materials containing various types of oligo/polySia chains are given in Figure 4a-d.

   a, b
   c, d

Figure 4. Separation of DMB-oligo/polySia by anion-exchange chromatography on a MonoQ HR 5/5 column and detection by fluorometry. Samples were derivatized under conditions specified for each compound. All derivatized sample solutions were adjusted to pH~13 with NaOH and kept at room temperature at least for 1 h before injection to cleave ester linkages formed. Elution with NaCl in 10 mM Tris-HCl (pH 8.0): 0 M, for 0-10 min, first linear gradient 0-0.14 M, between 10-20 min; second linear gradient 0.14-0.57 M, between 20-70 min. (a) Colominic acid. Sample (10 µg Neu5Ac) was derivatized in the medium containing 20 mM trifluorocetic acid for 2 h at 50°C. Injected amount: 1 µg Neu5Ac. (b) Rainbow trout PSGP (a glycoprotein containing [alpha]2-8-linked oligo/polyNeu5Gc). Sample (420 ng NeuGc) was derivatized as (a). Injected amount: 140 ng Neu5Gc. (c) PolySia-gp (Fraction 1sb) from sea urchin jelly. Sample (2.6 µg NeuGc) was derivatized in the medium containing 5 mM trifluoroacetic acid for 2 h at 50°C. Injected amount: 650 ng Neu5Gc. (d) ESP-Sia from sea urchin egg cell surface complex. Sample (1.1 µg Neu5Gc) was derivatized as (c). Injected amount: 450 ng Neu5Gc. Peaks are labeled with DP and S denotes 9-O-sulfate substitution at nonreducing terminal Neu5Gc.

For derivatization of [alpha]2-8-linked polySia chains the reaction medium containing 20 mM trifluoro-acetic acid gave good results (Figure 4 a and b) whereas 5 mM trifluoroacetic acid was used for [alpha]2-5-Oglycolyl-linked polyNeu5Gc chains (Figure 4c,d). We found that in 20 mM trifluoroacetic acid no detectable peak of higher oligomers (DP > 7) of [alpha]2-5-Oglycolyl-linked chains was observed (data not shown). The lability of Neu5Gc[alpha]2->5-Oglycolyl-Neu5Gc linkage towards acid was previously reported (Kitazume et al., 1994a,b). For [alpha]2-9-linked polyNeu5Ac derivatization in 10 mM trifluoroacetic acid was used to improve the yield of higher oligomers; however, the polymer with DP 20 was the largest detectable. The greater sensitivity to acid of [alpha]2-9-linked polyNeu5Ac than [alpha]2-8-linked polyNeu5Ac was previously reported (Jennings et al., 1985). The reaction at 50°C for 1-2 h gave satisfactory results for almost all compounds analyzed. Judging from the results obtained for (->8Neu5Ac[alpha]2->)n, n = 20-28 as a model compound polySia with DP 25 appeared to be the upper limit that can be resolved on the MonoQ column (data not shown), and this conclusion is in agreement with the earlier reports of separation of milligram quantities of underivatized oligo/polySia on MonoQ HR 5/5 columns under similar elution conditions (Hallenbeck et al., 1987; Kitazume et al., 1992). Therefore, the limitation of this method when applied to [alpha]2-8-linked polySia with high DP such as colominic acid is determined by the resolution power of the anion exchanger rather than the depoly-merization problem during derivatization under acidic conditions (Figure 4a). In general the amount of samples necessary for this analysis was one-tenth of that required in HPAEC-PED. Contrary to the HPAEC-PED method, the detection of the higher oligomers was not improved by increasing the amount of sample loaded larger than 1 µg (in total sialic acid). Because of its high sensitivity and high specificity of DMB-reagent to sialic acid, HPLC-FD is superior to the HPAEC-PED method for the polySia compounds of which DP is originally short (<25). Thus, in the case of rainbow trout PSGP the result shown in Figure 3b was obtained from about 300 ng of PSGP (140 ng Neu5Gc, injected amount) and decamer of Neu5Gc was detectable. In previous report 10 µg of PSGP was hydrolyzed in acetate buffer (pH 4.8) at 30°C for 39 h and the maximum DP of oligoNeu5Gc detected was 11 (Zhang et al., 1997), although by using different conditions of prehydrolysis (10 mM trifluoroacetic acid 15 min at 60°C) we detected a series of [alpha]2-8-linked oligoNeu5Gc up to DP13 by HPAEC-PED. Based on these and preparative anion-exchange chromatographic results (S.Inoue, unpublished observations; Kitazume et al., 1992) now we conclude that DP of polySia chains of rainbow trout PSGP is shorter than our previous estimation based on resolution of more than 20 peaks of sialooligosaccharide alditols liberated by alkaline borohydride treatment of PSGP and separated by DEAE-Sephadex A-25 column chromatography (Inoue and Iwasaki, 1980; Nomoto et al., 1982). In case of [alpha]2-5-Oglycolyl-linked Neu5Gc, the detection of higher oligomers was limited by the cleavage of the linkages under acidic conditions necessary for derivatization (Figure 4c). Nevertheless, this method is powerful when the material available is limited and especially when the oligo/polySia chains are originally short. Note that the chromatogram shown in Figure 4d was obtained from less than one-fourth the amount of ESP-Sia used in Figure 3b and clearly showed the presence of lower oligomers of [alpha]2-5-Oglycolyl-linked Neu5Gc and those capped with 9-O-sulfated Neu5Gc although the broad peak of 9-O-sulfated Neu5Gc monomer that was eluted between 32-40 min interfered with the analysis of this region.

Materials and methods

High-performance anion-exchange chromatography with pulsed electrochemical detection (HPAEC-PED)

A DX 500 ion chromatography system (Dionex, Sunnyvale, CA) was used with a CarboPac PA-100 column. An ED40 electrochem-ical detector was operated at integrated amperometry mode with a repeating potential sequence E1 = 0.05 V, t1 = 0.4 S: E2 = 0.75 V, t2 = 0.2S; E3 = -0.15 V, t3 = 0.4 S, and integration period between 0.2-0.4 S from the start as recommended by the manufacturer (Rohrer et al., 1998). We use the term pulsed electrochemical detection (PED), which encompasses all subtechniques generated by the instrument used (LaCourse, 1997), although the method used in this paper was basically same as that generated by pulsed amperometric detector (PAD). Samples in 100 µl amounts were injected manually or by using a Spectra System AS300 autosampler (Thermo Separation Products, San Jose, CA). Eluents were the mixture of water (A), 0.2 M NaOH (B), and 1 M NaNO3 (C), with initial conditions of A/B/C = 48/50/2. Concentration of NaOH was always kept at 0.1 M and oligo/polySia compounds were eluted at a flow rate of 1 ml/min with concentration gradient of NaNO3 as described in the figure captions (Zhang et al., 1997).

High performance liquid chromatography with fluorescent detection (HPLC-FD)

A Hewlett Packard HPLC system series 1100 with a fluorescence detector 1046A (set at 373 nm for excitation and 448 nm for emission) was used with a MonoQ HR 5/5 column (0.5 × 5 cm, Pharmacia, Sweden). Samples were eluted with 10 mM TrisHCl (pH 8.0) containing 0-0.7 M NaCl at a flow rate of 0.5 ml/min.

Oligo/polySia samples

Colominic acid (Na-salt, molecular weight range 10,000-12,000, DP ~ 35) was a product of Nacalai tesque (Kyoto, Japan). [alpha]2-9-Linked polyNeu5Ac (Bhattacharjee et al., 1975) was a gift from Dr. Harold Jennings, National Research Council of Canada, Canada. Sialic acid-rich glycoprotein (polySia-gp) containing [alpha]2-5-Oglycolyl-linked poly/Neu5Gc was isolated and fractionated from jelly of Hemicentrotus pulcherrimus by a method modified from the original (Kitazume et al., 1994a). Oligosialic acids with known degree of polymerization (DP) were prepared by controlled acid hydrolysis of the polymers and separated by preparative anion-exchange chromatography on DEAE-Sephadex A-25 by the method described previously (Nomoto et al., 1982). Sea urchin egg cell surface glycoprotein (ESP-Sia) and 9-O-sulfated oligoNeu5Gc samples prepared from ESP-Sia (Kitazume et al., 1996) were kindly given by Dr. Shinobu Kitazume-Kawaguchi (RIKEN, Saitama, Japan). Isolation of rainbow trout egg PSGP (polysialoglycoprotein) was as described previously (Nomoto et al., 1982).

Mild acid hydrolysis and derivatization

Unless otherwise stated, oligo/polySia samples analyzed by HPAEC-PED were subjected to prehydrolysis under mild conditions set for each analysis and described in the figure captions. All hydrolysates were adjusted to alkaline pH with 0.5 N NaOH before injection to cleave intramolecular ester linkages formed during acid hydrolysis (Cheng et al., 1998). For fluorometric HPLC analysis, samples were derivatized with DMB (1,2-diamino-4,5-methylenedioxybenzene, Dojinbo, Japan) under acidic conditions described for each series of oligo/polySia samples without prehydrolysis. Sample solutions were adjusted to alkaline pH (pH ~ 13) with NaOH after derivatization. Concentration of other reagents in the derivatization mixture was as described previously (Hara et al., 1987; Inoue et al., 1996).

Acknowledgments

We are indebted to Dr. Y.C.Lee (The Johns Hopkins University) for valuable information for HPAEC-PED analysis. For the terminology of PED, we thank Dr. W.R.LaCourse (University of Maryland, Baltimore County) for suggestion. This research was supported in part by NSC Grant 87-2311-B-001-122, the Grant from Academia Sinica, and National Health Research Institutes Grant DOH88-HR-805.

Abbreviations

Neu5Ac, N-acetylneuraminic acid; Neu5Gc, N-glycolylneuraminic acid; KDN, 2-keto-3-deoxy-d-glycero-d-galacto-nononic acid; Sia, sialic acid; HPLC, high-performance liquid chromatography; HPAEC, high-performance anion-exchange chromatography; PED, pulsed electrochemical detection; DP, degree of polymerization; DMB, 1,2-diamino-4,5-methylenedioxybenzene; FD, fluorescence detector; PSGP, [alpha]2-8-linked oligo/polyNeu5Gc chain-containing glycoprotein isolated from salmonid fish eggs; polySia-gp, [alpha]2-5-Oglycolyl-linked oligo/polyNeu5Gc chain-containing glycoprotein isolated from the jelly coat of sea urchin eggs; ESP-Sia, [alpha]2-5-Oglycolyl-linked oligoNeu5Gc chain-containing glycoprotein isolated from the sea urchin egg cell surface complex.

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