Abstract
Recent studies from many laboratories revealed remarkable structural, distributional, and functional diversities of oligo/polysialic acids (OSA/PSA) that exist in organisms ranging from bacteria to man. These diversities are further complicated by the fact that OSA/PSA spontaneously form lactones under even mildly acidic conditions. By using high performance anion-exchange chromatography (HPAEC) with nitrate eluents, we found that lactonization of alpha2,8-linked OSA/PSA (oligo/poly-Neu5Ac, oligo/poly-Neu5Gc and oligo/poly-KDN) proceeds readily, and the lactonization process displays three discrete stages. The initial stage is characterized by limited lactonization occurring between two internal sialic acid residues, reflected by a regular pattern of lactone peaks interdigitated with non-lactonized peaks on HPAEC. In the middle stage, multiple lactonized species are formed from a molecule with a given degree of polymerization (DP), in which the maximum number of lactone rings formed equals DP minus 2. At the final stage, completely lactonized species become the major components, resulting in drastic changes in the physicochemical properties of the sample. Interestingly, the smallest lactonizable OSA are tetramer, trimer, and dimer at the initial, middle, and final stages, respectively. At any of the stages, OSA/PSA of higher DP lactonize more rapidly, but all the lactone rings rapidly open up when exposed to mild alkali. Lactonized OSA/PSA are resistant to both enzyme- and acid-catalyzed glycosidic bond cleavage. The latter fact was utilized to obtain more high DP oligo/poly(alpha2,8-Neu5Gc) chains from a polysialoglycoprotein. Our results should be useful in preparation, storage, and analysis of OSA/PSA. Possible biological significance and bioengineering potentials of lactonization are discussed.
Highlights
We have developed a highly sensitive and efficient method for analysis of OSA/PSA using high performance anion-exchange chromatography (HPAEC) and pulsed amperometric detection (PAD) by utilizing either neutral or alkaline conditions for separation (27)
Lactonization of Colominic Acid—We have shown that colominic acid can be separated into a series of peaks by HPAEC using a neutral nitrate eluent (27)
These results suggest that there was no significant degradation of the high degree of polymerization (DP) homologues under the acidic condition used here
Summary
Materials—Quaternary methylamine anion exchanger is from Millipore Waters Chromatography Division (Milford, MA). Fractions containing intact PSGP showed the diagnostic peak patterns of oligo/poly-Neu5Gc by HPAEC (27) only after partial hydrolysis Such fractions were pooled and desalted on a Sephadex G-25 column (0.7 ϫ 19 cm) equilibrated and eluted with water, freezedried, and stored at Ϫ20 °C. 10 l of 1 M NaOH and 400 l of 3 mg/ml polylactone were mixed and kept at ambient temperature for 5 min to open all lactone rings. To the mixture was added 10 l of 4 M HCl and left at ambient temperature for 2 h (to induce lactonization) followed by dilution with water to a final concentration of 0.1 M HCl. For the control, equivalent amounts of solutions were used except that the NaOH and HCl were pre-mixed and diluted first before adding to the PSGP. The samples were heated at 80 °C for 15 min before they were neutralized and analyzed by HPAEC
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