肝素非还原端饱和结构的紫外吸收研究及在肝素寡糖测序中的应用

为了进一步探讨非还原端饱和结构的肝素寡糖在UV 232 nm的吸收情况, 制备了4种饱和结构的肝素二糖, 并用离子对反相液相色谱/离子阱飞行时间质谱(RPIP-LC/MS-IT-TOF)光电二极管阵列检测器分析了它们在UV 232 nm的吸收情况. 分析结果表明, 饱和结构的肝素二糖在UV 232 nm的检出限为9 μg(S/N=10), UV 232 nm/UV 206 nm约为不饱和结构肝素二糖UV 232 nm的7%~40%. 结果还表明, 肝素二糖UV 232 nm的吸收强度受亚硫酸基团(SO₃^2?)影响较大. 另外, 通过比较不饱和结构的肝素/硫酸类肝素(Hep/HS)标样二糖发现, 含N-未取代葡萄糖胺(GlcNH₃⁺)基团的二糖在UV 232 nm的吸收值较低. 最后, 通过简单的UV检测方法, 结合 HNO₂(pH=4.0)裂解法和RPIP-LC/MS-IT-TOF分析, 简化了含GlcNH₃⁺肝素六糖的测序方法. 本研究为以后用 HNO₂(pH=1.5)裂解法对混合组分N-硫酸化的肝素寡糖结构序列分析提供了可能.

Ultraviolet Absorbance of Saturated Structure in the Non-reducing End of Heparin/Heparan Sulfate Disaccharides and Its Application in Sequence of Heparin Oligosaccharides

It is well known that the saturated structure in the non-reducing end（NRE） of heparin/heparan sulfate（HS） oligosaccharides is lacking ultraviolet（UV） 232 nm absorbance（normally used for specific detection of C4―C5 unsaturated oligosaccharides）， which poses a significant challenge for structure analysis. However， we found that saturated heparin oligosaccharides can be detected at UV 232 nm with sufficient sensitivity in our previous research. In order to further study the UV 232 nm absorbance of the saturated structure， four saturated heparin disaccharides were prepared， and then their UV 232 nm absorptions were analyzed by ion-pair reversed-phase liquid chromatography ion-trap/time-of-flight mass spectrometry（RPIP-LC/MS-IT-TOF） with photodiode array detector. The results show that the saturated heparin disaccharides can be detected with sufficient sensitivity by absorbance at UV 232 nm， which still with 7%―40% of UV 232 nm intensity of heparin unsaturated disaccharides. It seems that the UV 232 nm intensity might be affected by the sulfate groups in the disaccharides. In addition， comparing to the common unsaturated heparin/HS disaccharides as references， the disaccharides with GlcNH₃⁺ residues also showed less sensitivity at UV 232 nm. Finally， we simplified the sequencing method of the N-unsubstituted dp6 oligosaccharides by a simple UV detection method， combined with pH 4.0 HNO₂ scission and RPIP-LC/MS-IT-TOF analysis. This strategy offers possibilities for sequencing at N-sulfated residues using alternative pH 1.5 HNO₂ scission.