核磁共振氢谱和高压液相分子排阻层析-十八角激光散射仪对肺炎链球菌荚膜多糖的结构及分子质量分析

Structure and molar mass analysis of pneumococcal capsular polysaccharides by 1 H NMR and HPSEC-MALLS

目的对6种血清型肺炎链球菌荚膜多糖进行结构和分子质量分析。方法核磁共振氢谱（1 H NMR）对6种血清型肺炎链球菌荚膜多糖的结构进行分析，通过各特征质子的化学位移来确定多糖结构的完整性及批间一致性；高压液相分子排阻层析-十八角激光散射仪（ HPSEC-MALLS）对荚膜多糖的分子质量进行分析，以确定其分子质量及分布情况。结果6种血清型肺炎链球菌荚膜多糖特征质子的化学位移与其标准化学位移一致；重均分子质量范围为7．182×104 g/mol （19A型）～1．273×106 g/mol（9V型）。结论 NMR和HPSEC-MALLS能够快速、高效地对各型肺炎链球菌荚膜多糖进行结构和分子质量分析。6种血清型肺炎链球菌荚膜多糖谱图中各个质子的化学位移完全符合该型肺炎链球菌荚膜多糖的化学结构，但荚膜多糖中存在含量不等的C多糖以及纯化过程中未去除彻底的醋酸盐类物质；由于荚膜多糖特性及纯化方式的不同，6种型肺炎链球菌荚膜多糖之间分子质量存在一定差异。

Objective To analyze the structures and molecular weight distributions of the capsular polysaccharides from 6 serotypes of pneumococcus .Methods The structures of pneumococcal capsular pol-ysaccharides of 6 serotypes were analyzed by 1 H nuclear magnetic resonance （ NMR） .Chemical shifts of all characteristic protons were investigated to analyze polysaccharide integrity and inter -assay consistency .High performance size exclusion chromatography-multi angle laser light scattering （ HPSEC-MALLS） was used to measure the molecular weights .Results The chemical shifts of all characteristic protons of the pneumococ-cal capsular polysaccharides of 6 serotypes were consistent with the standard chemical shift .The weight-aver-age molecular mass of the pneumococcal capsular polysaccharides ranged from 7.182×104 g/mol（for serotype 19A） to 1.273×106 g/mol（for serotype 9V）examined by HPSEC-MALLS.Conclusion The structures and molecular weight distributions of pneumococcal capsular polysaccharides could be rapidly and effectively ana -lyzed by 1 H NMR and HPSEC-MALLS.Moreover, C-PS and acetate contained in capsular polysaccharides could also be detected .HPSEC-MALLS is an applicable method for the quantitative analysis of molar mass distributions in different serotypes of pneumococcal capsular polysaccharides . Although 1 H NMR and HPSEC-MALLS have been accepted as the quality control measurements by WHO , to use them as the re-placements of the traditional QC method still needs further investigation .