Molecular size analysis of capsular polysaccharide preparations from Streptococcus pneumoniae

Purified capsular polysaccharide preparations from Streptococcus pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F were analyzed by high performance size exclusion chromatography (HPSEC) with multi-angle laser light scattering (MALLS), specific viscosity (SV), and refractive index (RI) detection to determine the molecular size and molar mass of each of the pneumococcal (Pn) polysaccharides. The Mw's of the polysaccharides ranged from a low of 606 kg/mol for Pn4 to a high of 1145 kg/mol for Pn9V, and the z-average radii of gyration ranged from 59 nm for Pn14 to 72 nm for Pn18C. Estimations of molar mass of the highly anionic polysaccharides (all but Pn14) by the universal calibration approach were unsuccessful, resulting in a 27-53% overestimate of the Mw's though application of Mark-Houwink-Sakurada coefficients calculated from the HPSEC-MALLS/SV/RI data resulted in estimates of Mw that were in agreement with the MALLS estimates for all but the Pn4 preparation. These results emphasize the need for direct measurement of both molecular size and intrinsic viscosity distributions for definitive characterization of the molar mass, hydrodynamic volume, rigidity, and drainage of complex biological polymers such as the pneumococcal polysaccharides.