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Direct and Indirect Methods for Molar-Mass Analysis of Fragments of the Capsular Polysaccharide ofHaemophilus influenzaeType b

1997; Elsevier BV; Volume: 250; Issue: 2 Linguagem: Inglês

10.1006/abio.1997.2224

1096-0309

Anello J. D’Ambra, Janice E. Baugher, Paul E. Concannon, Robert A. Pon, Francis Michon,

Glycosylation and Glycoproteins Research

Two methods are described for direct molar-mass measurement of low-molar-mass fragments obtained by oxidative cleavage of the capsular polysaccharide ofHaemophilus influenzaetype b. Absolute molar masses were determined by size-exclusion chromatography (SEC) with detection by multiangle laserlight-scattering photometry (MALLS) and differential refractometry (RI). The end-group structure of the polysaccharide fragments allowed the direct measurement of average chain length by quantitative1H NMR, from which molar masses were derived. Variation between the molar masses obtained by the two methods ranged from 5 to 7%. When molar masses estimated by indirect methods were compared to SEC-MALLS/RI data, significant deviations were observed. Analysis by SEC with secondary calibration with dextran standards gave molar masses that exceeded the SEC-MALLS/RI data by as much as 2.5-fold. Molar masses estimated by a combination of colorimetric assays varied from the SEC-MALLS/RI data by as much as 50%. These results demonstrated the applicability and superior accuracy of the direct methods of molar-mass determination of the polysaccharide fragments.