Characterization of group C meningococcal polysaccharide by light‐scattering spectroscopy. III. Determination of molecular weight, radius of gyration, and translational diffusional coefficient

Abstract

SynopsisGroup-specific polysaccharides isolated by means of a cetavlon procedure are immunogenic in man and induce protective immunity against meningococcal meningitis. Minute quantities of the polymers in solution can act as vaccines. We now report the first characterization of a fractionated (C-1) group C polysaccharide in 0.4M KC1 and 0.05M sodium acetate by means of light-scattering spectroscopy. Independent measurements of refractive index increments, absolute scattered intensities, angular scattering intensities and line widths as a function of scattering angles and delay times a t different concentrations using incident wavelengths of 632.8 nm from a He-Ne laser and of 488 nm from an argon-ion laser yield information on aggregation properties, molecular weight ( M r ) , radius of gyration ( r i ) t", translational diffusion coefficient (D):, and second virial coefficients A2 and B2 of C-1 polysaccharide.At relatively high ionic strength (0.4M KCI + 0.05M sodium acetate), we obtain for the C-1 polysaccharide in solution M, = 5.15 X lO5;(ri);" = 345 A, A2 = 1.25 X ml/g, ( 0 ) ; = 1.16 X cm2/sec with a corresponding Stokes radius of 240 A and B2 = 4.4 ml/g. A2and Bz are the second virial coefficients from intensity-and diffusion-coefficient measurements. The C-1 polysaccharide aggregates in solution and behaves hydrodynamically like random coils: Viscosity and sedimentation studies further confirm our conclusions that the fractionated C-1 polysaccharide aggregates in solution and EDTA can partially break up those aggregates. However, the system remains polydisperse even after adding an excess amount of EDTA. The weight-average molecular weight of the C-1 polysaccharide in solution depends upon ionic strength and exhibits a minimum a t 4 . 2 M KCI. Finally, viscosity, light-scattering, and sedimentation results all show that the aggregated macromolecular system behaves like random-coiled polymers with no measurable shape factors.

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“…30 %. It is known that these polysaccharides form aggregates [28], and it may be possible that higher aggregates were lost during the chromatographic process, such as on the guard column.…”

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“…Static light scattering can provide a measure of thermally induced aggregation of proteins. 22 These measurements were acquired simultaneously with intrinsic Trp fluorescence measurements using a second detector oriented 180 • from the fluorescence photomultiplier tube but still 90 • relative to the excitation source. Light scattering intensity at 295 nm was monitored as a function of temperature every 2.5 • C over 10 • C-85 • C. Buffer scans were subtracted from the sample readings prior to data analysis.…”

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“…and the high pointing and intensity stability of some laser beams has encouraged the use of laser sources in the traditional static light scattering experiments. Where the time averaged scattered intensities are studied [7].…”