Physical Characterization of Heparin by Light Scattering

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Dynamic and electrophoretic light scattering were used to measure the size and charge heterogeneity of a commercial preparation of heparin. For this preparation of porcine mucosal heparin (Mr = 10–20 kDa), the diffusion coefficient was 1.2 ± 0.5×10−8cm2/s and the mobility was 4.4 ± 0.9×10−4cm2/V s for an unfiltered solution at 22°C in distilled water. This diffusion constant is 2 orders of magnitude smaller than expected for a molecule the size of heparin. A fast diffusion component of 5.8 ± 1.0×10−7cm2/s, corresponding to the individual molecule, was observed in the presence of 2M NaCl, where single molecule motion is better observed. This indicates that a portion of the heparin population is in an aggregated state, which produces a higher scattering intensity than individual heparin molecules. The weight percentage of the aggregates was 5–10 as measured by high performance exclusion chromatography. These aggregates were stable up to a temperature of 75°C as measured by light scattering. This suggests that the aggregates are made up of tightly bound heparin molecules. From the diffusion coefficients we estimate the average aggregate to be made up of about 50 heparin monomers, and from the mobility, we estimate the electrophoretic charge on the aggregate to be about 1000 or about 20 electrophoretic charges for each heparin monomer. The electrophoretic light‐scattering measurements also indicate that the aggregate scattering species have very similar surface charge densities resulting from the aggregates being formed from heparin molecules. These results can provide a framework to interpret future light‐scattering data for various preparations of heparin.