Evaluation of Radius of Gyration and Intrinsic Viscosity Molar Mass Dependence and Stiffness of Hyaluronan

Abstract

Nine hyaluronan (HA) samples were fractionated by size-exclusion chromatography, and molar mass (M), radius of gyration (Rg), and intrinsic viscosity ([eta]) were measured in 0.15 M NaCl at 37 degrees C by on-line multiangle light scattering and viscometer detectors. Using such method, we investigated the Rg and [eta] molar mass dependence for HA over a very wide range of molar masses: M ranging from 4 x 10(4) to 5.5 x 10(6) g/mol. The Rg and the [eta] molar mass dependence found for HA showed a meaningful difference. The Rg = f(M) power law was substantially linear in the whole range of molar masses explored with a constant slope of 0.6. In contrast, the [eta] = f(M) power law (Mark-Houwink-Sakurada plot) showed a marked curve shape, and a linear regression over the whole range of molar masses does not make sense. Also the persistence length (stiffness) for HA was estimated. The persistence length derived by using both the Odijk's model (7.5 nm from Rg vs M data) and the Bohdanecky's plot (6.8 nm from [eta] vs M data) were quite similar. These persistence length values are congruent with a semistiff conformation of HA macromolecules.

Periodate oxidation and macromolecular compaction of hyaluronan

“…Analytical expressions are found in the articles of Mendichi et al [11] and Higashimura et al [12]. Using the theoretical values for M L (410 nm -1 ) [11] the results shown in Table 1 (column 2 and 3) were obtained. It may be noted the fit of the R G -M data to the model was not perfect, especially for D ox above 0.10, so data should be treated as 'apparent' or relative values.…”

“…In theory, such plots should be linear, providing the mass per unit length (M L ) and the persistence length (q) from a linear fit. The approach is identical to that applied earlier to oxidized alginates [3] and chitosans [5], as well as unoxidized hyaluronan [11]. The reader is referred to the latter articles for detailed equations and their validities.…”

“…Although partially oxidized hyaluronan is inhomogeneous with respect to chain flexibility along the chain due to the assumed random positioning of flexible 'hinges' resulting from oxidation, the wormlike chain model can be applied to calculate an 'apparent' persistence length. The Benoit-Doty model has been previously applied for semi-flexible polysaccharides [11,12], including their partially periodate oxidized derivatives [3]. The model provides the following analytical expression for the relationship between R G and M:…”

“…However, the curves are not linear as predicted by the MHS equation, but show curvature. According to Mendichi et al [11] this can be ascribed to '..the "partial draining" nature of charged chains changes with their length, that is, their hydrodynamic volume changes with M..'.…”

Effect of Bucillamine on Free-Radical-Mediated Degradation of High-Molar-Mass Hyaluronan Induced in vitro by Ascorbic Acid and Cu(II) Ions

“…Considering the high-molar-mass of the samples, a relatively low flow rate, 0.5 mL/min, was used to avoid shear-degradation of the polymer in the SEC columns. The experimental methodology for a reliable use of the SEC-MALS chromatographic system was described in detail [18][19][20][21]. The experimental conditions of the SEC-MALS system were the following: two Shodex (KB806 and KB805) columns from Tosoh Bioscience (Stuttgart, Germany); mobile phase: 0.20 mol/L aqueous NaCl; temperature: 35 °C; injection volume: 150 μL; polymer concentration: 0.4−1.0 mg/mL depending on the molar mass of the polymer sample.…”