1. Dielectric relaxation in Hyaluronic acid sodium salt S.Dolanski Babić 1,2, T.Ivek 1, T.Vuletić 1, S.Tomić 1 and R.Podgornik 3,4 1 Institut za fiziku, Zagreb, Hrvatska 2 Zavod za fiziku i biofiziku, Medicinski fakultet, Zagreb, Hrvatska 3 Department of Physics, University of Ljubljana, Ljubljana, Slovenia 4 J.Stefan Institute, Ljubljana, Slovenia

2. I Introduction II Samples III Low freq. Dielectric Spectroscopy IV Results V Plans

3. Introduction - Hyaluronan (hyaluronic acid or hyaluronate) is simple polymer of disaccharides themselves - composed of glucuronic acid and N-acetylglucosamine - linked together via alternating glycosidic bonds - Molecular weight of one monomer is 400 Da glucose

4. Introduction HYALURONAN IS- - found in all tissues and body fluids of vertebrates as well as in some bacteria - synthesized in the cellular plasma membrane - a major component of the synovial fluid and was found to increase the viscosity of the fluid - a major constituent in the vitreous of the human eye -an important component of articular cartilage, where it is present as a coat around each cell - is also a major component of skin, where it is involved in tissue repair Hyaluronan (HA) can be 25.000 monomers in length ~1 nm

5. Samples Hyaluronic acid Sodium salt from Streptococcus equi sp. (BioChemika Fluka) - molecular weights of HA* are in the range 300 kDa- 1900 kDa - polydisperse HA fragments in the range 750- 5000 monomers (0.75 -5 m) - pure water solutions in the range of 0.01-5 mg/mL - pure water: MilliPore, Milli-Q, 0.056 S/cm * K.Tamoto, Semin.Arthritis Rheum., Vol. 22 (1993)

6. Dielectric spectroscopy (40 Hz-100 MHz) Temperature control unit Temp. range:5°C - 60°C Stability: ± 0.1 K Impedance analyzer Agilent 4294 measures capacitance, C and conductance, G (real part) Y()= G()+iC() Chamber for liquid samples – water solutions - conductivity: 1.5-2000 S/cm - drops vol.: 50-200 L - reproducibility 0.8% - long term (2 h) 2% Pt

7. From complex conductance to complex dielectric function Y()=G()+iC() -G() and C() of DNA solutions are measured - These are subtracted for (G, C) of background (reference) NaCl solution with matching conductivity (at 100kHz) - This procedure enables to eliminate the electrode polarization effects, as well as other stray impedance effects.

8. From complex conductance to complex dielectric function (G-G NaCl, C-C NaCl ) =’()-i’’() FITS to a sum of two generalized Debye functions - relaxation process strength,  -  0, central relaxation time - symmetric broadening of the relaxation time distribution, 1 - 

9. Results -HA pure water solutions dielectric response spectra feature two modes (as DNA solutions) - two relaxation modes in kHz and MHz ranges

10. Results HF mode:   4, 1-  0.82 LF mode:   60 for c < 0.2 mg/mL   10 for c > 0.4 mg/mL 1-  0.75  HF  LF

11. Results semidilute regime above c*=0.001 mg/mL

12. Results =0.5=0.4

13. Results f HF - fraction of counterions participating in the HF process  HF - polarizability

14. Plans 1.Repeat the dielectric spectroscopy for Hyaluronic acid pure water solutions in the same range of HA concentration as reported here. 2. Hyaluronic acid in NaCl electrolyte (with added salt of fixed or varying concentration), similar range of HA concentration as reported here.