Structural characterisation of thiol-modified hyaluronans
Fourier-transform infrared spectroscopy and non-isothermal methods—chemiluminometry, differential scanning calorimetry, and differential thermogravimetry—were used to characterize potential structural changes of thiol-modified hyaluronans. Degradative conditions tested via rotational viscometry were first initiated applying oxidative Weissberger’s system in a reaction system under aerobic conditions. Several low-molecular-weight thiol compounds—cysteamine, l-cysteine, and N-acetyl-l-cysteine—were subsequently tested for their potential antioxidative effects against hyaluronan degradation. It was shown that different final values of dynamic viscosity of hyaluronan solutions were dependent on the thiol structure and its initial concentration. An idea has been put forward that together with the reduction of the hyaluronan molecular weight, which is a consequence of fragmentation, the degradation products might contain associated or even cross-linked structures. In the case of N-acetyl-l-cysteine application, the carbonaceous residue evidenced by differential thermogravimetry was increased when compared to that of intact hyaluronan.
KeywordsHyaluronan Rotational viscometry Fourier-transform infrared spectroscopy Non-isothermal methods
Though cellulose is the major component of biomass being extensively studied nowadays, not less is hyaluronan (HA) significant as the major component of extracellular matrix of vertebrates including humans.
HA is synthesized in the plasmatic membrane of various cells of vertebrates, and in tissues it functions in building-up of the extracellular matrices. Native HA macromolecules do not contain sulphate groups. HAs synthesized in vivo can reach the molecular weight values of up to 107 Da (Stern 2003).
High concentrations of HA in solution are very viscous (Kogan et al. 2007; Furth et al. 2008). The physiological level of HA in human synovial fluid (SF) is 2–4 mg/mL (Balazs et al. 1967). SF—a viscoelastic tissue—serves as a lubricant protecting cartilages against mechanical damage (Bastow et al. 2008). HA is characterized by an extraordinarily high rate of turnover. A 70 kg individual contains ≈15 g of this glycosaminoglycan. The one third of this amount turns over daily. The HA half-life in SF of healthy subjects is 12 h (Stern et al. 2007).
Under rheumatoid arthritis (RA) conditions the degradation of high-molecular-weight HA occurs due to the joint inflammation. Such a pathological situation is usually accompanied with the impairment and even the loss of the viscoelastic properties of SF. It was established that low-molecular-weight HA has different activities compared to the native high-molecular-weight biopolymer (Stern et al. 2007). While high-molecular-weight linear HAs are antiinflammatory with low angiogenic properties, the lower sized polymer fragments and branched or crosslinked HAs may have inflammatory, immunostimulatory, and highly angiogenic properties. HA fragments comprising 25–50 disaccharide units appear to function as endogenous danger signals (Stern 2004).
It is well-known that several endogenous thiols such as l-glutathione (GSH) act directly at scavenging of free radicals whose presence have been unambiguously evidenced at various diseases (RA and many others). GSH protects proteins against irreversible oxidation. The reaction between GSH and cysteine residues within a polypeptide chain is called S-glutathionylation (Hurd et al. 2005). Reversible formation of mixed disulphides prevents proteins in vivo from irreversible oxidations of their cysteine residues (Shenton and Grant 2003). S-Glutathionylated proteins may function as potential biomarkers of oxidative/nitrosative stress in some human diseases (Prakash et al. 2009). GSH has a special role not only as a biological antioxidant but also as a signalling molecule in the lung system regarding immunity and inflammation (Ghezzi 2011).
Recently, GSH has been tested for its significant antioxidative potential in the protection of high-molecular-weight HA against undesirable degradation (Hrabárová et al. 2009, 2012) induced by the Weissberger’s biogenic oxidative system (WBOS) (Weissberger and Luvalle 1944; Butt and Hallaway 1961). Prior to the application of non-isothermal methods, the Fourier-transform infrared (FT-IR) spectra of HAs recovered in a solid form after being treated via rotational viscometry revealed certain changes in comparison with the spectrum given for the intact HA, assuming thus a possible incorporation of thiol residue(s) into the HA macromolecule (Hrabárová et al. 2009).
Supposing that thiol compounds act as free radical scavengers and/or peroxide decomposers, our effort consisted in elucidation of their protective properties against the HA degradation induced by WBOS. A possible thiol incorporation into the HA macromolecule, recovered after degradation process in a solid form, was characterized via the methods such as CL, DSC and DTG. The CL method provides information about the relative efficiency of different antioxidants within a relatively large temperature interval. Moreover, when using an appropriate model of the HA degradation, the characterisation of the residual stability by the rate constant of the HA degradation may be more quantitative. As resulted from the DTG measurements, the possible extent of cross-linking may be deduced from the relative amount of carbonaceous residue that remains on the reaction pan. Finally, the DSC method which describes the extent of exothermic or endothermic process at higher temperatures may act as fingerprint of the structural changes in the HA macromolecules during their degradative fragmentation.
High-molecular-weight HA (P9710-2A; Mw = 808.7 kDa; Mw/Mn = 1.63) was kindly donated from Lifecore Biomedical Inc., Chaska, MN, USA. CAM, CYS, and NAC—all of analytical purity grade were purchased from Sigma–Aldrich Chemie GmbH, Steinheim, Germany.
The HA sample (2.5 mg/mL) was dissolved in 0.15 M aqueous NaCl solution for 24 h in the dark. The reagents were gradually added to the HA solution at the beginning of the reaction having final concentrations in the reaction system: 1.0 μM CuCl2, 100 μM thiol compound, and 100 μM ascorbate. The changes of dynamic viscosity of the final reaction mixture (8.0 mL) were on-line monitored for the period of 5 h via rotational viscometry at 25 °C (Hrabárová et al. 2009). Applying the solvent-exchange method for polymer recovery in a solid form, the reaction product was subsequently precipitated pouring the aqueous solution into 20 mL of 96 % ethanol under extensive stirring, and kept at +4 °C overnight. The next day, the precipitate was thoroughly washed out with ethanol, acetone, and dried in air. The yields of the recovered polymers ranged between 67 and 92 % of the initial HA amount. The dried polymeric products were obtained in the form of amorphous consistence. Such thiol-modified HAs as particulates were further used to characterize their potential structural changes due to degradation.
Fourier-transform infrared spectroscopy
FT-IR spectra were measured with Nicolet 6700 (Thermo Fisher Scientific, USA) spectrometer equipped with DTGS detector and Omnic 8.0 software. The spectra were collected in the middle region from 4,000 to 400 cm−1 at a resolution of 4 cm−1, the number of scans was 128. Diamond Smart Orbit ATR accessory was applied for measurement in solid state.
Differential scanning calorimetry
DSC measurements were performed using a Mettler-Toledo DSC 821e differential scanning calorimeter. Indium was used for calibration of temperature and heat of fusion. Glass transition temperature (Tg) and thermal stability of the samples were evaluated from the second heating of samples from room temperature up to 550 °C (10 °C/min) in a nitrogen atmosphere (50 mL/min). The first heating (from room temperature up to 170 °C) was used for water removal. Thermooxidative degradation was investigated in a temperature range from room temperature up to 550 °C (10 °C/min) in an oxygen flow (50 mL/min). At least three parallel runs were performed for each sample.
DTG measurements were performed using a Mettler-Toledo TGA/SDTA 851e instrument in a nitrogen or oxygen flow (30 mL/min) using a heating rate of 2.5 °C/min in a temperature range from room temperature up to 550 °C. Indium and aluminium were used for temperature calibration.
CL measurements were performed with a photon-counting instrument Lumipol 3 manufactured at the Polymer Institute of the Slovak Academy of Sciences. The sample was placed on an aluminium pan in the sample compartment. The gas flow (pure oxygen or nitrogen) through the sample cell was 3.0 L/h. The temperature in the sample compartment of the apparatus was raised from 40 up to 250 °C with the rate of 2.5 °C/min. The signal of the photocathode was recorded at 10 s data collection interval.
Evaluation of non-isothermal chemiluminescence records
Results and discussion
Based on the measurements of the dynamic viscosity changes (Fig. 1), it is evident that only slight changes may be observed in the case of CAM while NAC and CYS showed significantly lower protective effects against free-radical degradation of HA macromolecules. One should be aware that the results provided by the RV method may not only indicate the drop of antioxidative properties of the thiol additive but they may also indicate a potential occurrence of branching or even cross-linking during the HA modification. Then the resulting pattern of the viscosity changes could be the superposition of both processes.
Fourier-transform infrared spectroscopy
Physical properties of polysaccharides due to the hydrogen bond formation applying various drying methods are usually changed. This phenomenon can be studied by FT-IR for the –OH bond stretching regions. Besides free –OH groups, there are hydrogen bonds present as both inter- and intramolecular in polymer. Their ratio and interactions as a result of polymer modifications are therefore changed (Hromádková et al. 2003). As a consequence of the changed inter- and intramolecular hydrogen bond formation in the thiol-modified HA, the main maximum peak for the monosaccharide unit of polymer was shifted from 1,036 cm−1 as given for the intact HA to the lower values of wavenumber that was in the case of the thiol- modified HAs as follows: CYS 1,032 cm−1, CAM 1,030 cm−1, and NAC 1,026 cm−1 (Fig. 2). This was accompanied also with the increase of the half-width of the band. Among all the thiols, NAC exhibited the greatest influence on the shift of the main maximum peak that was 10 cm−1. As resulted from the changed ratio of inter- and intramolecular bond formation in polymer, band maximum position for stretching vibration ν(OH) bond was as well shifted from 3,330 cm−1 as given for the intact HA (Günzler and Gremlich 2002) to the lower frequencies 3,269 cm−1 as given for thiol-modified HAs accompanied with the broadening of the half-width of the band (Fig. 3).
It is known that FT-IR spectra of the stretching vibrations ν(C–H) bonds (–CH3, –CH2, –CH groups) have their absorption peaks in the wavenumber region 3,000–2,800 cm−1 (Günzler and Gremlich 2002). In this region we can observe a differential FT-IR spectrum obtained using a subtraction technique for the NAC-modified HA (Fig. 4). The increase of the C–H vibration intensities for individual groups was significant. Asymmetrical stretching vibration νas(C–H) bond and symmetrical stretching vibration νs(C–H) bond from the –CH3 group was at 2,959 cm−1 and 2,873 cm−1. The νas(C–H) bond was at 2,921 cm−1 and the νs(C–H) bond was at 2,854 cm−1 from the –CH2 group. Stretching vibration ν(C–H) bond from the C–H group was at 2,893 cm−1. The increase of the band intensities in the region of the C–H vibrations was evidenced from the differential FT-IR spectrum. Based on our results, we can thus assume the formation of a thiol-modified HA structure during viscometric processing.
The CL method is routinely used as a suitable analysis tool on in vitro study of spontaneous oxidative aging e.g. cellulosic materials (Rychlý et al. 2004). The same method was successfully applied to investigate structural changes in the HA macromolecules after accelerated oxidative aging in vivo (Rychlý et al. 2006).
Differential scanning calorimetry
Intact HA solutions were modified by the action of WBOS at the presence of the thiol compounds (CAM, CYS, and NAC) via the RV method. As a result of the degradation process, HAs were fragmented to a various final dynamic viscosity dependently on thiol applied. The results given by the FT-IR, CL, DSC, and DTG methods support the assumption that incorporation of a thiol moiety into the HA macromolecule forming associated or even cross-linked structures could be significant.
While decrease of dynamic viscosity was the result of the HA fragmentation during degradation, eventually combined with cross-linking of the HA macromolecules acting both in the opposite directions, the DSC signal in nitrogen revealed the disappearance of the second exotherm for the NAC-modified HA which indicates the additional fragmentation of higher-molecular-weight fraction. In the case of the NAC application, the carbonaceous residue evidenced by the DTG method was increased when compared to that of the intact HA.
The CL method appears to be an efficient tool for delimitation of the residual stability and the antioxidative efficiency of thiol-modified HAs along a relatively large temperature scale. At lower temperatures the corresponding link with the residual viscosity of HA and the rate constants of degradation may be clearly seen. Binding thiol moieties to the HA macromolecules during degradation was deduced from the shift of non-isothermal chemiluminescence records to higher temperatures when compared to that of the intact HA.
Intensities of both the CL and DSC signals are considerably stronger in oxygen than in nitrogen which may be taken as the proof of the participation of peroxyl radicals in the HA degradation.
The work was supported by the VEGA grant project Nos.: 2/0083/09, 1/0529/09, 2/0056/10, 1/0145/10, 2/0011/11 and 2/0147/12 of the Slovak Academy of Sciences. This contribution is the result of the project implementations: ITMS26220120054 and ITMS26240220040 supported by the Research & Development Operational Programme funded by the ERDF.
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