Kornelia Jumel

Simultaneous determinations of the molecular weight distributions of amyloses and the fine structures of amylopectins of native starches

Abstract Native A (wheat and waxy rice), B (potato), and C (cassava and sweet potato) types of starches were each debranched with isoamylase, and separated into amylose and amylopectin fractions by HPLC on size exclusion columns coupled on-line to multi-angle-laser-light-scattering and differential refractometer detectors. The absolute molecular weights of amyloses and chain length distributions of amylopectins were determined simultaneously, and pre-isolation of the amylopectin was not necessary. The molecular weights of debranched amylose from starches that have not been fractionated to separate amylose and amylopectin are significantly higher than published values for the undebranched fractionated amylose. The polymodal profiles of the refractive index chromatograms showed the complexity of the amylopectin structure of starches. The chain length distribution of amylopectin depends critically on the method for analysing the broad chromatogram when determined by either noting the minima/inflections or deconvoluting the overlapping amylopectin fraction into numerous normal/Gaussian distributions. Although the results from the former (conventional) method of analysis were comparable with the literature values, they did not appear to be as sensitive a technique for detecting differences as the multiple Gaussian approach. Overall, the study suggested that the amylopectin chain units might be more complex than originally envisaged and that different degrees of chain packing for the molecules can be inferred from this multiple component analysis.

Hydrodynamic characterisation of chemically degraded hyaluronic acid

Abstract Hyaluronic acid (HA) is a natural macromolecule with importance in the pharmaceutical, medical and cosmetic industries. The knowledge of its hydrodynamic properties is fundamentally important for further study on its applications. The aim of our study was to investigate and provide hydrodynamic parameters for six different molar mass samples chemically produced by depolymerisation of a high molar mass ‘parent’ HA using five different hydroxyl free radical concentrations. The main tools employed for these studies were size exclusion chromatography/multi-angle laser light scattering (SEC/MALLS), sedimentation velocity in the analytical ultracentrifuge and intrinsic viscosity. The results indicated that values for intrinsic viscosity, molar mass and sedimentation coefficient decreased with increasing hydroxyl free radical starting concentration. The six samples investigated here also represent a homologous series of a polysaccharide thus conformational information could be obtained, and the results indicate that HA adopts a ‘stiffish’ coil conformation in solution. Where appropriate our results were compared with previously published data.

Rapid size distribution and purity analysis of gastric mucus glycoproteins by size exclusion chromatography/multi angle laser light scattering

The weight average molar masses and molar mass distributions of two commercial pig gastric mucins and two fresh mucin preparations were determined by the technique of size exclusion chromatography coupled on-line to multi angle laser light scattering (SEC/MALLS). Both commercial samples exhibited much lower molar mass averages than the freshly prepared material and contained more impurities. On the other hand, a fresh mucin preparation examined after being stored frozen for 18 months revealed a slight increase in molar mass.

Characterization of pullulans produced from agro-industrial wastes

Abstract Pullulans produced from the Aureobasidium pullulans fermentation of various agro-industrial wastes were characterised in terms of carbohydrate analysis. heterogeneity and molecular weights. Grape skin pulp extract (GSPE), starch waste (SW), olive oil waste effluents (OW) and molasses (M) were used as substrates for the production of pullulan. A glucose based defined medium (D) was used for comparison purposes. Weight average molecular weights were determined using size exclusion chromatography/multiangle laser light scattering. In each of the ethanol precipitated substances the molecular weights were higher than that of the reference pullulan (Sigma), and the elution profiles indicated the polydisperse nature of the pullulans. When using D, the molecular weight of pullulan produced decreased with increasing fermentation time. Different substrates produced pullulans with different molecular weights. Of the substrates studied, D, GSPE and SW produced pullulans in high yields, which were relatively homogeneous, while OW alone or with M produced greatly heterogeneous ethanol agglutinating substances.

Effect of gamma irradiation on the macromolecular integrity of guar gum

Abstract Irradiation is becoming an increasingly important method of processing foodstuffs. Our understanding of its effects on the molecular integrity of food polysaccharides such as guar gum is however still very limited. In this study a range of techniques was used to determine absolute weight-average molecular weights and conformational parameters for a homologous series of irradiated guar gum samples. It was found that molar masses and viscosities of the irradiated samples decreased with increasing irradiation dose. However, this decrease was not linear but appeared to occur in two stages. Mark-Houwink parameters confirmed that there was no significant change in the gross conformation due to irradiation. Evaluation of the radiation yield through so-called G (seission) values gave an average value of 3.8 which is lower than expected for polysaccharides indicating some resistance to radiolytic degradation. The amount of guar gum recovered after sample work-up increased with increasing radiation dose. This suggests concentrated solution properties of guar gum are significantly influenced by non-hydrated/undissolved material.

A comparison of molecular mass determination of hyaluronic acid using SEC/MALLS and sedimentation equilibrium

Hyaluronic acid (HA) is a natural polysaccharide with importance in the pharmaceutical, medical and cosmetic industries. Determining factors in its final applications are its physicochemical properties, particularly molecular mass. A high molecular mass HA was degraded using five different hydroxyl free-radical starting concentrations chemically produced from ascorbic acid and hydrogen peroxide. The aims of the study were to investigate the effect of different hydroxyl free-radical concentrations on the chain length of HA and compare the molecular masses obtained from analytical ultracentrifugation using sedimentation equilibrium experiments and size exclusion chromatography/multi-angle laser light scattering (SEC/MALLS). The results indicated that their molecular masses varied, depending on the degree of hydroxyl free-radical starting concentration. Molecular mass values obtained from sedimentation equilibrium experiments for each sample showed the same trend as those obtained from the SEC/MALLS in the range of molecular masses studied. The molecular masses obtained from sedimentation equilibrium for high molecular mass samples from reciprocal plots of apparent weight average molecular mass against concentration gave values similar to those obtained by SEC/MALLS. In contrast, the molecular mass from conventional plots for high molecular mass samples were much lower than those from SEC/MALLS, even when high ionic strength buffers were used.

Amylose Content of Rice Starch

The amylose content separated from enzymatically debranched rice starches using Size Exclusion Chromatography Coupled to Multi-angle Laser-Light-Scattering (SEC-MALLS) ranged from 7-11 % of the total starch. This could be the true amylose content and is much lower than is obtained from iodine binding methods. The molecular weight and polydispersity value of the amyloses are also reported.

Positive co-operative activity and dimerization of the isolated ABC ATPase domain of HlyB from Escherichia coli

The ATPase activity of the ABC (ATP-binding cassette) ATPase domain of the HlyB (haemolysin B) transporter is required for secretion of Escherichia coli haemolysin via the type I pathway. Although ABC transporters are generally presumed to function as dimers, the precise role of dimerization remains unclear. In the present study, we have analysed the HlyB ABC domain, purified separately from the membrane domain, with respect to its activity and capacity to form physically detectable dimers. The ATPase activity of the isolated ABC domain clearly demonstrated positive co-operativity, with a Hill coefficient of 1.7. Furthermore, the activity is (reversibly) inhibited by salt concentrations in the physiological range accompanied by proportionately decreased binding of 8-azido-ATP. Inhibition of activity with increasing salt concentration resulted in a change in flexibility as detected by intrinsic tryptophan fluorescence. Finally, ATPase activity was sensitive towards orthovanadate, with an IC50 of 16 microM, consistent with the presence of transient dimers during ATP hydrolysis. Nevertheless, over a wide range of protein or of NaCl or KCl concentrations, the ABC ATPase was only detected as a monomer, as measured by ultracentrifugation or gel filtration. In contrast, in the absence of salt, the sedimentation velocity determined by analytical ultracentrifugation suggested a rapid equilibrium between monomers and dimers. Small amounts of dimers, but apparently only when stabilized by 8-azido-ATP, were also detected by gel filtration, even in the presence of salt. These data are consistent with the fact that monomers can interact at least transiently and are the important species during ATP hydrolysis.

Evaluation of meningococcal C oligosaccharide conjugate vaccines by size-exclusion chromatography/multi-angle laser light scattering

The mean molecular masses of three different meningococcal C saccharide (MenC)–protein conjugate vaccines and their constituent proteins were estimated using HPLC size‐exclusion chromatography (SEC) with multi‐angle laser light scattering (MALLS) and refractive‐index (RI) detection (SEC/MALLS). Chromatography of two CRM197 conjugates (MenC–CRM197‐A and MenC–CRM197‐B) and one tetanus toxoid (TT) conjugate (MenC–TT) was performed in PBS, pH 7.4, on TSK‐Gel® (TosoHaas) analytical columns [CRM197 is a non‐catalytic cross‐reacting mutant (CRM) of diphtheria toxin]. Analysis of the light‐scattering signal measured at 18 angles simultaneously, using the RI signal as a measure of concentration, gave absolute weight‐average‐molecular‐mass (M̄w) values for the CRM197 conjugates as follows: MenC‐CRM197‐A, ≈75000 g·mol−1 and MenC–CRM197‐B, ≈350000 g·mol−1, suggesting that MenC–CRM197‐A is a monomer (one carrier protein per conjugate molecule), while MenC‐CRM197‐B is largely composed of conjugates containing three or four CRM197 molecules. The MenC–TT conjugate eluted as a two‐component system with M̄w of 1.63×106 and 395000 g· mol−1, suggesting that some cross‐linked complexes contain up to six TT molecules. Comparison of results from MALLS/RI with those obtained using UV detection highlights the differences in size and relative composition of the various subpopulations of the MenC conjugates that can be obtained using different detection systems.

Molar mass and viscometric characterisation of hydroxypropylmethyl cellulose

The weight average molar masses and molar mass distributions of three different preparations (identical in chemical composition, but different in the average degree of polymerization) of hydroxypropylmethyl cellulose have been characterized by SEC/MALLS (size exclusion chromatography coupled to multiangle laser light scattering). The intrinsic viscosity and the viscosity in the semidilute region have also been measured. When the viscosity data is presented in the form of logarithmic plots of the zero shear specific viscosity against the coil overlap parameter (c[η]), a higher viscosity than would be expected from the usual ‘master curve’ relationships is found. This was particularly the case for the highest molar mass preparation (Mw = 2.2 × 106g/mol). It is suggested that the high viscosity is a consequence of enhanced intermolecular interactions as a result of solvent quality effects. Although the data is limited, the values of the Mark-Houwink-Kuhn-Sakurada exponent (0.4–0.5) suggested by the [η], Mw data set would be consistent with water being a poor solvent for this polysaccharide. A related factor may be interactions induced when the chains are aligned during drying which are not completely disrupted on hydration.