Size Exclusion Chromatography Coupled To Multi-angle Laser Light Scattering Research Articles

Pectin isolation and characterization from six okra genotypes

Pectin was isolated by aqueous extraction at pH 6.0 from the pods of six different okra genotypes (Abelmoschus esculentus L.). Genetic diversity was determined using fragment length analysis (FLA) of ten simple sequence repeat (SSR) markers. Physical and chemical evaluation of pectin was performed by means of FT-IR and NMR spectroscopy, sugar composition analysis (GC-MS), size exclusion chromatography coupled to multi-angle laser light scattering (SEC-MALLS), dilute solution viscometry and steady shear rheology assisted by principal component analysis (PCA). Each of the SSR markers detected on average 4.1 alleles and revealed unique genotypes for each sample. Extraction yield was between 11 and 14% resulting in pectin with galacturonic acid content between 43 and 63%, low degree of methyl-esterification (17–25%) and high degree of acetylation (20–40%). All samples were of high weight-average molar mass (Mw) (700–1700 × 103 g mol−1) and sugar composition analysis revealed the structural diversity of samples with HG/RG-I ratios ranging between 1.3 and 3.1. The present work shows that individual okra genotypes provide pectin with different structural properties that could potentially provide a new source of functional pectin for the food or pharmaceutical industries.

Effect of different drying methods on chemical and molecular structure of heteropolysaccharide–protein gum from durian seed

The functional properties and biological aspects of a natural biodegradable biopolymer depend on its chemical and molecular structure. In this study, the effect of different drying processes on the chemical and molecular structure of the natural biodegradable biopolymer from durian seed was investigated. The chemical structure was analyzed by assessing the carbohydrate profile, protein, amino acid composition, moisture, and ash. Molecular weight (Mw), number average molecular weight (Mn), Mw/Mn ratio and mass recovery were assessed by using a size-exclusion chromatography coupled to multi angle laser light-scattering (SEC-MALS). The present study revealed that main monosaccharides in the chemical structure of differently dried durian seed gums were galactose (50.1–64.9%), glucose (29.4–45.7%%), arabinose (0.11–0.89%), and xylose (0.019–0.86%). The protein analysis indicated the presence of a low amount of the protein fraction (3.2–3.9%) in the chemical structure of the biopolymer from durian seed. The most abundant amino acids in the chemical structure of durian seed gum were leucine (31.78–43.02%), lysine (6.23–7.78%), aspartic acid (6.45–8.58%), glycine (6.17–7.27%), glutamic acid (5.43–6.55%), alanine (4.60–6.23%), and valine (4.49–5.52). The current study exhibited that the biodegradable biopolymer from durian seed was a heteropolysaccharideprotein complex with medium Mw ranging from 1.06 × 105 to 1.15 × 105 (g/mol).

Chemical composition and molecular structure of polysaccharide-protein biopolymer from Durio zibethinusseed: extraction and purification process

BackgroundThe biological functions of natural biopolymers from plant sources depend on their chemical composition and molecular structure. In addition, the extraction and further processing conditions significantly influence the chemical and molecular structure of the plant biopolymer. The main objective of the present study was to characterize the chemical and molecular structure of a natural biopolymer from Durio zibethinus seed. A size-exclusion chromatography coupled to multi angle laser light-scattering (SEC-MALS) was applied to analyze the molecular weight (Mw), number average molecular weight (Mn), and polydispersity index (Mw/Mn).ResultsThe most abundant monosaccharide in the carbohydrate composition of durian seed gum were galactose (48.6-59.9%), glucose (37.1-45.1%), arabinose (0.58-3.41%), and xylose (0.3-3.21%). The predominant fatty acid of the lipid fraction from the durian seed gum were palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:2). The most abundant amino acids of durian seed gum were: leucine (30.9-37.3%), lysine (6.04-8.36%), aspartic acid (6.10-7.19%), glycine (6.07-7.42%), alanine (5.24-6.14%), glutamic acid (5.57-7.09%), valine (4.5-5.50%), proline (3.87-4.81%), serine (4.39-5.18%), threonine (3.44-6.50%), isoleucine (3.30-4.07%), and phenylalanine (3.11-9.04%).ConclusionThe presence of essential amino acids in the chemical structure of durian seed gum reinforces its nutritional value.

Biochemical and Biophysical Characterization of the Mg2+-induced 90-kDa Heat Shock Protein Oligomers

The 90-kDa heat shock protein (Hsp90) is involved in the regulation and activation of numerous client proteins essential for diverse functions such as cell growth and differentiation. Although the function of cytosolic Hsp90 is dependent on a battery of cochaperone proteins regulating both its ATPase activity and its interaction with client proteins, little is known about the real Hsp90 molecular mechanism. Besides its highly flexible dimeric state, Hsp90 is able to self-oligomerize in the presence of divalent cations or under heat shock. In addition to dimers, oligomers exhibit a chaperone activity. In this work, we focused on Mg(2+)-induced oligomers that we named Type I, Type II, and Type III in increasing molecular mass order. After stabilization of these quaternary structures, we optimized a purification protocol. Combining analytical ultracentrifugation, size exclusion chromatography coupled to multiangle laser light scattering, and high mass matrix-assisted laser desorption/ionization time of flight mass spectrometry, we determined biochemical and biophysical characteristics of each Hsp90 oligomer. We demonstrate that Type I oligomer is a tetramer, and Type II is an hexamer, whereas Type III is a dodecamer. These even-numbered structures demonstrate that the building brick for oligomerization is the dimer up to the Type II, whereas Type III probably results from the association of two Type II. Moreover, the Type II oligomer structure, studied by negative stain transmission electron microscopy tomography, exhibits a "nest-like" shape that forms a "cozy chaperoning chamber" where the client protein folding/protection could occur.

The kinetics of chitosan depolymerisation at different temperatures

The stability (in terms of molar mass) of chitosan potentially plays an important role in its behaviour and functional properties in a wide range of applications and therefore any changes over time must be understood. The weight-average molar masses and intrinsic viscosities of chitosan solutions at different temperatures (4, 25 and 40 °C) have been investigated using size exclusion chromatography coupled to multi-angle laser light scattering (SEC-MALLS) and a “rolling ball” viscometer respectively. The weight-average molar mass ( M w) and the intrinsic viscosity ([ η]) both decrease with increased storage time, although this phenomenon is more pronounced at elevated temperatures. Good correlation was found between the changes in molar mass and intrinsic viscosity with time and these parameters were used to determine the depolymerisation constant ( k) and the activation energy ( E a). Knowledge of the effect of storage conditions ( e.g. temperature) is important in the understanding the stability of chitosan solutions, but whether or not chitosan depolymerisation will be detrimental to its intended application will depend on the functional significance of the changes that occur.

Macromolecular conformation of chitosan in dilute solution: A new global hydrodynamic approach

Chitosans of different molar masses were prepared by storing freshly prepared samples for up to 6 months at either 4, 25 or 40 °C. The weight-average molar masses, M w and intrinsic viscosities, [ η] were then measured using size exclusion chromatography coupled to multi-angle laser light scattering (SEC-MALLS) and a “rolling ball” viscometer, respectively. The solution conformation of chitosan was then estimated from: (a) the Mark–Houwink–Kuhn–Sakurada (MHKS) power law relationship [ η] = kM w a and (b) the persistence length, L p calculated from a new approach based on equivalent radii [Ortega, A., & Garcia de la Torre, J. (2007). Equivalent radii and ratios of radii from solution properties as indicators of macromolecular conformation, shape, and flexibility. Biomacromolecules, 8, 2464–2475]. Both the MHKS power law exponent ( a = 0.95 ± 0.01) and the persistence length ( L p = 16 ± 2 nm) are consistent with a semi-flexible rod type (or stiff coil) conformation for all 33 chitosans studied. A semi-flexible rod conformation was further supported by the Wales–van Holde ratio, the translational frictional ratio and sedimentation conformation zoning.

Molar mass and solution conformation of branched α(1 → 4), α(1 → 6) Glucans. Part I: Glycogens in water

Solution molar masses and conformations of glycogens from different sources (rabbit, oyster, mussel and bovine) were analysed using sedimentation velocity in the analytical ultracentrifuge, size-exclusion chromatography coupled to multi-angle laser light scattering (SEC-MALLS), size-exclusion chromatography coupled to a differential pressure viscometer and dynamic light scattering. Rabbit, oyster and mussel glycogens consisted of one population of high molar mass (weight averages ranging from 4.6 × 10 6 to 1.1 × 10 7 g/mol) as demonstrated by sedimentation velocity and SEC-MALLS, whereas bovine glycogen had a bimodal distribution of significantly lower molar mass (1.0 × 10 5 and 4.5 × 10 5 g/mol). The spherical structure of all glycogen molecules was demonstrated in the slopes of the Mark–Houwink–Kuhn–Sakurada-type power-law relations for sedimentation coefficient ( s 20 ,w o ) , intrinsic viscosity ([ η]), radius of gyration ( r g, z ) and radius of hydration ( r H, z ), respectively, and was further supported by the ρ (= r g, z / r H, z ) function, the fractal dimension and the Perrin function. The degree of branching was estimated to be ∼10% from the shrinking factors, g′ (=[ η] branched/[ η] linear) and also h (=( f/ f o) branched/( f/ f o) linear), respectively, where ( f/ f o) is the translational frictional ratio, consistent with expectation.

Mutation of Two Intramembrane Polar Residues Conserved within the Hyaluronan Synthase Family Alters Hyaluronan Product Size

We identified two conserved polar amino acids within different membrane domains (MD) of Streptococcus equisimilis hyaluronan synthase (seHAS), Lys48 in MD2 and Glu327 in MD4. In eukaryotic HASs, the position of the Glu is very similar and the Lys is replaced by a conserved polar Gln. To assess whether Lys48 and Glu327 interact or influence seHAS activity, we investigated the effects of changing Lys48 to Arg or Glu and Glu327 to Lys, Asp, or Gln. Mutants, including a double switch variant with Lys48 and Glu327 exchanged, were expressed and assayed in Escherichia coli membranes. SeHASE327Q and seHASE327K were expressed at low levels, whereas seHASE327D and the Lys48 mutants were expressed well. The specific enzyme activities (relative to wild type) were 17 and 7% for the K48R and K48E mutants and 26 and 38% for the E327Q and E327D mutants, respectively. In contrast, seHAS(E327K) showed only 0.16% of wild-type activity but was rescued over 46-fold by changing Lys48 to Glu. Expression of the seHASE327K,K48E protein was also rescued to near wild-type levels. Based on size exclusion chromatography coupled to multiangle laser light scattering analysis, all the variants synthesized hyaluronan (HA) of smaller weight-average molar mass than wild-type enzyme (3.6 MDa); the smallest HA (approximately 0.6 MDa) was made by seHASE327K,K48E and seHASK48E. The results indicate that Glu327 within MD4 is a critical residue for the stability of seHAS, that it may interact with Lys48 within MD2, and that these residues are involved in the ability of HAS to synthesize very large HA.

Water-soluble polysaccharides with pharmaceutical importance from Durian rinds ( Durio zibethinus Murr.): isolation, fractionation, characterisation and bioactivity

Crude, pharmaceutically useful water-soluble polysaccharides have been isolated from durian rinds ( Durio zibethinus) by hot water extraction followed by ethanol precipitation. The polysaccharides were fractionated by anion exchange chromatography and size exclusion chromatography. Characterisation of the sub-fractions by methanolysis, methylation analysis and NMR spectroscopy revealed that the principal components were pectic polysaccharides with starch as a contaminant. Physical features namely molecular weight ( M w) and intrinsic viscosity of the main fractions were investigated by size exclusion chromatography coupled to multi angle laser light scattering (SEC/MALLS) and capillary viscometer, respectively. The main fractions were subjected to the complement-fixation assay and the relationship of the chemical features of the polysaccharide fractions with their activity was also considered.

Pressure cell assisted solution characterization of polysaccharides. 1. Guar gum.

To reduce time-dependent aggregation phenomena and achieve true "molecular" solution, the "pressure cell" solubilization method of Vorwerg and co-workers was applied to solutions of guar galactomannans (three samples of different molecular weights), using various heating, time, and pressure profiles. Physicochemical characterization of the guar samples before and after pressure cell treatment included measurements of intrinsic viscosity [eta] by capillary viscometry and M(w) and radius of gyration from size exclusion chromatography coupled to multiangle laser light scattering (SEC/MALLS). Heating the guar solutions (100-160 degrees C) without pressurization produced chain degradation with [eta] and M(w) values being reduced significantly, whereas this effect was reduced substantially for samples subject to initial pressurization ( approximately 5-10 bar). The constants in the Mark-Houwink-Sakurada equation, relating [eta] and M(w) were established and the characteristic ratio C(infinity) and chain persistence length L(p) were calculated using both the Burchard-Stockmayer-Fixman (BSF) method for flexible and semiflexible chains and the Hearst method more appropriate for stiffened chains. Definitive conclusions can now be drawn on the flexibility of the guar chain backbone, with L(p) approximately 4 nm from the BSF plot, in good agreement with previously published work using such geometric methods. This contrasts with the higher values obtained from extrapolation of data for polyelectrolytes with a similar backbone geometry, such as sodium carboxymethyl cellulose, to "infinite" ionic strength.

The effect of the degree of esterification on the hydrodynamic properties of citrus pectin

Estimation of the influence of the degree of esterification on the hydrodynamic properties of citrus pectins provides a simple demonstration of how chemical variation can influence structural properties of polysaccharides. Five citrus pectins with average degree of esterification 77.8, 65.0, 53.9, 37.8 and 27.9%, respectively were studied using capillary viscometry, sedimentation velocity, sedimentation equilibrium and size exclusion chromatography coupled to multi-angle laser light scattering (SEC-MALLS). Molecular weights (weight average) for all five pectin samples were within the range 190 000±30 000 g/mol as confirmed by the independent techniques of sedimentation equilibrium and SEC-MALLS. Estimates for the conformation dependent Wales–van Holde ( k s/[ η]) and frictional ( f/ f 0) ratios from the hydrodynamic data clearly indicates increasing chain stiffness with decreasing degree of esterification. It appears that both steric and electrostatic interactions are important in these conformational changes.

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.

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 ( M w = 2.2 × 10 6g/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], M w 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.