Macromolecular characterization of high ß-glucan oat lines.

Oat (Avena sativa) is a cereal grain rich in fibers, proteins, vitamins and minerals. Oats have been linked to several health benefits, such as lowering blood cholesterol levels, counteracting cardiovascular disease and regulating blood sugar levels. This study aimed to characterize two new oat lines with high ß-glucan content emanating from ethyl methyl sulphonate mutagenesis on the Lantmännen elite variety Belinda. Two of the mutated lines, and the mother variety Belinda, were profiled for ß-glucan, arabinoxylan, total dietary fiber and starch composition. In addition, total lipid and protein content, amino acid composition and ß-glucan molecular weights were analyzed. The high levels of ß-glucan resulted in a significant increase in total dietary fiber, but no correlation could be established between higher or lower levels of the assayed macromolecules, i.e., between arabinoxylan-, starch-, lipid- or protein levels in the mutated lines compared to the reference. The results indicate separate biosynthetic pathways for ß-glucans and other macromolecules and an independent regulation of the different polysaccharides studied. Therefore, ethyl methyl sulphonate mutagenesis can be used to increase levels of multiple macromolecules in the same line.

Development and validation of capillary electrophoresis sodium dodecyl sulfate (CE-SDS) method for purity analysis of pertussis toxin, filamentous haemagglutinin and pertactin antigens.

We report here the development and validation of CE-SDS method for purity analysis of Acellular Pertussis vaccine components viz. purified Pertussis toxin (PTx), purified Filamentous haemagglutinin (FHA), and Pertactinantigen (PRN). The method was found to be specific and showed excellent linearity at a concentration range of 15.62 µg/mL-1000 µg/mL for purified PTx, 31.25 µg/mL-1000 µg/mL for purified FHA, and 3.9 µg/mL-1000 µg/mL for PRN antigen. Method reported limit of quantification (LOQ) 31.25 µg/mL, 62.5 µg/mL, and 7.8 µg/mL for purified PTx, FHA, and PRN respectively. Method precision (repeatability and intermediate precision) for purity and molecular weight determination in product matrix was below 10% for all three proteins. Method comparability studies were performed with SDS-PAGE. CE-SDS demonstrated corroborating results with SDS-PAGE for the estimation of purity and molecular weight analysis. However, CE-SDS method exhibited better resolution capabilities for resolving all the sub-unit peaks of PTx and isoforms of purified FHA. CE-SDS method also demonstrated stability indicating potential and thus fits its intended purpose as an effective analytical tool for quality control of acellular pertussis-based vaccines.

Molecular weight determination of low molecular weight hyaluronic acid and chondroitin sulfate by gel permeation chromatography.

Low molecular weight (LWM) hyaluronic acid (HA) and chondroitin sulfate (CS) have a wide range of applications. To determine their molecular weight (MW), we developed a gel permeation chromatography (GPC) method, which is calibrated based on serrated peaks in the chromatograms. MW calibrants were obtained from the enzymolysis of HA and CS using hyaluronidase. The identical structure of calibrants and samples ensured the soundness of the method. The highest confidence MWs were up to 14,454 and 14,605 for HA and CS, respectively, and the standard curves showed very high correlation coefficients. Thanks to the changeless relationship between MW and its contribution to the GPC integral, the second calibration curves could be derived via one GPC column, also embodied correlation coefficients of >0.9999. The discrepancies of MW values were minuscule, and the measurement of a sample could be conducted in <30 min. The accuracy of the method was verified using LWM heparins, and the measured Mw values showed a 1.2 %-2.0 % error relative to pharmacopeia results. The MW results obtained for LWM-HA and LWM-CS samples were also consistent with the results obtained by multiangle laser light scattering. The method was also verified be able to measure the very low MWs.

Benchtop versus high field NMR: Comparable performance found for the molecular weight determination of lignin.

Lignin is a promising renewable biopolymer being investigated worldwide as an environmentally benign substitute of fossil-based aromatic compounds, e.g. for the use as an excipient with antioxidant and antimicrobial properties in drug delivery or even as active compound. For its successful implementation into process streams, a quick, easy, and reliable method is needed for its molecular weight determination. Here we present a method using 1H spectra of benchtop as well as conventional NMR systems in combination with multivariate data analysis, to determine lignin's molecular weight (Mw and Mn) and polydispersity index (PDI). A set of 36 organosolv lignin samples (from Miscanthus x giganteus, Paulownia tomentosa and Silphium perfoliatum) was used for the calibration and cross validation, and 17 samples were used as external validation set. Validation errors between 5.6% and 12.9% were achieved for all parameters on all NMR devices (43, 60, 500 and 600 MHz). Surprisingly, no significant difference in the performance of the benchtop and high-field devices was found. This facilitates the application of this method for determining lignin's molecular weight in an industrial environment because of the low maintenance expenditure, small footprint, ruggedness, and low cost of permanent magnet benchtop NMR systems.

Solvent-Independent Molecular Weight Determination of Polymers Based on a Truly Universal Calibration.

Diffusion-ordered NMR spectroscopy (DOSY) allows for accurate molecular weight calibration and determination that can be corrected for solvent influences. Polystyrene and poly(ethylene glycol) standards have been used to calibrate DOSY diffusion data for a variety of solvents, showing a high correlation of data when the bulk viscosity of the solvent is accounted for following the Stokes-Einstein equation. In this way, a type of universal calibration is introduced that allows for determinations of average molecular weight that are at least as accurate as those of traditional size-exclusion chromatography (SEC), if not better. Further, we demonstrate that DOSY calibrations can be used between laboratories, hence removing the need for individual calibration of setups as currently done.

A comparative study of CE-SDS, SDS-PAGE, and Simple Western: Influences of sample preparation on molecular weight determination of proteins.

The development of capillary electrophoresis, especially CE-SDS devices, has led CE-SDS to become an established tool in a wide range of applications in the analysis of biopharmaceuticals and is increasingly replacing its method of origin, SDS-PAGE. The goal of this study was to evaluate the comparability of molecular weight (MW) determination especially by CE-SDS and SDS-PAGE. For ensuring comparability, model proteins that have little or no posttranslational modifications and an IgG antibody were used. Only a minor influence of sample preparation conditions, including sample buffer, temperature conditions, and different reducing agents on the MW determination were found. In contrast, the selection of the MW marker plays a decisive role in determining the accurate apparent MW of a protein. When using different MW markers, the deviation in MW determination can exceed 10%. Interestingly, CE-SDS and 10% SDS-PAGE hardly differ in their trueness of MW determination. The trueness in relation to the reference MW for each protein was calculated. Although the trueness values for the model proteins considered range between 1.00 and 1.11 using CE-SDS, they range between 0.93 and 1.03 on SDS-PAGE, depending on the experimental conditions chosen.

Using Fractionation and Diffusion Ordered Spectroscopy to Study Lignin Molecular Weight.

Recent reports demonstrate that applications of the biopolymer lignin can be helped by the use of a fraction of the lignin which has an optimal molecular weight range. Unfortunately, the current methods used to determine lignin's molecular weight are inconsistent or not widely accessible. Here, an approach that relies on 2D DOSY NMR analysis is described that provides a measure of lignin's molecular weight. Consistent results were obtained using this well-established NMR technique across a range of lignins.

Electrospray Ionization with High-Resolution Mass Spectrometry as a Tool for Lignomics: Lignin Mass Spectrum Deconvolution.

The capability to characterize lignin, lignocellulose, and their degradation products is essential for the development of new renewable feedstocks. Electrospray ionization high-resolution time-of-flight mass spectrometry (ESI-HR TOF-MS) method was developed expanding the lignomics toolkit while targeting the simultaneous detection of low and high molecular weight (MW) lignin species. The effect of a broad range of electrolytes and various ionization conditions on ion formation and ionization effectiveness was studied using a suite of mono-, di-, and triarene lignin model compounds as well as kraft alkali lignin. Contrary to the previous studies, the positive ionization mode was found to be more effective for methoxy-substituted arenes and polyphenols, i.e., species of a broadly varied MW structurally similar to the native lignin. For the first time, we report an effective formation of multiply charged species of lignin with the subsequent mass spectrum deconvolution in the presence of 100 mmol L-1 formic acid in the positive ESI mode. The developed method enabled the detection of lignin species with an MW between 150 and 9000 Da or higher, depending on the mass analyzer. The obtained M n and Mw values of 1500 and 2500 Da, respectively, were in good agreement with those determined by gel permeation chromatography. Furthermore, the deconvoluted ESI mass spectrum was similar to that obtained with matrix-assisted laser desorption/ionization (MALDI)-HR TOF-MS, yet featuring a higher signal-to-noise ratio. The formation of multiply charged species was confirmed with ion mobility ESI-HR Q-TOF-MS. Graphical Abstract ᅟ.

Size exclusion chromatography of lignin: The mechanistic aspects and elimination of undesired secondary interactions.

Characterization of lignin and its degradation products, more specifically determination of their molecular weight (MW) distribution, is essential for assessment and applications of these potentially renewable phenolics. Several representative gel filtration and gel permeation systems were evaluated in this work focusing on understanding of undesired secondary non-SEC interactions while utilizing four sets of commercially available polymeric standards as well as low-MW lignin model compounds including diarene standards synthesized in-house. The gel permeation column with a nonpolar highly cross-linked porous polystyrene/divinylbenzene-based stationary phase provided the most effective separation by MW for both low and high MW model compounds. Notably, the column with a higher pore and lower particle size provided a better resolution towards polymeric standards, even though the particle size effect was downplayed in the earlier SEC studies of lignin. For two other evaluated gel filtration and gel permeation columns, the separation was strongly affected by functionalities of the analytes and correlated with the compounds' pKa rather than MW. We showed that the separation on the stationary phases featuring polar hydroxyl groups led to specific column-analyte secondary interactions, perhaps based on their hydrogen bonding with lignin. Further, the SEC column evaluation yielded similar results with two sets of chemically different standards. This setup may be used as a general approach to selecting an applicable column for lignin SEC analysis. We confirmed the obtained results with a different independent method implementing a novel approach for lignin number-average MW (Mn) calculation based on laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF-MS) data. The determined Mn corroborated the SEC results.

Molecular weight determination of a newly synthesized guanidinylated disulfide-containing poly(amido amine) by gel permeation chromatography.

A cationic gene delivery vector, guanidinylated disulfide-containing poly(amido amine) (CAR-CBA), was synthesized by Michael addition reaction between N,N'-cystaminebisacrylamide (CBA) and guanidine hydrochloride (CAR). Gel permeation chromatography (GPC) was used to evaluate the molecular weight of synthesized CAR-CBA. Polyethyleneimine (PEI) with molecular weight of 25 kDa was adopted as a reference, and polyethylene glycols (PEG) with different molecular weights were used to establish a standard curve for determining the molecular weight of CAR-CBA. The effects of two critical factors, namely columns and eluents, on the molecular weight measurement of CAR-CBA were investigated to optimize the GPC quantitative method. The results showed that Ultrahydrogel columns (120, 250) and HAc-NaAc (0.5 M, pH 4.5) buffer solution were the optimal column and GPC eluent, respectively. The molecular weight of the synthesized CAR-CBA was analyzed by the optimized GPC method and determined to be 24.66 kDa.

Comparison of Low-Molecular-Weight Heparins Prepared From Bovine Lung Heparin and Porcine Intestine Heparin.

Currently porcine intestine is the only approved source for producing pharmaceutical heparin in most countries. Enoxaparin, prepared by benzylation and alkaline depolymerization from porcine intestine heparin, is prevalent in the anticoagulant drug market. It is predicted that porcine intestine heparin-derived enoxaparin (PIE) will encounter shortage, and expanding its production from heparins obtained from other animal tissues may, therefore, be inevitable. Bovine lung heparin is a potential alternative source for producing enoxaparin. Critical processing parameters for producing bovine lung heparin-derived enoxaparin (BLE) are discussed. Three batches of BLEs were prepared and their detailed structures were compared with PIEs using modern analytical techniques, including disaccharide composition, intact chain mapping by liquid chromatography-mass spectrometry and 2-dimensional nuclear magnetic resonance spectroscopy. The results suggested that the differences between PIEs and BLEs mainly result from N-acetylation differences derived from the parent heparins. In addition, bioactivities of BLEs were about 70% of PIEs based on anti-factor IIa and Xa chromogenic assays. We conclude that BLE has the potential to be developed as an analogue of PIE, although some challenges still remain.

Characterization of ultrahigh-molecular weight cationic polyacrylamide using frit-inlet asymmetrical flow field-flow fractionation and multi-angle light scattering.

In this study, frit inlet asymmetrical flow field-flow fractionation (FlFFF) with multi-angle light scattering (MALS) and differential refractive index (DRI) detection is utilized for size separation, determination of molecular weight (MW), and conformation of ultrahigh-MW (10(7)-10(9) g/mol) cationic polyacrylamides (C-PAMs), a class of water-soluble copolymers based on acrylamide and vinyl-type comonomers with quaternary ammonium cations that are widely used in wastewater treatment and in paper industries. Linear and branched C-PAM copolymers prepared in two different polymerization methods (solution and emulsion) from varying amounts of crosslinking agent and initiator were size fractionated by FlFFF with field-programming. It was found experimentally that the linear copolymers from both polymerization methods were less than 10(8) g/mol in MW with compact, nearly spherical structures, while the branched C-PAM copolymers from the emulsion polymerization showed a significant increase in average MW up to ∼ 10(9)g/mol, which was about 20-fold greater than those from the solution method, and the branched copolymers had more compact or shrunken conformations. While both linear and branched copolymers less than 10(8) g/mol MW were well resolved in an increasing order of MW (normal mode), it was noted that branched copolymers prepared through emulsion polymerization exhibited significantly larger MWs of 10(8-)10(9) g/mol and eluted in the steric/hyperlayer mode, in which the elution order is reversed in an extreme run condition (strong initial field strength followed by a fast field decay during programming).

Preparation of hydrogel embedded polymer-growth factor conjugated nanoparticles as a diabetic wound dressing.

CONTEXT: Growth factors act in an integrated manner to promote the wound-healing process. However, probably due to early inactivation of these molecules in the wound site, their topical administration scarcely leads to a significant improvement in chronic wound repair. OBJECTIVES: With the aim of identifying improved therapeutics, a sodium carboxymethyl chitosan-recombinant human epidermal growth factor conjugate (NaCMCh-rhEGF) was developed. It is believed that conjugation will protect rhEGF against proteolysis and will mediate rhEGF release by α-amylase. MATERIAL AND METHODS: As hydrogels possess most of the desirable characteristics of an ideal dressing, we used our previously described chitosan-based hydrogel as a carrier for NaCMCh-rhEGF nanoparticles to make a novel wound dressing system. To evaluate the biological activity of NaCMCh-rhEGF and free rhEGF, the proliferation of fibroblasts was measured using a colorimetric assay. Additionally the stability of conjugated and free rhEGF against proteases was estimated. RESULT AND DISCUSSION: In vitro results revealed that the conjugated form exhibited more stability against proteolysis and also preserved its biological activity. Furthermore, in vivo studies were performed using an excision wound model on diabetic rats. After 15 d, the wound area in NaCMCh-rhEGF-hydrogel dressing group was significantly smaller than other groups and showed histological parameters equal to positive wound control group. CONCLUSION: A polymer conjugated rhEGF was developed that was more stable against proteases and reserved the biological activity of the drug. This dressing appears to be a competent candidate for chronic wound healing.

Preparation of hydrogel embedded polymer-growth factor conjugated nanoparticles as a diabetic wound dressing.

CONTEXT: Growth factors act in an integrated manner to promote the wound-healing process. However, probably due to early inactivation of these molecules in the wound site, their topical administration scarcely leads to a significant improvement in chronic wound repair. OBJECTIVES: With the aim of identifying improved therapeutics, a sodium carboxymethyl chitosan-recombinant human epidermal growth factor conjugate (NaCMCh-rhEGF) was developed. It is believed that conjugation will protect rhEGF against proteolysis and will mediate rhEGF release by α-amylase. MATERIAL AND METHODS: As hydrogels possess most of the desirable characteristics of an ideal dressing, we used our previously described chitosan-based hydrogel as a carrier for NaCMCh-rhEGF nanoparticles to make a novel wound dressing system. To evaluate the biological activity of NaCMCh-rhEGF and free rhEGF, the proliferation of fibroblasts was measured using a colorimetric assay. Additionally the stability of conjugated and free rhEGF against proteases was estimated. RESULT AND DISCUSSION: In vitro results revealed that the conjugated form exhibited more stability against proteolysis and also preserved its biological activity. Furthermore, in vivo studies were performed using an excision wound model on diabetic rats. After 15 d, the wound area in NaCMCh-rhEGF-hydrogel dressing group was significantly smaller than other groups and showed histological parameters equal to positive wound control group. CONCLUSION: A polymer conjugated rhEGF was developed that was more stable against proteases and reserved the biological activity of the drug. This dressing appears to be a competent candidate for chronic wound healing.

Determination of the content and the molecular weight of Mussel Polysaccharide by GPC / 中国生化药物杂志

Objective To develop a gel permeation chromatography method for determination of content and molecular weight ( Mw ) of Mussel Polysaccharide.Methods Using GPC method, the sample was separated with TSK-gel GMPWXL(7.8 mm ×300 mm) chromatography column which was set at 35℃.The mobile phase was 0.05 mol/mL NaNO3(including 0.05%Na2N3) and the flow rate was 0.6 mL/min.The detector was RID-20AT. Results The average molecular weight of the polysaccharide of Mytilus coruscus was 1 261 411 and the average content was 88.6%by using of the calibration curves of dextrans.The average molecular weight of the polysaccharide of Mytilus edulis was 1 244 062 and the average content was 87.4%. Conclusion The method established in this paper is simple and rapid, accurate and reproducible, which can be used for the quality control of Mussel Polysaccharide.

Viscometric study of chitosan solutions in acetic acid/sodium acetate and acetic acid/sodium chloride.

A viscometric study was carried out at 25°C to assess the physical-chemical behavior in solution and the mean viscometric molar mass (M¯v) of chitosan solutions with different deacetylation degrees, in two solvent mixtures: medium 1-acetic acid 0.3mol/L and sodium acetate 0.2mol/L; and medium 2-acetic acid 0.1mol/L and sodium chloride 0.2mol/L. Different equations were employed, by graphical extrapolation, to calculate the intrinsic viscosities [η] and the viscometric constants, to reveal the solvent's quality: Huggins (H), Kraemer (K) and Schulz-Blaschke (SB). For single-point determination, the equations used were SB, Solomon-Ciuta (SC) and Deb-Chanterjee (DC), resulting in a faster form of analysis. The values of ̄M¯v were calculated by applying the equation of Mark-Houwink-Sakurada. The SB and SC equations were most suitable for single-point determination of [η] and ̄M¯v and the Schulz-Blachke constant (kSB), equal to 0.28, already utilized for various systems, can also be employed to analyze chitosan solutions under the conditions studied.

Molecular weight estimation of esterase isoenzymes in closely related Drosophila Species (Diptera: Drosophilidae) in non-denaturing polyacrylamide gel electrophoresis

A method that allows the measure of molecular weight of two well-known and closely related esterases from Drosophila mojavensis and its sibling species, D. arizonae, is here described, using native polyacrylamide gel electrophoresis at several concentrations, applying Fergunson´s principles. These enzymes, namely EST-4 and EST-5, presented molecular weight values between 81 and 91 kDa. In spite of their distinct expression pattern through the insect's life cycle, they showed properties of isoenzymes codified by distinct structural genes, supporting the hypothesis of a rather recent gene duplication event that generated both in D. mojavensis and D. arizonae, as well as in other species of repleta group. The method is simple and adequate to be applied to preliminary molecular weight determination of other enzymes without any previous purification procedure.

Neste trabalho, um método que permite a estimativa do peso molecular de duas esterases conhecidas e intimamente relacionadas, encontradas em Drosophila mojavensis e sua espécie aparentada D. arizonae, é descrito. Este método é realizado utilizando a técnica de eletroforese em diferentes concentrações de gel e aplicando os princípios de Fergunson. As enzimas, denominadas EST-4 e EST-5, apresentaram pesos moleculares entre 81 e 91 kDa. Apesar de seus padrões diferenciados de expressão durante o ciclo de vida do inseto, elas demonstraram propriedades de enzimas codificadas por genes estruturais distintos, corroborando a hipótese de um evento de duplicação gênica recente que gerou ambas em D. mojavensis e D. arizonae, bem como em outras espécies do grupo repleta. O método proposto é simples e adequado para ser utilizado em estimativas preliminares de determinação de pesos moleculares de outras enzimas sem haver a necessidade de um procedimento prévio de purificação.