Multi-angle Laser Light-scattering Detection Research Articles

Characterization of sulfated polysaccharide from Laurencia obtusa and its apoptotic, gastroprotective and antioxidant activities

This study was designed to characterize the physico-chemical properties of the sulfated polysaccharide (SP) isolated from the red alga Laurencia obtusa and to evaluate its apoptotic, gastroprotective and antioxidant activities.The different macromolecular characteristics of SP were determined by size exclusion chromatography combined with multi-angle laser light-scattering detection (SEC-MALLS), Fourier transform infrared spectroscopy (FTIR) analysis and nuclear magnetic resonance spectroscopy (1H NMR and 13C NMR). The native molecular weight of the extracted polysaccharide is high (≥336,900 g·mol−1). It showed high amounts of sulfated groups (28.2%) and low levels of proteins. It was found to be a potent inducer of apoptosis on acute monocytic leukaemia THP-1cell lines with EC50 value of 53 μg·mL−1. Furthermore, a significant gastroprotective effect (p < 0.01) was also observed with a gastric ulcer inhibition of 63.44%, 78.42% and 82.15% at the doses 25, 50 and 100 mg·kg−1, respectively. In addition, SP significantly increased glutathione levels (GSH) and decreased the concentration of thiobarbituric acid-reactive substances (TBARS) in EtOH/HCl-damaged gastric mucosa in rats; it also exhibited an important antioxidant activity in vitro. Therefore, SP, derived from the red alga Laurencia obtusa, may have a potential therapeutic effect against acute myeloid leukaemia and a beneficial potential as gastroprotective and antioxidant natural product.

Synthesis and properties of highly soluble branched polyimide based on 2,4,6-triaminopyrimidine

A series of novel soluble polyimides was synthesized from commercially available 2,4,6-triaminopyrimidine (TAP), 2,2-bis(3,4-dicarboxylphenyl) hexafluoropropane dianhydride (6FDA), α,ω-aminopropylpoly(dimethylsiloxane) and 1,3-bis(3-aminophenoxy)benzene. TAP is the branched monomer, and polymerization process had been improved in three ways by using TAP to prepare soluble polyimides. The polyimides were characterized by Fourier transform infrared spectra, proton nuclear magnetic resonance, ultraviolet–visible spectrometer analysis, wide-angle X-ray diffraction, and size-exclusion chromatography with multiangle laser light-scattering detection. The branched polyimides showed excellent solubility both in strong polar solvents and in common low-boiling point solvents. Differential scanning calorimetric and thermogravimetric analyses showed high glass transition temperatures and excellent thermal stability for branched polyimides with moderate content of TAP. Polyimide membranes were formed at relatively low temperature, and the mechanical properties were tested. These results ensure that soluble polyimides with moderate content of TAP showed outstanding combined features and are desirable candidate materials for advanced applications.

Synthesis, characterization and properties of TAP-6FDA hyperbranched polyimides with different branching degrees

A series of hyperbranched polyimides were successfully synthesized by condensation polymerization of A2-type dianhydride monomer 2,2-bis(3,4-dicarboxylphenyl) hexafluoropropane dianhydride (6FDA) and B′B2-type triamine monomer 2,4,6-triaminopyrimidine (TAP). Polymers with different branching degrees (DB) and terminated groups were obtained by changing the monomer addition order and the monomer molar ratio. Fourier transform infrared spectroscopy and 1H NMR were used to verify the structure of the prepared polyimides, which indicated that the amino group still existed in all the products. The DB of the polymers indicated by 1H NMR increased from 30% to 79% with the molar ratio of TAP:6FDA decreasing from 1:1 to 1:2. The absolute molecular weights were measured by size-exclusion chromatography with multi-angle laser light-scattering detection, which suggested that the highest molecular weight would be obtained when the molar ratio of amino groups:anhydride groups of the monomers was 3:3.2. With the DB increasing, the d-spacing values indicated by wide angle X-ray diffraction increased from 5.15 Å to 5.68 Å and the UV − visible spectra of the polymers exhibited decreasing cut-off wavelengths. The 5% weight loss temperature in nitrogen increased with decreasing content of TAP monomer, and the glass transition temperatures of the obtained polyimides decreased from 282 °C to 258 °C with increasing DB. © 2013 Society of Chemical Industry

Composition and Molecular Size of Clostridium botulinum Type A Toxin–Hemagglutinin Complex

Pickett and Perrow have expressed several concerns regarding our recent publication on the stoichiometry of the Clostridium botulinum type A-Hall (Allergan) neurotoxin complex [1, 2]. These concerns demonstrate a misunderstanding regarding the purpose of our publication, which was focused on determining the subunit composition and homogeneity of this complex. Although the results in our publication were obtained with the C. botulinum type A neurotoxin complex used to manufacture BOTOX , it was not our goal to compare various type A neurotoxin complex products currently in clinical use. Based on current literature, knowledge regarding the number of each subunit present in the intact C. botulinum type A neurotoxin complex is extremely limited, even though the complex was purified to homogeneity more than 50 years ago [3, 4]. To date multiple reports exist in the literature suggesting that the 900 kDa complex is a dimer of a 500 kDa complex although there is limited evidence to support this [5, 6]. We established a quantitative procedure to determine the relative subunit composition and to confirm the molecular mass of our neurotoxin complex. As discussed in detail in our publication, we ensured that the established procedure was appropriate for this purpose by verifying both the recovery of the complex subunits as well as the overall consistency of the method. In addition, we clearly described our assumptions regarding the interpretation of the electropherograms, including our approach for handling the co-elution of the HA48 and the light chain subunits. We are, therefore, confident that our capillary electrophoresis method provides a valid procedure for quantifying the subunit stoichiometry of the neurotoxin complex. In contrast, sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS–PAGE) combined with mass spectrometry, as suggested by Pickett and Perrow, would not be suitable for this purpose since such an approach would only provide the molecular mass and the identity of proteins, and provide little if any information regarding the subunit stoichiometry. Analytical methods that evaluate the neurotoxin complex under denaturing conditions, including the capillary electrophoresis method described by us, cannot be used to establish whether the subunits form a single protein complex. We therefore, confirmed the integrity of the neurotoxin complex by size-exclusion HPLC in conjunction with multi-angle laser light-scattering detection. The molecular mass obtained for the neurotoxin complex shows that the different subunits identified in the electropherograms are part of a single complex and the results support the proposed subunit composition. The close agreement between the neurotoxin complex molecular mass values obtained by us and the complex mass values reported in earlier publications, while perhaps ‘‘unremarkable’’, confirms that the Allergan type A neurotoxin complex used to manufacture BOTOX consists of a homogeneous *900 kDa species containing one 150 kDa neurotoxin molecule per complex.

Applications of Multi-Angle Laser Light-Scattering Detection in the Analysis of Peptides and Proteins

The proliferation of new peptides and proteins requiring characterisation is a direct result of recent advances in genomics and proteomics, but protein aggregation is particular problem in the biotechnology industry, where aggregation is encountered throughout the lifetime of a therapeutic protein, including during refolding, purification, sterilization, shipping, and storage process. To ensure that it meets quality standards, the size, molecular weight and/or molecular weight distribution, and aggregate state must be accurately determined. Traditional analytical methods for determining molecular weight include size-exclusion chromatography (SEC), gel electrophoresis, analytical ultracentrifugation and time-of-flight mass spectrometry. These technologies are time-consuming (some take days), provide data based on relative standards, or cannot characterise very high molecular weight aggregates. Laser light-scattering (LS) detection coupled with SEC system have been used for over a decade to determine the size and molecular weight of bio-molecules such as proteins, peptides, polysaccharides, oligonucleotides, and antibodies, the method of choice being for molar mass determinations and the study of self-association and heterogeneous interaction under native, equilibrium conditions in solution. The purpose of the current review is to describe and discuss the capability of the SEC/LS system to determine absolute molecular weight of proteins and their complexes and the association state of the conjugate, either with itself or with protein receptor/ligands. For this, the "two or three detector" methods, each with its advantages and limitations, can be used to calculate the molecular weight of a simple protein or glycoprotein, and the stoichiometry of their complexes. Also, some alternative techniques for determining the molecular weight are discussed in this review. Applications of all these methodologies are described.

Measurement of uncertainty in peptide molecular weight determination using size-exclusion chromatography with multi-angle laser light-scattering detection and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The molecular weight of biopolymers such as peptides or proteins is vital information for understanding their physical/chemical properties. The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and size-exclusion chromotography–multi-angle laser light scattering (SEC–MALLS) techniques each with its advantages and limitations were used for molecular weight determination of the SPf66 peptide. The precision of each method was studied using a two-factor fully-nested design with all the analyses performed by the same operator on a single instrument. The overall precision corresponded to the time-different intermediate precision (i.e. time and repeatability conditions). In the case of SEC–MALLS, all potential uncertainty components were carefully evaluated in an Ishikawa diagram, then included and mathematically combined with the uncertainty arising from the accuracy assessment to provide the overall uncertainty. In this case, the refractive-index increment with solute concentration value (d n/d c) provided the most significant contribution to the combined uncertainty. A method for its quantitation is proposed. The accuracy of method B (SEC–MALLS) against reference method A (MALDI-TOF-MS) was assessed using the interval testing hypothesis to limit the risk of unacceptable bias. The results indicate that the bias of B is higher than the limit established at 5%, and is therefore not traceable to A under the studied conditions.

Characterization of antimalarial SPf66 peptide using MALDI–TOF MS, CD and SEC

SPf66 is the first chemically synthesized peptide to elicit a partial protective immune response against malaria. Size-exclusion chromatography (SEC) with multi-angle laser light-scattering (MALLS) detection and hydrogen/deuterium (H/D) exchange monitored by (matrix-assisted laser desorption/ionization) MALDI–TOF (time-of-flight) mass spectrometry (MS) were used to assess the conformation and stability in aqueous solution after storage at different temperatures. Moreover, the feasible conformational changes of this peptide were also measured by circular dichroism (CD)-spectroscopy. The absolute molecular weight of SPf66 monomer and dimer species were 4765 and 8960 Da using SEC with MALLS detection, and 4643 and 9490 Da by MALDI–TOF MS, the discrepancy being between both methods lower than 5.7%, a value quite close to those found in other proteins. The results from H/D exchange monitored by MALDI–TOF MS and CD-spectroscopy show that the SPf66 monomer lacks ordered structure, whereas the SPf66 dimer species presents segments of secondary structure as a determined by CD measurements.

Characterization of submicrometer aqueous iron(III) colloids formed in the presence of phosphate by sedimentation field flow fractionation with multiangle laser light scattering detection.

Iron colloids play a major role in the water chemistry of natural watersheds and of engineered drinking water distribution systems. Phosphate is frequently added to distribution systems to control corrosion problems, so iron-phosphate colloids may form through reaction of iron in water pipes. In this study, sedimentation field flow fractionation (SdFFF) is coupled on-line with multiangle laser light scattering (MALLS) detection to characterize these iron colloids formed following the oxygenation of iron(II) in the presence of phosphate. The SdFFF-MALLS data were used to calculate the hydrodynamic diameter, density, and particle size distribution of these submicrometer colloids. The system was first verified with standard polystyrene beads, and the results compared well with certified values. Iron(III) colloids were formed in the presence of phosphate at a variety of pH conditions. The colloids' hydrodynamic diameters, which ranged from 218 +/- 3 (pH 7) to 208 +/- 4 nm (pH 10), did not change significantly within the 95% confidence limit. Colloid density did increase significantly from 1.12 +/- 0.01 (pH 7) to 1.36 +/- 0.02 g/mL (pH 10). Iron(III) colloids formed at pH 10 in the presence of phosphate were compared to iron(III) colloids formed without phosphate and also to iron(III) colloids formed with silicate. The iron(III) colloids formed without phosphate or silicate were 0.46 g/mL more dense than any other colloids and were >6 times more narrowly distributed than the other colloids. The data suggest competitive incorporation of respective anions into the colloid during formation.