Multi-angle Laser Light Scattering Analysis Research Articles

Molecular Weight Determination of Aloe Polysaccharides Using Size Exclusion Chromatography Coupled with Multi-Angle Laser Light Scattering and Refractive Index Detectors

Background: Size exclusion chromatography (SEC)/refractive index (RI) were used to determine molecular weight (MW) and molecular weight distributions (MWD) of polysaccharides. In aloe product research and quality control, commercially available pullulan and dextran are most commonly employed as calibration standards. Significant difference in the MW and MWD were found in literature when different methods were used. Objectives: This study was to investigate the traditional methods and more recent technologies used to determine the MW and MWD of Aloe vera polysaccharides. Methods: In this study, multi-angle laser light scattering (MALS) detection was studied on three polysaccharides, 1, 2, and 3, that were isolated and purified from A. vera leaf. The chemical structures of 1-3 were characterized as 1, 4-β-linked glucomannans by monosaccharide composition and glycosidic linkage analysis. Absolute MW and root-mean-square radius were determined by MALS on the isolated aloe polysaccharides. The conditions to obtain reliable results from MALS measurement were examined. Results: MALS analysis demonstrates that the 1, 4-β-linked glucomannan adopt the conformation of random coils or hard spheres in the analytical environment of a 0.1 M NaCl solution. Non-size exclusion effects and interactions between polysaccharide molecules were also observed in some aloe polysaccharides in the current analysis. The weight-average MW obtained by MALS measurement for 1, 2, and 3 are 55, 129, and 962 kDa, respectively. Comparing the results with SEC/RI calibrated by pullulan and dextran standards, marked differences in the MWD are found. Both overestimated the MW of 1 and 2 by factors of 4.4 and 4.2, and 2.4 and 1.6, when using dextran and pullulan calibration, respectively. Using pullulan calibration underestimated the MW of 3 by a factor of 3.1, but a similar result was obtained from dextran calibration compared to MALS measurement. The two isolated aloe polysaccharides were employed to be broad calibration standards or to be combined with narrow polydispersity pullulan calibration standards. Several aloe samples were tested using the different calibration curves, and the determined MWs were compared with the results obtained by MALS measurement. Conclusions: The results clearly indicated that until true polysaccharide standards become available MW and MWD's will be simply relative to the standards employed and the technologies used.

Isolation, structural characterization and immunological activity of an exopolysaccharide produced by Bacillus licheniformis 8-37-0-1

A strain of Bacillus licheniformis 8-37-0-1 with high exopolysaccharide (EPS) production ability was isolated and identified based on morphological and physiological characteristics and phylogenetic analysis of 16S rDNA sequences. A new type of EPS was isolated from the strain fermentation broth by enzymolysis, isopropanol precipitation, anion-exchange, and gel-filtration chromatography. The new EPS was determined as homogeneous, with a molecular weight of 2.826 × 10 4, as determined by High-Performance Size-Exclusion Chromatography Multi-Angle Laser Light Scattering analysis. Its structural characteristics were investigated and elucidated by methylation analysis, partial acid hydrolysis, gas–liquid chromatography mass spectrometry, Fourier transform infrared, and nuclear magnetic resonance spectroscopy. Based on obtained data, the EPS was found to be a levan containing a (2 → 6)-linked backbone with a single β- d-fructose at the C-1 position every seven residue, on average, along the main chain. Preliminary in vitro tests revealed that EPS could significantly stimulate the proliferation of spleen lymphocyte.

Heme Ligand Binding Properties and Intradimer Interactions in the Full-length Sensor Protein Dos from Escherichia coli and Its Isolated Heme Domain

Dos from Escherichia coli is a bacterial gas sensor protein comprising a heme-containing gas sensor domain and a phosphodiesterase catalytic domain. Using a combination of static light scattering and gel filtration experiments, we established that, as are many other sensor proteins, the full-length protein is dimeric. The full-length dimer (association constant <10 nm) is more stable than the dimeric heme domain (association constant approximately 1 mum), and the dimer interface presumably includes both sensor and catalytic domains. Ultrafast spectroscopic studies showed little influence of the catalytic domain on kinetic processes in the direct vicinity of the heme. By contrast, the properties of ligand (CO and O(2)) binding to the heme in the sensor domain, occurring on a microsecond to second time scale, were found to be influenced by (i) the presence of the catalytic domain, (ii) the dimerization state, and in dimers, (iii) the ligation state of the other subunit. These results imply allosteric interactions within dimers. Steady-state titrations demonstrated marked cooperativity in oxygen binding to both the full-length protein and the isolated heme domain, a feature not reported to date for any dimeric sensor protein. Analysis of a variety of time-resolved experiments showed that Met-95 plays a major role in the intradimer interactions. The intrinsic binding and dissociation rates of Met-95 to the heme were modulated approximately 10-fold by intradimer and sensor-catalytic domain interactions. Dimerization effects were also observed for cyanide binding to the ferric heme domains, suggesting a similar role for Met-95 in ferric proteins.

Rice Starch, Amylopectin, and Amylose: Molecular Weight and Solubility in Dimethyl Sulfoxide-Based Solvents

Dimethyl sulfoxide (DMSO), with either 50 mM LiBr, 10% water, or both, was used as solvent for multi-angle laser-light scattering (MALLS) batch mode analysis of rice starch, and amylopectin and amylose weight-average molecular weight (Mw). DMSO/50 mM LiBr was a better solvent for these measurements than was DMSO/10% water, based on this solvent's ability to dissolve starch and to reduce the size of starch aggregates. Starch concentration decreased and amylose:amylopectin ratio increased when starch suspended in DMSO was centrifuged or filtered prior to size-exclusion chromatography (SEC)-MALLS analysis. A higher amylose:amylopectin ratio made starch more soluble, and the higher this ratio, the lower the Mw of eluted amylopectin. For SEC analysis of Mw, fractions of starch amylopectin and amylose dispersed in DMSO-based solvents yielded better results than starch dispersed directly into the solvents, because dispersion of these fractions decreased starch aggregation. When these two starch components were fractionated and then dissolved separately in DMSO/50 mM LiBr, the Mw of dispersed amylopectin ranged from 40 to 50 million, and that of amylose was ca. 3 million, whereas starch from three rice varieties of varying amylose content ranged from 60 to 130 million. We recommend that SEC evaluation of amylopectin and amylose be accomplished with fractionated samples as in this study; such evaluations were superior to evaluations of natural mixtures of amylopectin and amylose.

Evaluation and comparison of commercially available Aloe vera L. products using size exclusion chromatography with refractive index and multi-angle laser light scattering detection

Raw materials supplied as Aloe vera L. (sometimes referred to as Aloe barbadensis) samples often contain different composition of low and high molecular weight components when analyzed by size exclusion chromatography. One major reason for variable compositions of commercial A. vera L. materials is that they are produced by different manufacturing techniques. Consistent composition of matter based upon a given standard has been difficult to define. In addition, the method of quantifying and characterization of these commercially available materials has not been agreed upon within the industry. The end user, whether a researcher, a manufacturer, a marketing arm of industry or the consumer, should know that they are receiving a consistent product. A blind study of 32 various A. vera L. samples from different manufacturers, and a prepared sample of fresh A. vera L. gel with the commercial, biologic drug Acemannan Immunostimulant™, were analyzed for content of high molecular weight (polysaccharides) material by size exclusion chromatography with refractive index detection (SEC/RI) and SEC/RI coupled with multi-angle laser light scattering (MALLS) detection. Results from the SEC/RI analysis showed significant variation in the high molecular weight content, and the MALLS analysis also showed significant variation versus SEC/RI. In addition, HPLC analysis of the anthraquinone content showed that all samples contained significantly less than that of the raw, unwashed aloe gel. The variation of results from all analysis is attributed to differing methods in which the samples were processed by the different manufacturers.

Factors Influencing the Characterization of Gluten Proteins by Size‐Exclusion Chromatography and Multiangle Laser Light Scattering (SEC‐MALLS)

ABSTRACTThe use of multiangle laser light scattering (MALLS) in conjunction with size exclusion chromatography (SEC) was investigated for characterizing wheat proteins. Four solvent systems including 50% acetonitrile and 0.1% trifluoroacetic acid, 50 mM sodium phosphate (NaPhos) pH 2.5, 500 mM acetic acid, and 50 mM NaPhos pH 7.0 + 1% SDS were evaluated for protein extraction as well as for use as SEC mobile phases for MALLS analysis. The dn/dc values for wheat proteins were measured in each solvent. Except for the SDS‐based mobile phase, gluten proteins showed dn/dc values of 0.16–0.20 that were similar to values reported for other proteins. When analyzed in the SDS solvent, gluten proteins showed dn/dc values of 0.32, similar to that found for other SDS‐protein complexes. While all solvents showed similar resolution when used as mobile phases in SEC analysis, the SDS solvent extracted the most protein (≈82%) in the unreduced form. This solvent system also displayed no concentrationdependent or electrostatic effects during MALLS analysis. SDS‐soluble and insoluble protein complexes were characterized by MALLS. Mw distributions of 8.1 × 107 Da were found for the SDS‐insoluble protein complexes. The effect of the column void volume was also examined as was data analysis parameters such as fitting method and peak placement.

Molecular Characterization of Wheat Amylopectins by Multiangle Laser Light Scattering Analysis

ABSTRACTWeight‐average molecular weight (Mw) and chain length of eight amylopectins isolated from one Australian, two United States, and five Korean wheats were measured using multiangle laser light scattering (MALLS) and refractive index (RI) detectors operated in a microbatch mode, and in a high‐performance size‐exclusion chromatography (HPSEC) mode. The Mw of amylopectins measured in the microbatch mode ranged from 29 × 106 to 349 × 106. Three amylopectins (Geuru, Tapdong and WW) showed significantly high Mw values over 200 × 106. The Mw measured by HPSEC mode with MALLS‐RI detectors (42 × 106 to 73 × 106) were significantly less than those obtained in the microbatch mode with exception of dark northern spring hard wheat (DNS) amylopectin, indicating the possible variation of Mw by the analysis mode. Root‐mean square of the radius of gyration (Rg) also was greater when the microbatch mode was used (122–340 vs. 95–116 nm). Chain length distributions of debranched amylopectins of different cultivars, measured by the HPSEC‐MALLS‐RI system, were similar. Weight average degrees of polymerization (DPw) of A, B1, and larger B chains (B≥2) had ranges of 13–22, 26–46, and 58–73, respectively, and mass ratios of A and B chains ranged from 0.7 to 1.1.

Quaternary structure of the extracellular haemoglobin of the lugworm Arenicola marina: a multi-angle-laser-light-scattering and electrospray-ionisation-mass-spectrometry analysis.

To elucidate the quaternary structure of the extracellular haemoglobin (Hb) of the marine polychaete Arenicola marina (lugworm) it was subjected to multi-angle laser-light scattering (MALLS) and to electrospray-ionisation mass spectrometry (ESI-MS). It was also subjected to SDS/PAGE analysis for comparative purposes. MALLS analysis gave a molecular mass of 3648 +/- 24 kDa and a gyration radius of 11.3 +/- 1.7 nm. Maximum entropy analysis of the multiply charged electrospray spectra of the native, dehaemed, reduced and carbamidomethylated Hb forms, provided its complete polypeptide chain and subunit composition. We found, in the reduced condition, eight globin chains of molecular masses 15952.5 Da (a1), 15974.8 Da (a2), 15920.9 Da (b1), 16020.1 Da (b2), 16036.2 Da (b3), 16664.8 Da (c), 16983.2 Da (d1), 17033.1 Da (d2) and two linker chains L1, 25174.1 Da, and L2, 26829.7 Da. In the native Hb, chains b, c, d occur as five disulphide-bonded trimer subunits T with masses of 49560.4 Da (T1), 49613.9 Da (T2), 49658.6 Da (T3), 49706.8 Da (T4), 49724.5 Da (T5). Linker chains L1 and L2 occur as one disulphide-bonded homodimer 2L1 (D1) of 50323.1 Da and one disulphide-bonded heterodimer L1-L2 (D2) of 51 981.5 Da. Polypeptide chains a and d possess one free cysteine residue and chains d possess an unusual total of five cysteine residues. Semi-quantitative analysis of ESI-MS data allowed us to propose the following model for the one-twelfth protomer: [(3a1)(3a2)2T] (T corresponding to either T3, T4 or T5). From electron micrograph data T1 and T2 are probably located at the centre of the molecule as mentioned in previous studies. The Hb would thus be composed of 198 polypeptide chains with 156 globin chains and 42 linker chains, each twelfth being in contact with 3.5 linker subunits, providing a total mass of 3682 kDa including haems in agreement with the experimental molecular mass determined by MALLS. From ESI-MS relative intensities and the model proposed above, the globin/linker ratio gave 0.71:0.29 and 0.73:0.27, respectively. The estimation of haem content by pyridine haemochromogen and by cyanmethaemoglobin (HiCN) methods also support the globin chain number provided by ESI-MS.

Absolute on-line molecular mass analysis of basic fibroblast growth factor and its multimers by reversed-phase liquid chromatography with multi-angle laser light scattering detection

The multi-angle laser light scattering (MALLS) detection method was combined with reversed-phase high-performance liquid chromatography to analyze multimerization of basic fibroblast growth factor (bFGF) formed by oxidation of bFGF with air or with 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB). This analysis provided the absolute molecular mass and the mean square radius for each eluted protein fraction of each slice of the chromatogram. It was shown that depending on the oxidation conditions, bFGF forms different multimetric forms, from dimers to hexamers. It was found that these multimers have varied conformations of the same molecular mass, but different structure. Molecular mass and size analyses provided molecular conformation of the aggregates; the results indicated the formation of rod-like rigid structures. The MALLS analysis confirmed that, during oxidation, each bFGF monomer bound sequentially to form the extended multimer. The proposed scheme of bFGF oxidation with DTNB revealed that the difference in the aggregate structural forms was probably due either to the presence of covalently bound residues of nitrobenzoic acid in the products of oxidation, or to the participation of sulfhydryl groups in disulfide bond formation.