Size Exclusion Chromatography/multi-angle Laser Light Scattering Research Articles

Use of Multi-Angle Laser Light Scattering and Size-Exclusion Chromatography to Characterize the Molecular Weight and Types of Aggregates Present in Commercial Whey Protein Products

In this study, a range of commercial whey protein products were characterized by the use of size-exclusion chromatography coupled with a multi-angle laser light scattering (MALLS) detector. The MALLS system detected some very large-sized material that eluted close to void volume in all samples; this material was hardly detected by concentration detector. It was demonstrated by chitosan treatment that this peak was very small lipid globules or phospholipids, which gave the residual “cloudy” appearance in upper layers after ultracentrifugation of whey products. Composition, molecular weight, and the photo diode array (PDA) spectrum (200 to 400nm) of the major protein peaks, including: β-lactoglobulin (BLG), α-lactalbumin (ALA), bovine serum albumin (BSA), immunoglobulin G (IgG) and some minor components were analyzed. The molecular weight of BLG, ALA, BSA, and IgG peaks in whey protein isolates (WPI) were 2.3 to 3.7×104, 1.4 to 1.6×104, 4.8 to 6.7×104, and 1.2 to 2.5×105 Da, respectively. Compared with WPI, WPC has similar major proteins, but more large-sized residual lipid material and different minor constituents such as lactose and nonprotein nitrogen, depending on various commercial samples and protein content. An improved TCA-precipitation method was applied to quantify glycomacropeptide (GMP) in whey proteins, which demonstrated that there was a very low concentration of GMP in WPI manufactured using an ion-exchange process. The molecular weight of GMP was found to be ∼8600Da. Size-exclusion chromatography MALLS was demonstrated to be a powerful technique for detailed analysis of the molecular weight of various proteins, aggregates and minor components, such as GMP, in whey protein products.

Hydrodynamic characterisation of chemically degraded hyaluronic acid

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.

Comparison of Molecular Weight and Molecular Weight Distributions of Softwood and Hardwood Lignosulfonates

Lignosulfonates obtained from spruce (Picea abies), aspen (Populus sp.) and two species of Eucalyptus (E. globulus and E. grandis) were characterized by aqueous size exclusion chromatography (SEC) combined with in-line multi-angle laser light scattering (MALLS). In general, the hardwood lignosulfonates were shifted to lower molecular weights (M w = 5.700–12.000 g/mol) as compared to softwood lignosulfonates (M w = 36.000–61.000 g/mol). Lignosulfonates from E. grandis were further fractionated to obtain fractions of different molecular weights (3.500–30.000 g/mol). The degree of sulfonation increased with decreasing M w for the fractions as previously found for fractions of spruce lignosulfonate (Fredheim, G.; Braaten S.M.; Christensen, B.E. Molecular weight determination of lignosulfonates by size exclusion chromatography and multi-angle laser lightscattering. J. Chromatogr. 2002, 942, 191–199). The relationship between the intrinsic viscosity (in 0.1 M NaCl) and molecular weight was essentially the same for spruce and E. grandis lignosulfonate fractions, with an estimated Mark–Houwink–Sakurada (MHS) exponent of 0.36. This value, combined with the low intrinsic viscosities, confirms that lignosulfonates are compact structures in aqueous solution. Based on the SEC–MALLS results a SEC-method using a UV-detector (SEC–UV) was developed, where lignosulfonate fractions were used as broad molecular weight calibration standards.

Further characterization of a high molecular weight glycoprotein antigen from the yeast Saccharomyces cerevisiae

A high molecular weight glycoprotein antigen was isolated by size exclusion chromatography on Sepharose 4B from an extract of the yeast Saccharomyces cerevisiae. The glycoprotein antigen Sc 500 was shown to be identical to the antigen termed gp200 previously isolated (Heelan et al., 1991). The MW of Se 500 was determined to be about 500 kDa by size exclusion chromatography on Superose 6 and 460 kDa ± 20k Da by size-exclusion chromatography/multi-angle laser light scattering (SEC/MALLS). Sc 500 contained 90% mannose and traces of N-acetylglucosamine. The amino acid composition revealed that serine and threonine were the most abundant amino acids of the protein part. By alkaline borohydride treatment some, but not all bonds between protein and carbohydrate were broken. This indicates that the main type of linkage between protein and carbohydrate is O-glycosidic and that a minor type is of N-glycosidic nature. Methylation analysis revealed that the mannose residues were connected by 1 → 2 and 1 → 3 linkages with 1 → 2, 1→ 6 linked branch points. Purified Sc 500 was subjected to a series of chemical and enzymatic modifications followed by studies of antibody binding activity. Treatments with both periodate and alkaline sodium borohydride reduced the human serum IgA, IgG and monoclonal IgM antibody binding activity of Sc 500 whereas trypsin and pronase did not affect its ability to bind these antibodies. The mannosidase Manα1 → 2,3,6Man reduced the IgM binding to Sc 500 while the other enzymes included in this experiment (Manα1→2 Man, Manβ1 →4GlcNAc and PNGase F) had no effect on the antibody binding.