Isolation and Self-Association Studies of Beta-Lactoglobulin

Abstract

The aim of this study was to investigate isolated β-lactoglobulin (β-LG) from the whey protein isolate (WPI) solution using the column chromatography with SP Sephadex. The physicochemical characterization (self-association, the pH stability in various salt solutions, the identification of oligomeric forms) of the protein obtained have been carried out. The electrophoretically pure β-LG fraction was obtained at pH 4.8. The fraction was characterized by the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/TOF MS) technique. The use of the HCCA matrix indicated the presence of oligomeric β-LG forms, while the SA and DHB matrices enabled the differentiation of A and B isoforms in the sample. The impact of sodium chloride, potassium chloride, ammonium sulfate, and sodium citrate in dispersion medium on β-LG electrophoretic stability in solution was also studied. Type of the dispersion medium led to the changes in the isoelectric point of protein. Sodium citrate stabilizes protein in comparison to ammonium sulfate. Additionally, the potential of capillary electrophoresis (CE) with UV detection using bare fused capillary to monitor β-LG oligomerization was discussed. Obtained CE data were further compared by the asymmetric flow field flow fractionation coupled with the multi-angle light scattering detector (AF4-MALS). It was shown that the β-LG is a monomer at pH 3.0, dimer at pH 7.0. At pH 5.0 (near the isoelectric point), oligomers with structures from dimeric to octameric are formed. However, the appearance of the oligomers equilibrium is dependent on the concentration of protein. The higher quantity of protein leads to the formation of the octamer. The far UV circular dichroism (CD) spectra carried out at pH 3.0, 5.0, and 7.0 confirmed that β-sheet conformation is dominant at pH 3.0, 5.0, while at pH 7.0, this conformation is approximately in the same quantity as α-helix and random structures.

Synthesis and physicochemical characterization of zinc-lactoferrin complexes

“…For instance, for A-type lipase of Candida antarctica pI value at 4 was determined by electrophoretic mobility, whereas the isoelectric focusing technique showed the pI value as 7.5 (Salgın et al, 2012). Moreover, the studies performed in our group have shown that depending on the ions present in the solution the determined pI values for β-LG were from 5.3 to 7.6 within the same technique (Gołębiowski et al, 2020). The study indicates the high importance of the effect of specific proteinion interactions on the obtained results.…”

Complex Protein Retention Shifts with a Pressure Increase: An Indication of a Standard Partial Molar Volume Increase during Adsorption?

“…Figure 5 shows that there is a moderate increase in the β-Lg negative charge with a pH value rise from 5.2 to 8.5. Unfortunately, contrary to lower pH values where the protein is present in a monomeric form, the dimeric form predominantly occurs in the discussed pH range, 28 resulting in a significantly different size and also surface charge distribution. For this reason, any differences in the retention trend could not be attributed solely to a change of interaction strength, but structural changes must also be considered, making a correct interpretation challenging.…”

“… 26 More importantly, it has a high dipole moment 27 and a broad isoelectric point between 4.8 and 5.9. 28 This enables retention on an anion and cation exchange column at certain mobile phase pH values within this range. The same conditions in the mobile phase and the same pressure (same standard partial molar volume in the mobile phase and charge distribution) enabled the study of the change in the retention time trend with pressure solely due to the differences in the adsorption processes.…”

“…It is a globular protein with 162 amino acids, of which 18 residues can exhibit a positive charge and 25, a negative charge, depending on the pH, while the remaining are always neutral . More importantly, it has a high dipole moment and a broad isoelectric point between 4.8 and 5.9 . This enables retention on an anion and cation exchange column at certain mobile phase pH values within this range.…”

The Study on Molecular Profile Changes of Pathogens via Zinc Nanocomposites Immobilization Approach

“…These values are obtained using the multi-angle light scattering (MALS) technique. Determining the radius of gyration, as well as the molar mass distribution of biocolloids and biopolymers, is an important issue in the field of physicochemical description of nanoparticles [29][30][31]. The applied analytical method allowed us to obtain ZnO particle size values before and after immobilization with ampicillin and tetracycline.…”