Isolation and Self-Association Studies of Beta-Lactoglobulin

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...

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Bibliographic Details
Published in:	International journal of molecular sciences 2020-12, Vol.21 (24), p.9711
Main Authors:	Gołębiowski, Adrian, Pomastowski, Paweł, Rodzik, Agnieszka, Król-Górniak, Anna, Kowalkowski, Tomasz, Górecki, Marcin, Buszewski, Bogusław
Format:	Article
Language:	eng
Subjects:	Ammonium Ammonium sulfate Animals asymmetric flow field flow fractionation (AF4) b-lactoglobulin Capillary electrophoresis Cattle Chromatography Circular dichroism Citric acid Column chromatography Dichroism Dimers Dispersion Electrophoresis Electrophoresis, Capillary - methods Fractionation Hydrogen-Ion Concentration Investigations Ions Isoforms Lactoglobulin Lactoglobulins - chemistry Lactoglobulins - isolation & purification Lactoglobulins - metabolism Light scattering Mass spectrometry Mass Spectrometry - methods Mass spectroscopy oligomeric forms Oligomerization Oligomers pH effects Potassium chloride Protein Multimerization protein stability Proteins Saline solutions Self-association Sodium chloride Sodium citrate Solvents Stability Whey Whey protein Whey Proteins - chemistry β-lactoglobulin (β-LG)
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Summary:	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.
ISSN:	1422-0067 1661-6596 1422-0067