Isolation and Self-Association Studies of Beta-Lactoglobulin.

Marcin Górecki,Paweł Pomastowski,Tomasz Kowalkowski,Bogusław Buszewski,Agnieszka Rodzik,Adrian Gołębiowski,Anna Król-Górniak

doi:10.3390/ijms21249711

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.

Highlights

Β-LG is an important source of the essential and branched-chain amino acids
We investigated conformation of protein in these conditions by applying circular dichroism (CD) spectroscopy
Isolation of β-LG from whey protein isolate (WPI) Solution by Chromatographic Column β-lactoglobulin was isolated in a 0.2 M citrate buffer at the linear pH gradient by column chromatography

Summary

Introduction

Β-LG is an important source of the essential and branched-chain amino acids (leucine, isoleucine, and valine). WPC can be obtained as a result of modern membrane-based separation technologies such as ultra-filtration for protein concentration, diafiltration (DF) to remove lactose, minerals and low molecular weight compounds. Depending on their concentration, there are WPCs containing 35%, 50%, 65%, and 80% (w/w) of proteins. Β-LG isolation can be performed by many routes [14,15], e.g., the precipitation by denaturing agents (salts, acids, acetone, temperature) These approaches can lead to undesirable precipitation of other proteins. In solution β-LG exists in different oligomeric states, which depend on the pH, the ionic strength, temperature, and protein concentration [17]. In neutral pH conditions, the dimeric state is stabilized by hydrogen bonds between the surface AB loop and the anti-parallel β-sheet [27]

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