Baric Oligomerization in α-Lactalbumin/β-Lactoglobulin Mixtures

Abstract

The influence of high pressure on α-lactalbumin(ALA)/β-lactoglobulin (BLG) mixtures of various compositions was studied at pH 8.5 (50 mM Tris) by gel-permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) without 2-mercaptoethanol. High-molecular-weight disulfide-linked protein oligomers formed at a pressure of 10 kbar (1000 MPa) if the weight fraction of BLG (W BLG 0) in the protein mixture exceeded 0.2. The maximum yield of these oligomers, of the order 80−85%, was observed at W BLG 0 ≥ 0.4. Conversion of both proteins in the oligomers was roughly the same. The estimates of the oligomerization yield obtained by gel-permeation chromatography and SDS-PAGE agreed well. SDS-PAGE results indicated that the formation of intermolecular disulfide bonds is necessary for the oligomerization. Thus, the oligomerization of pressure-denatured ALA and BLG is driven by thiol ↔ disulfide exchange in which the free thiol group of BLG acts as an initiator. Keywords: High pressure; α-lactalbumin; β-lactoglobulin; protein aggregation; gel-permeation chromatography; SDS-PAGE; intermolecular disulfide bonds; thiol ↔ disulfide exchange

Changes in structure and functional properties of whey proteins induced by high hydrostatic pressure: A review

“…α-LA forms a MG state under a variety of conditions, including at low pH, at low salt concentrations in the absence of Ca 2+ (neutral pH) [68][69][70], or after pressure treatment [25,46,71]. However, the stability of α-LA towards high pressure is greater than that of β-LG, probably due to the lack of free sulfhydryl groups in α-LA [46,73].…”

“…This renaturation effect is not observed in WPC, which might be due to heat-induced interactions with β-LG and BSA. In the mixture of α-LA and β-LG, during HHP treatment (1000 MPa, 30 min), β-LG promoted the oligomerization of α-LA [73]. In this case, mixing and denaturation of β-LG with α-LA resulted in the formation of a large heterogeneous population of oligomers including β-LG or α-LA/β-LG dimmers [74].…”

Influence of stabilizing bonds on the texture properties of high-pressure-induced whey protein gels

“…Thus whey protein denaturation and gelation was studied under various conditions and in systems like milk (Felipe, Capellas, & Law, 1997a;Felipe, Gervilla, Guamis, & Law, 1997b;Law et al, 1998;Arias, Lopez-Fandino, & Olano, 2000;Hinrichs, 2000;Scollard, Beresford, Needs, Murphy, & Kelly, 2000), whey protein concentrate (WPC) (Camp van, Messens, Clement, & Huyghebaert, 1997b;Johnston, Rutherford, & McCreedy, 2002), whey protein isolate (WPI) (Hinrichs, Rademacher, & Kessler, 1996;Hinrichs, 2000;Michel et al, 2001), and isolates of the individual whey proteins (Dumay, Kalichevsky, & Cheftel, 1994;Funtenberger, Dumay, & Cheftel, 1995;Jegouic, Grinberg, Guingant, & Haertle, 1996;Galazka, Ledward, Summer, & Dickinson, 1997;Jegouic et al, 1996;Olsen, Ipsen, Otte, & Skibsted, 1999;Grinberg & Haertle, 2000). The degree of denaturation was determined as solubility at pH 4.6, either as the amount of total non-soluble protein (Funtenberger et al, 1995;Camp van et al, 1997b) or by measuring the individual whey protein fractions and aggregates by means of RP-HPLC (Hinrichs et al, 1996;Arias et al, 2000;Scollard et al, 2000;Hinrichs, 2000;Michel et al, 2001), gel permeation chromatography (Dumay et al, 1994;Jegouic et al, 1996;Camp van et al, 1997b;Felipe, Capellas, & Law, 1997a, b;Jegouic et al, 1997;Law et al, 1998;Grinberg & Haertle, 2000) or S...…”

“…Denaturation of b-lactoglobulin runs faster than a-lactalbumin. However, the denaturation of the pressure resistant a-lactalbumin is accelerated when adding low-molecular thiol reducers (cystein, mercaptoethanol, DTT) or b-lactoglobulin (Jegouic et al, 1996(Jegouic et al, , 1997Grinberg & Haertle, 2000).…”

“…Intermolecular disulfide bonds can be formed through oxidation of thiol groups and/or thiol-disulfide exchange (Funtenberger et al, 1995;Galazka et al, 1997;Iametti et al, 1997;Dumay et al, 1998;Famelart et al, 1998). b-lactoglobulin can also react with a-lactalbumin via thiol-disulfide exchange (Jegouic et al, 1997;Grinberg & Haertle, 2000). The contribution of disulfide bonds to the aggregation of whey proteins was well examined by addition of thiol blocking agents (NEM, DTNB) before pressure treatment (Camp van & Huyghebaert, 1995a;Tanaka, Tsuri, Kobayashi, & Kunugi, 1996;Iametti et al, 1997), and comparative SDS-PAGE with and without mercaptoethanol as reducing agent (Johnston & Murphy, 1995;Tanaka et al, 1996;Camp van and Huyghebaert, 1995b;Kanno et al, 1998).…”