Stable monomeric intermediate with exposed Cys-119 is formed during heat denaturation of β-lactoglobulin

Abstract

The role of the free sulfhydryl group of beta-lactoglobulin in the formation of a stable non-native monomer during heat-treatment of beta-lactoglobulin solutions was investigated. Two concomitant events occurred at the earlier stage of heating: unfolding of native globular monomer and intramolecular sulfhydryl/disulfide exchange reaction. Thus, two denatured monomeric species were formed: a non-native monomer with exposed Cys-121 (Mcys121) which became reversible after cooling, and a stable non-native monomer with exposed Cys-119 (Mcys119) which exhibited both a larger hydrodynamic conformation than native monomer and low solubility at pH 4.7. The results also show that the formation of these monomeric species throughout heat-induced denaturation of native beta-lg monomers is faster than their subsequent aggregation. A mechanism describing the behavior of beta-lg denaturation/aggregation during heat-treatment under selected conditions (5.8 mg/ml, low ionic strength, pH 6.6, 85 degrees C) is presented.

Reversible Unfolding of Bovine β-Lactoglobulin Mutants without a Free Thiol Group

“…Irreversible denaturation has been thought to arise from thiol-disulfide exchange caused by the free thiol of Cys-121. Indeed, the formation of intermolecular disulfide bridges by heat denaturation at neutral pH has been reported previously (27)(28)(29)(30). Even at pH 2.1, an incomplete recovery of CD spectra was observed in samples treated with 7 M urea (22).…”

Nanotechnology and Food Allergy

“…It is present as a mixture of monomers and dimers at neutral pH, dissociating on heating to 70 ° C [37] and appears to adopt a partially folded state following thermal denaturation [38] , before forming thread -like aggregates around 50 nm in diameter [39] . Heat -induced unfolding of β -Lg reveals the buried Cys 121 , which is then able to catalyze disulfi de interchange with other disulfi des in β -Lg to form a non -native monomer in which Cys 119 is exposed [40] .…”

The Effect of Molecular Chaperone, Alpha-Crystallin, on the Heat-Induced Aggregation of Beta-lactoglobulin

“…Beta-lactoglobulin is a major whey protein of milk [1][2][3] , which at physiological pH values exists as a dimer [4,5] . Each monomer has a molecular weight of 18.3 kDa, contains 162 amino acid residues, two disulphide bonds (C106-C119 and C66-C160) and one free cysteine residue (C121) [6][7][8] .…”

“…Upon heating, betalactoglobulin dissociates into monomers, leading to exposure of hydrophobic groups and the free sulfhydryl group [9,10] . This then gives rise to aggregation and/or polymerization [4,5,9,11,12] . Beta-lactoglobulin is sensitive to heating at 60-100°C.…”

“…Beta-lactoglobulin is sensitive to heating at 60-100°C. During heating, the protein undergoes an initial dissociation of dimers followed by conformational changes which increase the exposure of previously buried hydrophobic groups and the thiol group [4,9,12,13,14] . Alpha-Crystallin is the major protein component of the mammalian eye lens, making up 50% of its dry weight [15,16] .…”