Proteolysis of bovine β‐lactoglobulin during thermal treatment in subdenaturing conditions highlights some structural features of the temperature‐modified protein and yields fragments with low immunoreactivity

Abstract

Bovine b-lactoglobulin was hydrolyzed with trypsin or chymotrypsin in the course of heat treatment at 55, 60 and 65°C at neutral pH. At these temperatures b-lactoglobulin undergoes significant but reversible structural changes. In the conditions used in the present study, b-lactoglobulin was virtually insensitive to proteolysis by either enzyme at room temperature, but underwent extensive proteolysis when either protease was present during the heat treatment. Hightemperature proteolysis occurs in a progressive manner. Mass spectrometry analysis of some large-sized breakdown intermediates formed in the early steps of hydrolysis indicated that both enzymes effectively hydrolyzed some regions of b-lactoglobulin that were transiently exposed during the physical treatments and that were not accessible in the native protein. The immunochemical properties of the products of b-lactoglobulin hydrolysis were assessed by using various b-lactoglobulin-specific antibodies, and most epitopic sites were no longer present after attack of the partially unfolded protein by the two proteases.Keywords: bovine b-lactoglobulin; limited proteolysis; partial unfolding; thermal treatment; reduced immunoreactivity.The globular protein b-lactoglobulin is found in the whey fraction of the milk of many mammals, but is absent from human milk. In spite of numerous physical and biochemical studies, its function is still not clearly understood [1,2]. The crystal structure of bovine b-lactoglobulin (BLG) shows a marked similarity with the plasma retinol binding protein and the odorant binding protein, that all belong to the lipocalin superfamily [2][3][4][5].Denaturation of BLG by physical means is a complex phenomenon, that occurs through a series of intermediate steps, whose kinetics and equilibrium depend on the treatment conditions, on the protein concentration, and on the interaction with other components when complex systems such as milk and whey are considered. Most of the steps occurring below a given intensity threshold of physical treatment (temperatures below 60-65°C, or pressures below 600 MPa [6,7]) are fully reversible in solutions of the pure protein at neutral pH. Transient BLG conformers are formed by either physical treatment in the same conditions, and the properties of these conformers have been investigated in some detail [7][8][9][10].Limited proteolysis represents a common and powerful tool for the investigation of protein structure, including transient conformational states of proteins generated during folding or unfolding (reviewed in [11]). This approach has not been popular for use with BLG in view of its structural toughness, which makes native BLG quite insensitive to most proteases under nondenaturing conditions [12][13][14][15][16], in particular at pH values lower than 7.5, where the wellknown Tanford transition of the protein structure occurs. Most proteolytic studies on unfolded BLG only addressed the products of severe thermal treatment, i.e. above the temperature threshold for irreversible structural modificat...

Hydrolysis of β-lactoglobulin by trypsin under acidic pH and analysis of the hydrolysates with MALDI–TOF–MS/MS

“…Compared to native b-Lg, these fragments had similar b-strands but no a-helices. Molinari et al (1996) found the same phenomenon while Iametti et al (2002) sub-denatured b-Lg during thermal treatment and hydrolysed it with trypsin. They obtained fragments in the region of f(41-70).…”

“…The most interesting influences are attributed to temperature and pressure. Through high temperature and high pressure the protein structure is changed, leading to the exposure to the enzyme of more cleavage sites (Chicon, Belloque, Alonso, Martin-Alvarez, & Lopez-Fandino, 2008;Chicon, Belloque, Recio, & Lopez-Fandino, 2006;Iametti et al, 2002). Thus hydrolysis may be quicker although cases of enzyme inhibition were reported with protein heat denaturing (Cheison, Wang, & Xu, 2007) over time.…”

“…Many reports demonstrate the resistance of b-Lg to proteases (Chen, Hardin, & Swaisgood, 1993;Guo, Fox, Flynn, & Kindstedt, 1995;Iametti et al, 2002;Schmidt & van Markwijk, 1993). Using limited proteolysis (by lowering the temperature) with immobilised trypsin, Chen et al (1993) found two large polypeptide fragments, f(48-101) and f(41-100), which were thought to be from the core domain of b-Lg and resistant to trypsinolysis.…”

Unfolding Intermediate in the Peroxisomal Flavoprotein d-Amino Acid Oxidase

“…The source temperature was 200°C, and the capillary and orifice voltages were 3.6 kV and 40 V, respectively. Mass scale calibration was performed using the multiple charged ions from a separate injection of horse heart myoglobin (20). Mass spectra were elaborated using the software MassLynx 2.0, furnished with the spectrometer.…”

Electrostatics of folded and unfolded bovine β-lactoglobulin

“…A peculiar example is connected with Alzheimer's disease (AD): fibroblasts derived from AD patients express an altered conformational status of p53, and the exposure to nanomolar concentrations of amyloid b 1-40 induces the expression of an unfolded p53 protein isoform in fibroblasts derived from non-AD subjects (Lanni et al 2007). Even more common, the occurrence of misfolded proteins during industrial processes [as exemplified by protein aggregation by heat and other physical treatments (Iametti et al 1996;Iametti et al 2002)] and in the field of protein-based nanotechnology [as exemplified by the assembly of fibrils, tubules, and other nanostructures from partially denatured bovine b-lactoglobulin (BLG) (Rasmussen et al 2007)]. …”