Bovine β-lactoglobulin was hydrolyzed with trypsin or chymotrypsin before, during and after treatment at 600 MPa and pH 6·8 for 10 min at 30, 37 and 44 °C. The extent of β-lactoglobulin hydrolysis under pressure was noticeably higher than at atmospheric pressure, particularly when chymotrypsin was used. Addition of proteases at ambient pressure to previously pressure-treated β-lactoglobulin gave only a modest increase in proteolysis with respect to the untreated protein. Products of enzyme hydrolysis under pressure were separated by reverse-phase HPLC, and were found to be different from those obtained at atmospheric pressure when chymotrypsin was used. The residual immunochemical reactivity of the products of combined pressure-enzyme treatment was assessed on the unresolved hydrolysates by ELISA tests using polyclonal and monoclonal antibodies, and on individual hydrolytic fractions by Western Blotting using sera of paediatric patients allergic to whey proteins in cow milk. The immunoreactivity of the whole hydrolysates was related to their content of residual intact β-lactoglobulin, and no immunochemical reactivity was found for all the products of chymotrypsin hydrolysis under pressure. The results indicate that chymotrypsin effectively hydrolysed hydrophobic regions of β-lactoglobulin that were transiently exposed during the pressure treatments and that were not accessible in the native protein or in the protein that had been previously pressure treated.
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...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.