Whey proteins: targets of oxidation, or mediators of redox protection.

Bovine whey proteins are highly valued dairy ingredients. This is primarily due to their amino acid content, digestibility, bioactivities and their processing characteristics. One of the reported bioactivities of whey proteins is antioxidant activity. Numerous dietary intervention trials with humans and animals indicate that consumption of whey products can modulate redox biomarkers to reduce oxidative stress. This bioactivity has in part been assigned to whey peptides using a range of biochemical or cellular assays in vitro. Superimposing whey peptide sequences from gastrointestinal samples, with whey peptides proven to be antioxidant in vitro, allows us to propose peptides from whey likely to exhibit antioxidant activity in the diet. However, whey proteins themselves are targets of oxidation during processing particularly when exposed to high thermal loads and/or extensive processing (e.g. infant formula manufacture). Oxidative damage of whey proteins can be selective with regard to the residues that are modified and are associated with the degree of protein unfolding, with α-Lactalbumin more susceptible than ß-Lactoglobulin. Such oxidative damage may have adverse effects on human health. This review summarises how whey proteins can modulate cellular redox pathways and conversely how whey proteins can be oxidised during processing. Given the extensive processing steps that whey proteins are often subjected to, we conclude that oxidation during processing is likely to compromise the positive health attributes associated with whey proteins.

Effect of Methylglyoxal-Induced Glycation on the Composition and Structure of ß-Lactoglobulin and α-Lactalbumin.

Glycation, and particularly reactions between aldehydes and nucleophiles (thiols, amines), can initiate changes in the structure, solubility, composition, hydrophobicity, conformation, function, and susceptibility to proteolysis of proteins. This can have adverse consequences for mammals, plants, foodstuffs, and pharmaceuticals. Low-molecular-mass dialdehydes such as methylglyoxal (MGO) are much more reactive than parent glucose and therefore potentially highly damaging. These are present at significant levels in some foods. This study investigated whether and how MGO exposure, with or without concurrent heat exposure, affected the major whey proteins ß-lactoglobulin and α-lactalbumin. MGO diminished the formation of heat-induced, reducible, intermolecular disulfide cross-links for both proteins, with this being associated, at least in part, with alternative thiol consuming reactions of MGO. At long incubation times, nonreducible protein cross-links were formed in a dose-dependent manner, with LC-MS/MS and UPLC analysis showing the presence of methylglyoxal-lysine dimers (MOLD). UPLC analysis revealed MGO-dependent consumption of specific amino acids in the order Cys > Arg > Lys > Trp for both proteins, with α-lactalbumin affected to a greater extent than ß-lactoglobulin. SDS-PAGE revealed altered protein mobility consistent with modification of charged residues. MGO exposure also resulted in increased binding of the hydrophobic dye, 8-anilino-1-naphthalene sulfonic acid, consistent with limited protein unfolding. Overall, these data are consistent with rapid reaction of MGO residues at Cys residues (when available) and surface accessible Arg and Lys residues, with formation of adducts and cross-linked materials. These alternative reactions of dialdehydes diminish direct heat-induced (disulfide) cross-link formation and result in limited protein unfolding.

Effect of Oxidation and Protein Unfolding on Cross-Linking of ß-Lactoglobulin and α-Lactalbumin.

Oxidation and heat treatment can initiate changes in the amino acid composition, structure, solubility, hydrophobicity, conformation, function, and susceptibility to proteolysis of proteins. These can result in adverse consequences for mammals, plants, foodstuffs, and pharmaceuticals. This study investigated whether and how individual or combined treatment with heat, a commonly encountered factor in industrial processing, and H2O2 alters the structure and composition of two major milk whey proteins, α-lactalbumin and ß-lactoglobulin, and mixtures of these. Thermal treatment induced reducible cross-links in isolated ß-lactoglobulin, but not isolated α-lactalbumin under the conditions employed. Cross-linking occurred at lower temperatures and to a greater extent in the presence of low concentrations of H2O2. H2O2 did not induce cross-linking in the absence of heat. Mixtures of α-lactalbumin and ß-lactoglobulin showed similar behavior, except that mixed α-lactalbumin-ß-lactoglobulin dimers were detected. Cross-linking was associated with formation of sulfenic acids (RS-OH species), oxidation of methionine residues, cleavage of disulfide bonds in α-lactalbumin, altered conformation of disulfide bonds in ß-lactoglobulin, alterations in the fluorescence intensity and maximum emission wavelength of endogenous tryptophan residues, and binding of the hydrophobic probe 8-anilinonaphthalenesulfonate. These data are consistent with increased unfolding and subsequent aggregation of the protein, with these changes being maximized in the presence of both heat and H2O2. The enhanced aggregation detected with H2O2 is consistent with additional pathways to aggregation above that induced by heat alone. These mechanistic insights provide potential strategies for modulating the extent and nature of protein modification induced by thermal and oxidant treatment.

Protein cysteine oxidation in redox signaling: Caveats on sulfenic acid detection and quantification.

Oxidation of critical signaling protein cysteines regulated by H2O2 has been considered to involve sulfenic acid (RSOH) formation. RSOH may subsequently form either a sulfenyl amide (RSNHR') with a neighboring amide, or a mixed disulfide (RSSR') with another protein cysteine or glutathione. Previous studies have claimed that RSOH can be detected as an adduct (e.g., with 5,5-dimethylcyclohexane-1,3-dione; dimedone). Here, kinetic data are discussed which indicate that few proteins can form RSOH under physiological signaling conditions. We also present experimental evidence that indicates that (1) dimedone reacts rapidly with sulfenyl amides, and more rapidly than with sulfenic acids, and (2) that disulfides can react reversibly with amides to form sulfenyl amides. As some proteins are more stable as the sulfenyl amide than as a glutathionylated species, the former may account for some of the species previously identified as the "sulfenome" - the cellular complement of reversibly-oxidized thiol proteins generated via sulfenic acids.

Key role of cysteine residues and sulfenic acids in thermal- and H2O2-mediated modification of ß-lactoglobulin.

Oxidation results in protein deterioration in mammals, plants, foodstuffs and pharmaceuticals, via changes in amino acid composition, fragmentation, aggregation, solubility, hydrophobicity, conformation, function and susceptibility to digestion. This study investigated whether and how individual or combined treatment with heat, a commonly encountered factor in industrial processing, and H2O2 alters the structure and composition of the major whey protein ß-lactoglobulin. Thermal treatment induced reducible cross-links, with this being enhanced by low H2O2 concentrations, but decreased by high concentrations, where fragmentation was detected. Cross-linking was prevented when the single free Cys121 residue was blocked with iodoacetamide. Low concentrations of H2O2 added before heating depleted thiols, with H2O2 alone, or H2O2 added after heating, having lesser effects. A similar pattern was detected for methionine loss and methionine sulfoxide formation. Tryptophan loss was only detected with high levels of H2O2, and no other amino acid was affected, indicating that sulfur-centered amino acids are critical targets. No protection against aggregation was provided by high concentrations of the radical scavenger 5, 5-dimethyl-1-pyrroline N-oxide (DMPO), consistent with molecular oxidation, rather than radical reactions, being the major process. Sulfenic acid formation was detected by Western blotting and LC-MS/MS peptide mass-mapping of dimedone-treated protein, consistent with these species being significant intermediates in heat-induced cross-linking, especially in the presence of H2O2. Studies using circular dichroism and intrinsic fluorescence indicate that H2O2 increases unfolding during heating. These mechanistic insights provide potential strategies for modulating the extent of modification of proteins exposed to thermal and oxidant treatment.

Antioxidative Mechanisms of Sulfite and Protein-Derived Thiols during Early Stages of Metal Induced Oxidative Reactions in Beer.

The radical-mediated reactions occurring during the early stages of beer storage were studied by following the rate of oxygen consumption, radical formation as detected by electron spin resonance spectroscopy, and concentrations of the antioxidant compounds sulfite and thiols. Addition of either Fe(III) or Fe(II) had similar effects, indicating that a fast redox equilibrium is obtained between the two species in beer. Addition of iron in combination with hydrogen peroxide gave the most pronounced levels of oxidation due to a direct initiation of ethanol oxidation through generation of hydroxyl radicals by the Fenton reaction. The concentration of sulfite decreased more than the thiol concentration, suggesting that thiols play a secondary role as antioxidants by mainly quenching 1-hydroxyethyl radicals that are intermediates in the oxidation of ethanol. Increasing the temperature had a minor effect on the rate of oxygen consumption.