RESUMEN
Whey proteins are widely used as ingredients in the form of aggregates to obtain certain functionalities in food applications. The aim of this study was to understand how UV illumination generates aggregates of α-lactalbumin (α-LA) as an alternative to heat treatments traditionally used for industrial production of protein aggregates. Absorption of UV light by α-LA caused cleavage of disulfide bonds and release of thiol groups, which resulted in primarily disulfide-mediated aggregation. This process mediated efficient aggregation with up to 98% monomer conversion into aggregates through formation of intermolecular disulfide bonds, while only minor levels of nonreducible cross-links were observed. SDS-PAGE analysis revealed that illumination led to formation of dimeric, trimeric, and oligomeric forms of α-LA. LC-MS/MS analysis showed that all of the four native disulfide bonds in α-LA were cleaved by UV illumination but to different extents, and the extent of cleavage was found to be higher in the absence of calcium. Seventeen different non-native disulfides were formed after 24 h of UV illumination. Two dityrosine bonds were identified (Tyr103-Tyr103 and Tyr36-Tyr103) alongside ditryptophan (Trp118-Trp118) and tyrosine-tryptophan (Tyr50-Trp60) cross-links. In addition, Trp60, Trp118, Cys73, Cys91, Cys120, Phe80, Met90, His68, and His107 were found to be oxidized up to 12% as compared to a nonilluminated control. Our work illustrates that light exposure can be used for generation of α-LA aggregates, but optimization of the illumination conditions is required to reduce oxidative damage to Trp, Cys, Phe, Met, and His residues.
Asunto(s)
Lactalbúmina/química , Secuencias de Aminoácidos , Animales , Bovinos , Cromatografía Liquida , Lactalbúmina/efectos de la radiación , Agregado de Proteínas/efectos de la radiación , Conformación Proteica/efectos de la radiación , Espectrometría de Masas en Tándem , Rayos UltravioletaRESUMEN
Bovine α-lactalbumin (α-La) is a major food allergen found in milk and is characterized by high conformational stability because of its four disulfide bridges and being calcium bound. This study aimed to describe the influence of gamma irradiation on the structure and potential allergenicity of α-La. The prepared α-La was irradiated at doses of 1-10 kGy. The changes in structure were characterized through SDS-PAGE, circular dichroism spectroscopy, ultraviolet absorption spectroscopy, and fluorescence spectroscopy. The potential allergenicity of the irradiated α-La was evaluated in vitro through IgG/IgE inhibition ELISA and the human basophil KU812 degranulation assay. The results showed that the secondary and tertiary structures of α-La significantly changed and caused extensive protein denaturation and aggregation. IgG and IgE binding properties remarkably decreased, and the degranulation capacity of basophils weakened. The results suggested that structural damage of α-La induced by irradiation significantly reduces the potential allergenicity of α-La.
Asunto(s)
Alérgenos/química , Lactalbúmina/química , Leche/química , Alérgenos/inmunología , Alérgenos/efectos de la radiación , Animales , Basófilos/inmunología , Calcio/química , Línea Celular Tumoral , Preescolar , Dicroismo Circular , Disulfuros/química , Relación Dosis-Respuesta en la Radiación , Electroforesis en Gel de Poliacrilamida , Femenino , Hipersensibilidad a los Alimentos/inmunología , Hipersensibilidad a los Alimentos/prevención & control , Irradiación de Alimentos , Rayos gamma , Humanos , Inmunoglobulina E/inmunología , Inmunoglobulina G/inmunología , Lactante , Lactalbúmina/inmunología , Lactalbúmina/efectos de la radiación , Masculino , Peso Molecular , Unión Proteica , Desnaturalización Proteica , Conejos , Espectrometría de FluorescenciaRESUMEN
Free radicals and other oxidation products were characterized on α-lactalbumin with electron spin resonance (ESR), immuno-spin trapping, and mass spectrometry (MS) after riboflavin-mediated oxidation. Radicals were detected using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in immuno-spin trapping with both enzyme-linked immunosorbent assay (ELISA) and Western blotting and further characterized with mass spectrometry. A DMPO-trapped radical was identified at His68 and another at one of the tyrosine residues, Tyr50 or Tyr36, respectively, generated by a type II or I mechanism. Not all tyrosyl radicals were trapped, as the secondary oxidation product, 3,4-dihydroxyphenylalanine (DOPA), was detected by mass spectrometry at Tyr18 and Tyr50. A further oxidation of DOPA resulted in the DOPA o-semiquinone radical, which was characterized by ESR. Both surface exposure and the neighboring residues in the local environment of the tertiary structure of α-lactalbumin seem to play a role in the generation of DMPO trapped radicals and secondary oxidation products.
Asunto(s)
Histidina/análogos & derivados , Lactalbúmina/química , Riboflavina/química , Tirosina/análogos & derivados , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres/análisis , Radicales Libres/química , Histidina/análisis , Histidina/química , Histidina/efectos de la radiación , Lactalbúmina/efectos de la radiación , Luz , Oxidación-Reducción , Riboflavina/efectos de la radiación , Espectrometría de Masa por Ionización de Electrospray , Detección de Spin , Espectrometría de Masas en Tándem , Tirosina/análisis , Tirosina/química , Tirosina/efectos de la radiaciónRESUMEN
Goat alpha-lactalbumin (GLA) contains four tryptophan (Trp) residues and four disulfide bonds. Illumination with near-UV light results in the cleavage of disulfide bridges and in the formation of free thiols. To obtain information about the reaction products, the illuminated protein was carbamidomethylated and digested with trypsin and the peptides were analyzed by mass spectrometry. Peptides containing Cys120Cam, Cys61Cam, or Cys91Cam were detected, as well as two peptides containing a new Cys-Lys cross-link. In one, Cys6 was cross-linked to Lys122, while the cross-link in the second was either a Cys91-Lys79 or Cys73-Lys93 cross-link; however, the exact linkage could not be defined. The results demonstrate photolytic cleavage of the Cys6-Cys120, Cys61-Cys77, and Cys73-Cys91 disulfide bonds. While photolysis of Cys6-Cys120 and Cys73-Cys91 disulfide bonds in GLA has been reported, cleavage of the Cys61-Cys77 disulfide bonds has not been previously detected. To examine the contribution of the individual Trp residues, we constructed the GLA mutants, W26F, W60F, W104F, and W118F, by replacing single Trp residues with phenylalanine (Phe). The substitution of each Trp residue led to less thiol production compared to that for wild-type GLA, showing that each Trp residue in GLA contributed to the photolytic cleavage of disulfide bridges. The specificity was expressed by the nature of the reaction products. No cleavage of the Cys6-Cys120 disulfide bridge was detected when the W26F mutant was illuminated, and no cleavage of the Cys73-Cys91 disulfide bridge was seen following illumination of W26F or W104F. In contrast, Cys61Cam, resulting from the cleavage of the Cys61-Cys77 disulfide bridge, was found following illumination of any of the mutants.
Asunto(s)
Disulfuros/efectos de la radiación , Lactalbúmina/efectos de la radiación , Fotólisis/efectos de los fármacos , Triptófano/efectos de la radiación , Rayos Ultravioleta , Secuencia de Aminoácidos , Animales , Disulfuros/química , Cabras , Lactalbúmina/genética , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Mutación Puntual , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Illumination of goat alpha-lactalbumin (GLA) with 280 or 295 nm light results in tryptophan-mediated photolysis of disulfide bonds within the protein. The photolysis is not dependent on the absence or presence of Ca(2+) and is observed as well on illumination of native and of partially unfolded GLA. However, photolysis of native GLA results in a partial unfolding of the protein. The latter phenomenon is most clearly observed on fluorescence measurements at low temperatures (near 3 degrees C). The photolysis induces some dimerization and oligomerization, but most GLA molecules remain monomeric. To obtain more information about the reaction products, the illuminated protein is treated with iodoacetamide to label the free thiol groups, it is fragmented with trypsin, and the fragments are analyzed by mass spectrometry. Via this approach, we observe that the cleavage of disulfide bonds is restricted to Cys6-Cys120 and Cys73-Cys91 bonds. The photolytic cleavage of either of these disulfide bonds results in the formation of a single free thiol, a phenomenon restricted to Cys120 and Cys91, respectively. We also found indications that a thioether linkage is formed between Cys73 and Trp60. The alkylsulfenylation of Trp60 presumably results from a combination of primary thiyl and tryptyl radicals.
Asunto(s)
Disulfuros/química , Lactalbúmina/química , Fotólisis , Triptófano/química , Animales , Dimerización , Disulfuros/efectos de la radiación , Cabras , Hidrólisis/efectos de la radiación , Lactalbúmina/efectos de la radiación , Oxidación-Reducción/efectos de la radiación , Fotones , Espectrometría de Fluorescencia , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Triptófano/efectos de la radiaciónRESUMEN
Alpha-lactalbumin was irradiated in the lyophilized state in air at ambient temperature. The irradiated protein was examined by size exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis, circular dichroism, and microcalorimetry. Irradiation induced the loss of aromatic amino acids and of helicity so that fragmentation and aggregation products were obtained. The thermodynamic properties of the protein were also modified. The irradiated protein had lower stability, however, the temperature at which denaturation occurred process remained constant.