化学
色氨酸
激进的
酪氨酸
氨基酸
荧光
酪蛋白
光化学
生物化学
量子力学
物理
作者
Eduardo Fuentes‐Lemus,Eduardo Silva,Pablo Barrías,Alexis Aspée,Elizabeth Sánchez Escobar,Lasse G. Lorentzen,Luke Carroll,Fabian Leinisch,Michael J. Davies,Camilo López-Alarcón
标识
DOI:10.1016/j.freeradbiomed.2018.06.005
摘要
The present work examined the role of Tyr and Trp in oxidative modifications of caseins, the most abundant milk proteins, induced by peroxyl radicals (ROO•). We hypothesized that the selectivity of ROO• and the high flexibility of caseins (implying a high exposure of Tyr and Trp residues) would favor radical-radical reactions, and di-tyrosine (di-Tyr) and di-tryptophan (di-Trp) formation. Solutions of α- and β-caseins were exposed to ROO• from thermolysis and photolysis of AAPH (2,2'-azobis(2-methylpropionamidine)dihydrochloride). Oxidative modifications were examined using electrophoresis, western blotting, fluorescence, and chromatographic methodologies with diode array, fluorescence and mass detection. Exposure of caseins to AAPH at 37 °C gave fragmentation, cross-linking and protein aggregation. Amino acid analysis showed consumption of Trp, Tyr, Met, His and Lys residues. Quantification of Trp and Tyr products, showed low levels of di-Tyr and di-Trp, together with an accumulation of carbonyls indicating that casein aggregation is, at least partly, associated with secondary reactions between carbonyls and Lys and His residues. AAPH photolysis, which generates a high flux of free radicals increased the extent of formation of di-Tyr in both model peptides and α- and β- caseins; di-Trp was only detected in peptides and α-casein. Thus, in spite of the high flexibility of caseins, which would be expected to favor radical-radical reactions, the low flux of ROO• generated during AAPH thermolysis disfavours the formation of dimeric radical-radical cross-links such as di-Tyr and di-Trp, instead favoring other O2-dependent crosslinking pathways such as those involving secondary reactions of initial carbonyl products.
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