Oxidative consequences of UV irradiation on isolated milk proteins: Effects of hydrogen peroxide and bivalent metal ions Dana Scheidegger, Geraldine Larsen, Silvia Clara Kivatinitz * Departamento de Química Biol ogica-CIQUIBIC, Centro de Química Aplicada, Facultad de Ciencias Químicas, Universidad Nacional de C ordoba, Medina Allende y Haya de La Torre, Ciudad Universitaria, X5000HUA C ordoba, Argentina article info Article history: Received 12 June 2015 Received in revised form 1 December 2015 Accepted 6 December 2015 Available online 30 December 2015 abstract b-Casein (b-CN), b-lactoglobulin (b-LG), and a-lactalbumin (a-LA), were subjected to UV irradiation, alone or in the presence of hydrogen peroxide, hydrogen peroxide plus copper, or iron. Disappearance of tryptophan (Trp) was fastest for a-LA and slowest for b-CN. Hydrogen peroxide accelerated loss of Trp, while addition of iron did not further increase the speed of Trp reduction, but the addition of copper did. Oxidation products were higher for b-CN than for b-LG following UV irradiation alone or in the presence of hydrogen peroxide or hydrogen peroxide plus iron. In the presence of copper, oxidation rates were similar for both proteins. UV photo-oxidation caused the loss of the native polypeptides, the rate of this being faster for b-LG than for b-CN. These results are compatible with the concept that formation of dityrosine and N-formylkynurenine were favoured by mobility in the native protein. © 2016 Elsevier Ltd. All rights reserved. 1. Introduction Photo-oxidation of proteins has been subject of increasing in- terest in the last decade, centred mainly in effects on living cells or in relation to health (Kivatinitz, 2012; Pattison, Rahmanto, & Davies, 2012). Several studies have been performed involving irradiation with UV-A, the predominant form of irradiation on the earth's surface, which affects living cells (Pattison & Davies, 2006), biological fluids like serum (Carbone et al., 2008) and milk (Dalsgaard & Larsen, 2008). This type of irradiation mostly affects a few amino acid residues (e.g., tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe), histidine (His), methionine (Met), cysteine (Cys) and disulphide (cystine) bonds). UV-C irradiation affects the same amino acids with a high incidence of dityrosine bond for- mation and the creation of aggregates (Balasubramanian, 2005; Correia, Neves-Petersen, Jeppesen, Gregersen, & Petersen, 2012; Kivatinitz, Pelsman, Alonso, Bagatolli, & Quiroga, 1997; Scheideg- ger, Pecora, Radici, & Kivatinitz, 2010). UV-C technology is being used in food industry as a non-thermal sterilisation method, and has been applied in continuous operation (Falguera, Pag an, Garza, Garvín, & Ibarz, 2011). Photo-oxidation by UV-C of whole and skim milk proteins has been studied by our group. Changes in the primary structure, due to destruction of Trp, together with oxidation of other amino acids, resulting in dityr- osine (DT), N-formylkynurenine (NFK) and carbonylation products (PC) have been observed. Major changes in molecular mass, ag- gregation, oligomerisation and fragmentation were detected by SDS-PAGE (Scheidegger et al., 2010). Oligomers were formed by dityrosine or disulphydryl bonds. The involvement of copper and iron in dityrosine formation by H 2 O 2 has been assessed in peptides, and it has been concluded that only copper promotes dityrosine bond formation, this effect being proportional to the concentration of copper (MacGregor, Kato Marshall, Nevell & Shute 2011). The effects of copper and iron ions on the formation of HO  radicals by metal-catalysed Fenton- type reactions are different; Cu 1þ reacts directly with H 2 O 2 , whereas Fe 2þ needs a chelating agent (Kocha, Yamaguchi, Ohtaki, Fukuda, & Aoyagi, 1997). Also, the effect of copper on the oxida- tive degradation of albumin caused by H 2 O 2 differed from that of iron, because copper ions bind to albumin and induce site-specific degradation, while the iron system induces non-specific degrada- tion of albumin (Kocha et al., 1997). Dityrosine formation has been described in lipoproteins and amyloid protein (Capeillere-Blandin, Delaveau, & Descamps-Latscha, 1991; Yan, Lodge, Traber, Matsugo, & Packer, 1997) and it has been demonstrated that the formation of dityrosine was specific for H 2 O 2 /Cu and independent of the con- centration of dissolved oxygen for lens proteins (Kato, Kitamoto, Kawai, & Osawa, 2001). The extent of dityrosine formation in milk * Corresponding author. Tel.: þ54(351) 535 3855. E-mail address: skivatinitz@gmail.com (S.C. Kivatinitz). Contents lists available at ScienceDirect International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj http://dx.doi.org/10.1016/j.idairyj.2015.12.005 0958-6946/© 2016 Elsevier Ltd. All rights reserved. International Dairy Journal 55 (2016) 64e71