Quantitation of Protein Cysteine–Phenol Adducts in Minced Beef Containing 4-Methyl Catechol
2020
Arsad, Siti Suriani | Zainudin, Mohd Asraf Mohd | De Gobba, Cristian | Jongberg, Sisse | Larsen, Flemming H. | Lametsch, René | Andersen, Mogens L. | Lund, Marianne N.
Thiol groups of cysteine (Cys) residues in proteins react with quinones, oxidation products of polyphenols, to form protein–polyphenol adducts. The aim of the present work was to quantify the amount of adduct formed between Cys residues and 4-methylcatechol (4MC) in minced beef. A Cys–4MC adduct standard was electrochemically synthesized and characterized by liquid chromatography–mass spectrometry (LC–MS) as well as NMR spectroscopy. Cys–4MC adducts were quantified after acidic hydrolysis of myofibrillar protein isolates (MPIs) and LC–MS/MS analysis of meat containing either 500 or 1500 ppm 4MC and stored at 4 °C for 7 days under a nitrogen or oxygen atmosphere. The concentrations of Cys–4MC were found to be 2.2 ± 0.3 nmol/mg MPI and 8.1 ± 0.9 nmol/mg MPI in meat containing 500 and 1500 ppm 4MC, respectively, and stored for 7 days under oxygen. The formation of the Cys–4MC adduct resulted in protein thiol loss, and ca. 62% of the thiol loss was estimated to account for the formation of the Cys–4MC adduct for meat containing 1500 ppm 4MC. Furthermore, protein polymerization increased in samples containing 4MC as evaluated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and the polymerization was found to originate from protein–polyphenol interactions as evaluated by a blotting assay with staining by nitroblue tetrazolium.
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