Quantification of radicals formed during heating of β-lactoglobulin with glucose in aqueous ethanol

Heating β-lactoglobulin alone, or in the presence of glucose, at moderately elevated temperatures (50, 70 and 80°C) in 30% aqueous ethanol at pH 6.0, 7.0 and 8.0 led to an iron catalysed formation of highly reactive radicals at concentrations of up to 1.0 mmol/mol protein as trapped by α-(4-pyridyl N-oxide)-N-tert-butylnitrone (POBN) and quantified by ESR spectroscopy. Oxidation of β-lactoglobulin was favoured by increasing temperature and high pH conditions, whereas presence of glucose decreased oxidation, indicating that oxidation of β-lactoglobulin involves either amine or thiol side chain group blocked as oxidation substrate by reaction with glucose as a reducing sugar. The oxidation of β-lactoglobulin was hampered by the phenolic antioxidant 4-methylcatechol and to an even larger extent by the oxidised quinone form 4-methyl-1,2-benzoquinone, suggesting that it is the quinone forms of phenolic antioxidants which compete with reducing sugars in protecting proteins against oxidation.