Direct determination of methionine sulfoxide in milk proteins by enzyme hydrolysis/high-performance liquid chromatography

A direct and simultaneous HPLC/UV determination of methionine and methionine sulfoxide in enzyme-hydrolyzed milk proteins is described. Protein hydrolysis is accomplished by a three-enzyme (pronase, leucine aminopeptidase, prolidase) 20-h/37 °C digestion. A gradient elution reversed-phase HPLC system with UV detection at 214 nm and 280 nm is then used to determine the quantitative releases of methionine sulfoxide, methionine, tyrosine, and tryptophan. The ease of methionine oxidation by a wide variety of oxidants, coupled with the quantitative release of both methionine and its sulfoxide by the three-enzyme hydrolysis, renders the approach valuable for identifying oxidized milk proteins. The relatively simple method proved accurate and precise in its application to commercial milk products, finding methionine sulfoxide levels as high as 74% of the total methionine.