RP-HPLC method for simultaneous quantification of free and total thiol groups in native and heat aggregated whey proteins

Disulfide formation of whey proteins during heat treatment via thiol oxidation is important with regard to techno-functional properties. Due to the formation of other oxidation products than disulfides, the decrease in free thiol concentration is not proportional to the disulfide formation. Thus, in order to evaluate thiol reactivity and disulfide concentration both parameters are required. Currently applied methods focus mainly on the loss of free thiols using the spectrophotometric Ellman's assay. Next to that, we improved an existing RP-HPLC assay using the thiol reagent 4,4′-Dithiodipyridine (DTDP) to quantify free thiols as well as total (free thiols and disulfide bonds) thiols of native and heat-treated whey proteins. Thereby, the sample preparation technique, the sample handling, and the analysis technique were optimized. Thus, the paper provides a simple RP-HPLC method for quantification of thiol oxidation reactions to determine heat-induced changes in the structure of whey proteins. In addition, the method should be applicable to other protein systems due to the method validation by proteins of different amounts of free and total thiols in their structure. • Simple RP-HPLC method for quantification of free and total thiols using 4,4′-Dithiodipyridine (DTDP). • High recovery rates for free and total thiols. • High stability within 24 h.