Direct colorimetric assay of free thiol groups and disulfide bonds in suspensions of solubilized and particulate cereal proteins

Abstract

A direct colorimetric method that simultaneously combines measurement of solubilized and insoluble thiol groups and disulfide bonds in corn meal-based materials is described. Ellman's reagent, 5,5'-dithiobis (2-nitrobenzoic acid), which reacts specifically with thiol groups, or disodium 2-nitro-5-thiosulfobenzoate, which reacts with cysteine and thiol groups formed after reduction of disulfide bonds with sodium sulfite, were reacted directly with corn meal in the presence of surfactants (urea and/or sodium dodecyl sulfate), releasing the soluble chromophore 2-nitro-5-thiobenzoate. After a clarification step to remove suspended material, absorbance at 412 nm was read. This assay was highly reproducible, and measurements agreed with direct amino acid analysis. Twin-screw extrusion of corn meal at 150 degrees C at moisture levels of 16 and 18% had no significant effect on cysteine or disulfide bond levels. Other possible changes such as disulfide bond rearrangements could not be determined by the mixed-phase assay. This method provides a rapid and convenient means for screening thiol and disulfide levels in insoluble proteinaceous materials