Direct Colorimetric Assay of Free Thiol Groups and Disulfide Bonds in Suspensions of Solubilized and Particulate Cereal Proteins' KIN-YU CHAN and BRUCE

Cereal Chem. 70(1):22-26 A direct colorimetric method that simultaneously combines measurematerial, absorbance at 412 nm was read. This assay was highly reproment of solubilized and insoluble thiol groups and disulfide bonds in ducible, and measurements agreed with direct amino acid analysis. Twincorn meal-based materials is described. Ellman's reagent, 5,5'-dithiobis screw extrusion of corn meal at 1501C at moisture levels of 16 and 18% (2-nitrobenzoic acid), which reacts specifically with thiol groups, or had no significant effect on cysteine or disulfide bond levels. Other possible disodium 2-nitro-5-thiosulfobenzoate, which reacts with cysteine and thiol changes such as disulfide bond rearrangements could not be determined groups formed after reduction of disulfide bonds with sodium sulfite, by the mixed-phase assay. This method provides a rapid and convenient were reacted directly with corn meal in the presence of surfactants (urea means for screening thiol and disulfide levels in insoluble proteinaceous and/ or sodium dodecyl sulfate), releasing the soluble chromophore materials. 2-nitro-5-thiobenzoate. After a clarification step to remove suspended Disulfide bonds are thought to play an important role in the texture of cereal-based products. However, because of the hydrophobic and insoluble nature of cereal proteins, quantification of thiol and disulfide bonds has proven difficult. Complete extraction of corn meal protein with sodium dodecyl sulfate (SDS), urea, and a reducing agent leads to cleavage of disulfide bonds. Furthermore, since extraction with SDS and urea results in only partial solubilization of the protein, assays based on solubilization followed by measurement of thiol and disulfide content often yield highly variable results and underestimates of true thiol and disulfide group content. A technique that avoids this initial protein solubilization step would largely eliminate these problems. This article describes a solid-phase assay that simultaneously combines quantification of soluble and particulate thiol and disulfide groups in cereal-based proteins. The principle of this method is to suspend the entire sample in urea and to react it with a color reagent that will simultaneously react with both soluble and insoluble proteins, with release of a soluble chromophore. Ellman's reagent, 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), which reacts specifically with thiol groups (Ellman 1958, Riddles et al 1983), and disodium 2-nitro-5-thiosulfobenzoate (NTSB2-), which is used to quantify disulfide group content (Thannhauser et al 1987), are ideally suited for this purpose, since reaction with either results in the release of the 2-nitro-5-thiobenzoate anion (NTB2), which is soluble in aqueous solution. Following the removal of insoluble material by clarification steps, absorbance at 412 nm is then read. This method was used to assess the effects of twin-screw extrusion processing on thiol and disulfide levels in corn meal. MATERIALS AND METHODS Materials Corn meal (12% moisture, 7% protein, 0.7% oil, 0.5% fiber, 0.4% ash, and 79.4% N-free extract) was obtained from Lauhoff Grain Co., Danville, IL. DTNB and NTSB2were obtained from Aldrich Chemical Co., Milwaukee, WI. Extrusion was conducted in a Brabender type 2003 single-screw extruder with an axially ground barrel; length-to-diameter ratio, 20; screw diameter, 1.9 cm (0.75 in.); screw length, 38.1 cm (15 in.); and compression ratio, 1:3. Solid-Phase Assay for Free Thiol Content Colorimetric reactions were conducted under the conditions described by Ellman (1958, 1959). Unless otherwise indicated, 'New Jersey Agricultural Experiment Station publication D-10544-1-92. 2 Department of Food Science, New Jersey Agricultural Experiment Station, Cook College, Rutgers University, New Brunswick 08903-0231. @1993 American Association of Cereal Chemists, Inc. samples (30 mg ground to 40 mesh and dried in vacuo) were suspended in 1.0 ml of reaction buffer consisting of 8M urea, 10 mM DTNB, 3 mM ethylene-diaminetetraacetic acid (EDTA), and 0.2M Tris-HCl, pH 8.0. SDS (1%) was present where indicated. Samples were incubated under N2 for various intervals. To remove particulate matter, samples were centrifuged at 13,600 X g for 10 min in a microcentrifuge. A 0. 1-ml aliquot of supernatant was removed and diluted with 0.9 ml of 8M urea, 1% SDS, 3 mM EDTA, and 0.2M Tris-HCl, pH 8.0. This solution was centrifuged at 13,600 X g, and its absorbance was read at 412 nm. Solid-Phase Assay for Total Sulfhydryl Group Content Colorimetric reactions were conducted under the conditions described by Thannhauser et al (1987). Unless otherwise indicated, samples (30 mg ground to 40 mesh and dried in vacuo) were suspended in 1.0 ml of reaction buffer consisting of 8M urea, 0.1M sodium sulfite, 3 mM EDTA, 0.2M Tris-HCl, pH 9.5, and 10 mM NTSB2, synthesized from DTNB in the presence of sodium sulfite and 02 as described in Thannhauser et al (1987). SDS (1%) was added where indicated. To remove particulate matter, samples were centrifuged at 13,600 X g in a microcentrifuge for 10 min. A 0.1-ml aliquot of supernatant was removed and diluted with 0.9 ml of 8M urea, 1% SDS, 0.1M sodium sulfite, 3 mM EDTA, and 0.2M Tris-HCl, pH 8.0. This solution was centrifuged at 13,600 X g, and its absorbance was read at 412 nm. Disulfide group content was calculated as the difference between thiol group content before and after reduction of disulfide bonds with sulfite. Total cysteine was calculated as [-SH] + 2[-S-S-]. Two-Step Method for Sulfhydryl-Disulfide Assay Unless otherwise indicated, samples (60 mg ground to 40 mesh and dried in vacuo) were extracted under N2 with 2.0 ml of buffer consisting of 8M urea, 3 mM EDTA, and 0.2M Tris-HCl, pH 8.0. On the basis of a time course of protein solubilization (Fig. 1 A), extractions were conducted for 3 hr under N2. To remove particulate matter, samples were centrifuged at 16,000 X g for 30 min in a Sorvall RC5C preparative centrifuge (Sorvall Instruments, Du Pont, Wilmington, DE). Aliquots of 0.2 ml of supernatant were removed and brought to 1.0 ml with appropriate DTNBand NTSB2-containing reaction mixtures (see above) for spectrophotometric determination of thiol and disulfide groups.