Direct colorimetric assay of free thiol groups and disulfide bonds in suspensions of solubilized and particulate cereal proteins
Direct colorimetric assay of free thiol groups and disulfide bonds in suspensions of solubilized and particulate cereal proteins
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发表时间：
1993-09
期刊：
影响因子：
2.4
通讯作者：
Kin-Yu Chan;B. P. Wasserman
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作者：
Kin-Yu Chan;B. P. Wasserman
谷物化学。 70(1):22-26 一种直接比色法，同时结合测量材料，读取 412 nm 处的吸光度。该测定高度评价了可溶性和不溶性的硫醇基团和二硫键，并且测量结果与直接氨基酸分析一致。描述了基于双玉米粉的材料。 Ellman 试剂，5,5'-二硫双螺杆挤出玉米粉，温度为 1501C，水分含量为 16% 和 18%（2-硝基苯甲酸），与硫醇基团发生特异性反应，或者对半胱氨酸或二硫键水平没有显着影响。其他可能与半胱氨酸和硫醇变化（例如二硫键重排）发生反应的2-硝基-5-硫代磺基苯甲酸二钠，无法通过混合相测定确定用亚硫酸钠还原二硫键后形成的基团。该方法提供了在表面活性剂（用于筛选不溶性蛋白质和/或十二烷基硫酸钠中的硫醇和二硫化物水平的尿素手段）存在下直接与玉米粉反应的快速且方便的方法，释放可溶性发色团材料。 2-硝基-5-硫代苯甲酸酯。经过澄清步骤去除悬浮的二硫键后，二硫键被认为在谷物产品的质地中发挥着重要作用。然而，由于谷物蛋白的疏水性和不溶性，硫醇和二硫键的定量已被证明是困难的。用十二烷基硫酸钠 (SDS)、尿素和还原剂完全提取玉米粉蛋白会导致二硫键断裂。此外，由于用 SDS 和尿素提取仅导致蛋白质部分溶解，因此基于溶解然后测量硫醇和二硫键含量的测定通常会产生高度可变的结果，并低估真实的硫醇和二硫键含量。避免这种初始蛋白质溶解步骤的技术将在很大程度上消除这些问题。本文描述了一种固相测定，该测定同时结合了谷物蛋白中可溶性和颗粒性硫醇和二硫基的定量。该方法的原理是将整个样品悬浮在尿素中，并与显色剂反应，显色剂同时与可溶性和不溶性蛋白质反应，释放可溶性发色团。 Ellman 试剂 5,5'-二硫代双（2-硝基苯甲酸）（DTNB）与硫醇基团发生特异性反应（Ellman 1958，Riddles 等人 1983），以及 2-硝基-5-硫代磺基苯甲酸二钠（NTSB2-），用于定量二硫基团含量（Thannhauser 等人 1987），非常适合此目的目的，因为与任一反应都会导致释放 2-硝基-5-硫代苯甲酸阴离子 (NTB2)，该阴离子可溶于水溶液。通过澄清步骤去除不溶物质后，读取 412 nm 处的吸光度。该方法用于评估双螺杆挤出加工对玉米粉中硫醇和二硫化物含量的影响。材料和方法 材料 玉米粉（12% 水分、7% 蛋白质、0.7% 油、0.5% 纤维、0.4% 灰分和 79.4% 无氮提取物）购自伊利诺伊州丹维尔的 Lauhoff Grain Co.。 DTNB 和 NTSB2 获自威斯康星州密尔沃基市的 Aldrich Chemical Co.。挤出是在带有轴向磨削机筒的 Brabender 2003 型单螺杆挤出机中进行的；长径比，20；螺杆直径，1.9 厘米（0.75 英寸）；螺丝长度，38.1 厘米（15 英寸）；压缩比为1:3。游离硫醇含量的固相测定 比色反应在 Ellman (1958, 1959) 描述的条件下进行。除非另有说明，“新泽西农业实验站出版物 D-10544-1-92”。 2 罗格斯大学库克学院新泽西农业实验站食品科学系，新不伦瑞克 08903-0231。 @1993 American Association of Cereal Chemists, Inc. 样品（30 mg 研磨至 40 目并真空干燥）悬浮于 1.0 ml 反应缓冲液中，该缓冲液由 8M 尿素、10 mM DTNB、3 mM 乙二胺四乙酸 (EDTA) 和 0.2M Tris-HCl（pH 8.0）组成。注明的地方存在 SDS (1%)。样品在 N2 下孵育不同的时间间隔。为了去除颗粒物，样品在微量离心机中以 13,600 X g 离心 10 分钟。取出 0. 1-ml 等份上清液并用 0.9 ml 8M 尿素、1% SDS、3 mM EDTA 和 0.2M Tris-HCl（pH 8.0）稀释。该溶液在 13,600 X g 下离心，并在 412 nm 处读取其吸光度。总巯基含量的固相测定比色反应在Thannhauser 等人(1987)描述的条件下进行。除非另有说明，将样品（30 mg 研磨至 40 目并真空干燥）悬浮于 1.0 ml 反应缓冲液中，该缓冲液由 8M 尿素、0.1M 亚硫酸钠、3 mM EDTA、0.2M Tris-HCl、pH 9.5 和 10 mM NTSB2 组成，在亚硫酸钠和 02 存在下由 DTNB 合成，如 Thannhauser 等人所述（1987）。在指定位置添加 SDS (1%)。为了去除颗粒物，将样品在微量离心机中以 13,600 X g 离心 10 分钟。取出 0.1 ml 等份上清液并用 0.9 ml 8M 尿素、1% SDS、0.1M 亚硫酸钠、3 mM EDTA 和 0.2M Tris-HCl（pH 8.0）稀释。该溶液在 13,600 X g 下离心，并在 412 nm 处读取其吸光度。二硫基含量计算为用亚硫酸盐还原二硫键之前和之后的硫醇基含量之差。总半胱氨酸计算为[-SH] + 2[-S-S-]。硫氢基-二硫化物测定的两步法 除非另有说明，样品（60 mg 研磨至 40 目并真空干燥）在 N2 下用 2.0 ml 由 8M 尿素、3 mM EDTA 和 0.2M Tris-HCl（pH 8.0）组成的缓冲液提取。根据蛋白质溶解的时间进程（图 1 A），在 N2 下进行提取 3 小时。为了去除颗粒物，样品在 Sorvall RC5C 制备型离心机（Sorvall Instruments，Du Pont，Wilmington，DE）中以 16,000 X g 离心 30 分钟。取出 0.2 ml 上清液的等分试样，并用适当的含有 DTNB 和 NTSB2 的反应混合物（见上文）调至 1.0 ml，用于分光光度法测定硫醇和二硫化物基团。
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.