DTNB (Ellman's reagent) is an abbreviation for 5,5'-Dithiobis(2-nitrobenzoic acid). When it reacts with thiol compounds, colorless DTNB changes into yellow 5-thio-2-nitrobenzoic acid. This transformation is useful because 5-thio-2-nitrobenzoic acid has its maximum absorption at 412 nm, ensuring that DTNB's absorption spectrum doesn't interfere with thiol determination. Consequently, DTNB is commonly employed to measure thiol content in proteins and peptide tissues. It can also be used for monitoring organophosphorus pesticide poisoning through acetylcholinesterase assays[1-3].
DTNB modifies the SH group of the enzyme, enabling the determination of the number of SH groups in each molecule and their contribution to the activity of the enzyme[4]. Using a hydroxycinnamoyl-CoA:l-DOPA hydroxycinnamoyl transferase as a model, DTNB has little to no effect on the transferase reaction and can be used to provide a good estimate of hydroxycinnamoyl amide formation, thus allowing for the quick and easy collection of reaction rate data and determination of transferase kinetic parameters[5]. DTNB modification facilitated crystallization of CcbP by inducing polar interactions in the crystal lattice. DTNB-mediated cysteine modification was demonstrated to have little effect on the overall structure and the Ca2+ binding of CcbP. DTNB modification may provide a simple and general approach for protein modification to improve the success of crystallization screening[6].
参考文献:
[1]. ELLMAN GL, COURTNEY KD, et,al. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol. 1961 Jul;7:88-95. doi: 10.1016/0006-2952(61)90145-9. PMID: 13726518.
[2]. ELLMAN GL. Tissue sulfhydryl groups. Arch Biochem Biophys. 1959 May;82(1):70-7. doi: 10.1016/0003-9861(59)90090-6. PMID: 13650640.
[3]. Tietze F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem. 1969 Mar;27(3):502-22. doi: 10.1016/0003-2697(69)90064-5. PMID: 4388022.
[4]. Nagaoka T, Hachimori A, et,al. DTNB modification of SH groups of isocitrate dehydrogenase from Bacillus stearothermophilus purified by affinity chromatography. J Biochem. 1977 Jan;81(1):71-8. doi: 10.1093/oxfordjournals.jbchem.a131452. PMID: 14937.
[5]. Sullivan ML, Bonawitz ND. Spectrophotometric determination of reaction rates and kinetic parameters of a BAHD acyltransferase using DTNB (5,5'-dithio-bis-[2-nitrobenzoic acid]). Plant Sci. 2018 Apr;269:148-152. doi: 10.1016/j.plantsci.2018.01.012. Epub 2018 Jan 31. PMID: 29606213.
[6]. Fan XX, Zhou YF, et,al. Ellman's reagent in promoting crystallization and structure determination of Anabaena CcbP. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2012 Nov 1;68(Pt 11):1409-14. doi: 10.1107/S1744309112034938. Epub 2012 Oct 30. PMID: 23143261; PMCID: PMC3515393.
DTNB (Ellman's reagent)是5,5'-二硫比斯(2-硝基苯甲酸)的缩写。当它与硫醇类化合物反应时，无色的DTNB变成黄色的5-硫-2-硝基苯甲酸。5-硫代-2-硝基苯甲酸在412 nm处有最大吸收，DTNB的吸收光谱不会干扰硫醇的测定。因此，DTNB通常用于测量蛋白质和肽组织中的硫醇含量。通过乙酰胆碱酯酶测定，也可用于有机磷农药中毒的监测[1-3]。
DTNB可以修饰酶的SH基团，能够确定每个分子中SH基团的数量及其对酶活性的贡献[4]。以羟肉桂酰辅酶a:l-DOPA羟肉桂酰转移酶为模型，DTNB对转移酶反应几乎没有影响，可以很好地估计羟肉桂酰酰胺的形成，从而可以快速方便地收集反应速率数据和确定转移酶动力学参数[5]。DTNB改性通过诱导晶格中的极性相互作用促进了CcbP的结晶。DTNB介导的半胱氨酸修饰被证明对CcbP的整体结构和Ca2+结合几乎没有影响。DTNB修饰可为蛋白质修饰提供一种简单而通用的方法，从而提高结晶筛选的成功率[6]。
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DTNB使用说明 [1]: |
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标准溶液(10mm):避光处理，首先称出0.198克DTNB，在50 mM Na2HPO4 (pH 7.0)中配制50 mL溶液。将溶液保存在棕色或琥珀色的瓶子中，并保存在阴凉(4℃)，黑暗的地方备用。 |
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秀丽隐杆线虫总谷胱甘肽的测定 [2]: |
DTNB solution: 2 mg DTNB;3 ml KPE buffer; 现用现配，放冰上，用铝箔包裹（避光）。 KPE buffer (pH 7.5): ?6.8 g KH2PO4加500 ml无菌水，4℃保存;溶液B: 11.4 g K2HPO4-3H2O加500 ml无菌水，4℃保存;4℃保存，可保存1个月。 Glutathione reductase (GR) solution: 40 μl GR (250 U/ml);3 ml KPE buffer; 现用现配，放冰上。 GSH standards: Stock 2 mM GSH: :6.14 mg谷胱甘肽于10 ml KPE缓冲液中，1ml分装，在-20℃下保存1个月。从40 μM GSH (20 μl原液在1 ml KPE缓冲液中)开始连续稀释至0.625 μM。 NADPH solution: 2 mg NADPH;3 ml KPE buffer; 现用现配，放冰上，用铝箔包裹（避光）。 |
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用DTNB修饰酶[3]: |
仅供参考，请根据您的实验具体需要进行修改。 |
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参考文献: [1]. ELLMAN GL, COURTNEY KD, et,al. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol. 1961 Jul;7:88-95. doi: 10.1016/0006-2952(61)90145-9. PMID: 13726518. [2]. Caito SW, Aschner M. Quantification of Glutathione in Caenorhabditis elegans. Curr Protoc Toxicol. 2015;64(618):6.18.1-6.18.6. doi: 10.1002/0471140856.tx0618s64. PMID: 26309452; PMCID: PMC4545653. [3]. Nagaoka T, Hachimori A, Takeda A, Samejima T. DTNB modification of SH groups of isocitrate dehydrogenase from Bacillus stearothermophilus purified by affinity chromatography. J Biochem. 1977 Jan;81(1):71-8. doi: 10.1093/oxfordjournals.jbchem.a131452. PMID: 14937. |
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