ellman测巯基

埃尔曼的试剂，5,5'二硫代双-（二硝基苯甲酸），5克分子量：396.35CAS号：69-78-3使用半胱氨酸标准精确量化巯基程序材料的制备1 反应缓冲液：0.1M的磷酸钠，pH 8.0，含有1 mM的EDTA半胱氨酸标准品，分子量136.2 ：配成一定浓度梯度3 Ellman试剂溶液：1mL反应缓冲液4毫克溶解DTNB程序3在各试管中加入250μL各浓度的标准工作液或待测物溶液注：对于未知的样品需要稀释，以便使其浓度标准曲线的工作范围之内（理想的浓度为0.1-1.0）。

4在室温混合和反应15分钟。

在412 nm处测量吸光度。

根据得到数值生成一个标准曲线，测定待测样品由标准曲线计算SH含量摩尔吸光系数的程序精确量化的巯基的材料的制备1 反应缓冲液：0.1M的磷酸钠，pH 8.0，含有1 mM的EDTAEllman试剂溶液：1mL反应缓冲液4毫克溶解DTNB测量吸光度去若干试管，每管加入50μL的Ellman试剂溶液和2.5mL反应缓冲液。

加入250μL待测样品,空白添加250μL反应缓冲液在室温混合和反应15分钟。

分光光度计412nm测定吸光度。

根据TNB的摩尔消光系数(14,150)计算巯基含量该国能在412nm的合成，有一个相对比较激烈的吸光度同时二硫化物。

由于蛋白质的巯基，以国能形成化学计量为1:1，国能形成可以用来评估硫醇数目。

在变性剂的情况下，唯一能够找到的硫醇的反应，而残留的chaotropic代理人在场的总数减少胱氨酸目前可以衡量的。

经处理后减少的蛋白质与chaotropes及DTNB能够产生的半胱氨酸总数（半胱氨酸巯基加半胱氨酸-β-半胱氨酸）。

- -反应是敏感的碱性pH值（俄亥俄州）与RS竞争），酸性pH值（二硫化物可以打破），氧（R的巯基再氧化），以及温度（热致变色）。

因此，通常的反应是进行了过多的DTNB的蛋白质，在中性pH值固定的温度，erature，有时在厌氧条件。

此外，国能是敏感的各种离子的缓冲，所以采用消光系数硫醇数量计算必须正确匹配的反应条件。

这也是进行了存在的真实反应尿素或盐酸胍的变性剂（盐酸胍）。

下表概述在DTNB的和国能在各种条件下的一些有用的数据：-------------------------------------------------- ------------------------复方最大的lambda（pH值?7）灭绝：（以磷酸盐）（以磷酸三）（以盐酸胍）--------- ----------------- -------------------- ---- ------ ----------DTNB的324纳米17780 16600 ----国能---- 412纳米14,150 13,700-------------------------------------------------- ------------------------消光系数以每摩尔每厘米长度的路径，30degC单位国能在降低盐酸胍存在的灭绝是由于在移位拉姆达从412到422最大纳米。

在DTNB的灭绝是在412纳米212/M/cm，规模虽小但可衡量的。

=-=-=-=-=-=-=-==协议==-=-=-=-=-=-=-=联合解决方案：蛋白质溶液（本地或以前减少，脱盐）（?5 - 0.1M的磷酸钾缓冲液，pH值7.4 40uM）DTNB的股票（20毫米0.1M的KPO4，pH值7.4，分子量396.35; 7.93毫克/mL）（DTNB的很慢进入的解决办法;涡定期在2-3小时内实现完整的解决方案，这将是很轻，颜色黄色）尿素或盐酸胍（高纯度，结晶固体）主要设备：双光束分光光度计动力学模式设置在412nm恒温细胞，30degreesC程序：示例1 = 1.0mL细胞蛋白质溶液+ 50μL20毫米DTNB的引用单元1 = 1.0mL+ 50μL DTNB的缓冲区记录吸光度前，DTNB的除了“零”。

跟随在412 nm的反应在30degC不断。

记录过去的时候，反应达到最大值。

如果任何被视为重要的下坡最大吸收后到达，这将需要更正为了获得可访问硫醇的正确数目。

在第二个实验中，添加结晶盐酸胍到您的蛋白质溶液，以达到最终浓度约4米。

请注意体积变化以及由此产生的蛋白显着的稀释解决方案！示例2 = 1.0mL细胞蛋白质溶液（4 M盐酸胍）+ 20 mM的DTNB的50μL 引用单元2 = 1.0mL缓冲液（4 M盐酸胍）+ 50μL20毫米DTNB的同样，在30摄氏度的纪录外径412纳米，跨越高峰吸收。

本实验将产生硫醇总数减少。

样品细胞3 = 1.0mL预变性，减少，蛋白质和透析+ 50μL20毫米DTNB的引用单元3 = 1.0mL缓冲液（4 M盐酸胍）+ 20 mM的DTNB的50μL同样，记录OD值随时间在30degC 412 nm到峰值吸收和超越。

本实验将产生的蛋白质的半胱氨酸残基的总数。

=-=-=-=-=-=-=-=-=-=数据分析= ==-=-=-=-=-=-=-=-=-=计算硫醇数修改（或浓度除外）使用在412 nm的最大吸收测量（或修正值除外）啤酒的法律，以及适当的消光系数;鸿沟这个值由精确已知的摩尔反应蛋白浓度比色皿三个实验的每一个。

这将使你获得硫醇，总半胱氨酸巯基，总半胱氨酸巯基以及半胱氨酸-β-半胱氨酸。

这样做，每个实验中重复或一式三份，平均分别复制。

如果您的数字统计整数，你就大功告成了。

如果不考虑重复该议定书，但采用厌氧（脱气）缓冲区的所有反应。

=-=-=-=-=-=-=-=-=-=-=一些参考= ==-=-=-=-=-=-=-=-=-=-=“甲硫醇为确定低浓度比色法”埃尔曼，吉尔（1958）拱。

生化。

Biophys。

74，443-450。

“重新评估埃尔曼的试剂”谜语，密码，布莱克利，汤顿和泽纳，二（1983）方法酶学。

91，49-60。

对兔血红素结合半胱氨酸残身份“研究肝微粒体细胞色素P - 450同工酶2“黑色，SD和库恩，美兆（1985）生物化学，Biophys。

水库。

Commun。

128，82-89。

Hi All,I had a couple of folks ask for my opinion and method for use of Ellmen'sreagent (DTNB) in the detemination of protein thiols (Cys-SH). Below, I've included a protocol, references, and general information that should behelpful to all who anticipate using this method. Best Regards, Shaun=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-== Use of Ellman's Reagent for Determination of Protein Thiols ==-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-==-=-=-=-=-=-=-== Background ==-=-=-=-=-=-=-=Ellman's reagent or 5,5'-dithiobis(2-nitrobenzoate) (DTNB) is a symmetricalaryl disulfide which readily undergoes the thiol-disulfide interchangereaction in the presence of a free thiol:- - -COO COO COO\___ ___/ \___/ \ / \ | - / \ |O N- O -S-S- O -N O + Cys-S ----> O N- O -S-S-Cys (Mixed2 \___/ \___/ 2 | 2 \___/ | disulfide)(Excess) +-COO\___/ \ -(2-nitro-5-O N- O -S thiobenzoate)\___/(TNB)The TNB dianion has a relatively intense absorbance at 412nm compared toboth disulfides. Because the stoichiometry of protein thiol to TNB formedis 1:1, TNB formation can be used to assess the number of thiols present.In the absence of denaturants, only accessible thiols will react, whereasin the presence of chaotropic agents the total number of reduced Cys residues present can be measured. Reduction of the protein followed by treatmentwith chaotropes and DTNB can yield the total number of cysteines (Cys-SHplus Cys-S-S-Cys).--The reaction is sensitive to alkaline pH ( OH) competes with R-S ),acidic pH (disulfides can be broken), oxygen (reoxidation of R-SH), and temperature (thermochromism). Consequently, the reaction is usuallycarried out with an excess of DTNB to protein, at neutral pH, fixed temp-erature, and sometimes under anaerobic conditions. Furthermore, TNB is sensitive to various buffer ions, so the extinction coefficient used tocalculate number of thiols must be properly matched to the reactionconditions. This is also true of reactions carried out in the presence ofthe denaturants urea or guanidine (GuHCl). The following table summarizes some useful data on DTNB and TNB under various conditions:--------------------------------------------------------------------------Compound lambda max (pH~7) Extinction: (in PO4) (in Tris) (in GuHCl) --------- ----------------- -------------------- ---------- ----------DTNB 324 nm 17,780 16,600----TNB 412 nm 14,150 ----13,700--------------------------------------------------------------------------Extinction coefficients are in units of per Molar per cm path length, 30degC Decreased TNB extinction in the presence of GuHCl is due to a shift in thelambda max from 412 to 422 nm. The extinction of DTNB at 412 nm is 212/M/cm, small but measurable.=-=-=-=-=-=-=-== Protocol ==-=-=-=-=-=-=-=Stock Solutions:Protein solution (native or previously reduced and desalted)(~5-40uM in 0.1M potassium phosphate buffer, pH 7.4) DTNB stock (20 mM in 0.1M KPO4, pH 7.4; MW 396.35; 7.93 mg/ml)(DTNB is very slow to go into solutions; vortex periodicallyover the period of 2-3 hours to achieve complete solution,which will be very light yellow in color)Urea or Guanidine-HCl (high purity, crystalline solids)Major Equipment:Dual beam spectrophotometer set in kinetic mode at 412nmThermostated cells, 30degreesCProcedure:Sample cell 1 = 1.0 mL protein solution + 50 uL 20mM DTNBReference cell 1 = 1.0 mL buffer + 50 uL DTNBRecord the absorbance before addition of the DTNB as "zero". Followthe reaction at 412 nm at 30degC continuously. Record past the time whenthe reaction reaches a maximum. If any significant down slope is seenafter the maximum absorbance is reached, this will need to be corrected forin order to get the correct number of accessible thiols.In the second experiment, add crystalline Guanidine-HCl to yourprotein solution to achieve a final concentration of about 4M. Note thevolume change and the resulting significant dilution of your proteinsolution!Sample cell 2 = 1.0 mL protein solution (+4M GuHCl) + 50 uL 20 mM DTNBReference cell 2 = 1.0 mL buffer (+4M GuHCl) + 50 uL 20mM DTNBAgain, record OD 412 nm at 30 degC up to and beyond peak absorbance.This experiment will yield the total number of reduced thiols.Sample cell 3 = 1.0 mL pre-denatured,reduced, and dialyzed protein+ 50 uL 20mM DTNBReference cell 3 = 1.0 mL buffer (+4M GuHCl) + 50 uL 20 mM DTNBAgain, record OD 412 nm at 30degC vs time to peak absorbance and beyond. This experiment will yield the total number of Cys residues in the protein.=-=-=-=-=-=-=-=-=-== Data Analysis ==-=-=-=-=-=-=-=-=-=Calculate the number of thiols modified (or concentration thereof) usingthe maximum absorbance at 412 nm measured (or the corrected value thereof), Beer's Law, and the appropriate extinction coefficient; divide this valueby the accurately known molar protein concentration in the reaction cuvettefor each of the three experiments. This will give you accessible thiols,total Cys-SH, and total Cys-SH plus Cys-SS-Cys. Do each experiment in duplicate or triplicate and average the respective replicates. If yournumbers are statistically integers, you're done. If not, consider repeatingthe protocol, but using anaerobic (degassed) buffers for all reactions.=-=-=-=-=-=-=-=-=-=-== Some References ==-=-=-=-=-=-=-=-=-=-="A colorimetric method for determining low concentrations of mercaptans" Ellman, G.L. (1958) Arch. Biochem. Biophys. 74, 443-450. "Reassessment of Ellman's reagent" Riddles, P.W., Blakeley, R.L., and Zerner, B. (1983) Methods Enzymol. 91, 49-60."Studies on the identity of the heme-binding cysteinyl residue in rabbitliver microsomal cytochrome P-450 isozyme 2" Black, S.D. and Coon, M.J. (1985) Biochem, Biophys. Res. Commun. 128, 82-89.。