Tris (2-carboxyethyl) phosphine (TCEP) 是一种用于分子生物学和蛋白质生物化学研究的还原试剂。

TCEP Chemical Structure (PubChem ID: 119411)

在制备用于凝胶电泳的蛋白样品时，研究者经常在变性蛋白中加入TCEP。TCEP也可用于蛋白质的长期储存和样品的制备，在许多其他应用中，例如:

• -蛋白质偶联conjugation或标记
• 毛细管电泳-激光诱导荧光检测(CE-LIF)
• 蛋白质色谱纯化
• 在DNA和RNA分离纯化

蛋白质中的巯基和二硫键

肽和蛋白质由氨基酸组成，以肽键连接在一起。

巯基或硫醇(R-SH)存在于含有氨基酸半胱氨酸残基的蛋白质中。当两个巯基相互靠近时，它们可以通过氧化反应形成二硫桥(R-S-S-R ‘)。

重复的半胱氨酸残基和二硫桥通常存在于膜结合受体的胞外结构域。许多细胞外或分泌蛋白和多肽也有二硫桥，包括一些激素、酶、血浆蛋白、抑制剂和毒液蛋白。这种二硫桥结构对蛋白质的生物学功能以及蛋白质二级和三级结构的稳定具有重要意义。

蛋白的变性. 还原剂的加入使二硫键断裂并使蛋白质变性

为了研究蛋白质的结构和功能，研究人员经常将每个二硫键作为一个酶片段来分析，并研究一个键与其他键之间的连接。当半胱氨酸残基紧密聚在一起时，很难将二硫键之间的肽链分开。

加入还原剂破坏二硫键是解决这一问题的一种方法。还原剂是将一个(或多个)电子提供给另一个化合物的化合物。还原剂的例子有TCEP、DTT、2-巯基乙醇和2-巯基乙胺。

TCEP 和还原反应

TCEP是一种有效的二硫键裂解试剂。TCEP在水溶液中稳定，反应性强，对二硫化物结构有选择性。

Nucleophilic Substitution by the Phosphorus Atom of TCEP. 1. The phosphorus atom attacks one sulfur atom along the S-S bond. 2. A thioalkoxyphosphonium cation and a sulfhydryl anion are formed. 3. A rapid hydrolysis releases the second sulfhydryl molecule and the phosphine oxide.

There are two main steps in the breaking of disulfide bridges and forming a free sulfhydryl using TCEP:

1. cleavage of the S-S bond
2. oxidation of the phosphine and release of sulfhydryl

TCEP Reaction. 1. Cleavage of S-S bond and formation of a compound containing a disulfide bridge structure. 2. Release of sulfhydryl molecules and formation of oxidized phosphine.

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TCEP or DTT?

• Compared to DTT and other reducing agents, TCEP has a more neutral odor and TCEP is more resistant to oxidation by air.

• TCEP is a useful reductant with a wide pH range (1.5-8.5) and it is more stable than DTT at pH above 7.5 (biological pH).

• TCEP reaction is irreversible, whereas DTT reaction is reversible.

• Compared to DTT, TCEP is preferred for labeling cysteine residues with maleimides. DTT contains thiols and shows reactivity with maleimides, which reduces the labeling efficiency.

• You can use either TCEP or DTT to label with iodoacetamide. TCEP and DTT only lower labeling efficiency of iodoacetamide by a very small amount. But the ratio of dye over iodoacetamide, pH, and temperature may affect the efficiency of iodoacetamide labeling.

• TCEP is more stable than DTT in the presence of metal ions (such as Fe3+ and Ni2+). You can use TCEP instead of DTT to perform metal-ion affinity chromatography.

• In the presence of EGTA, DTT’s stability increases, but TCEP’s stability decreases. The TCEP instability is due to metal chelators (such as EGTA) catalyzing TCEP oxidation.

• You can use either TCEP or DTT to label with iodoacetamide. TCEP and DTT only lower labeling efficiency of iodoacetamide by a very small amount. But the ratio of dye over iodoacetamide, pH, and temperature may affect the efficiency of iodoacetamide labeling.

Note: TCEP does not contain thiols so there is no need to remove it from the labeling reaction. But TCEP may react with maleimides under certain conditions, such as acidic conditions, at 20°C, and too much TCEP for labeling of proteomic samples.

If you decide to remove excess TCEP before the addition of maleimides, you can use:

- dialysis

- TCEP-immobilized resin

- column chromatography

-4-azidobenzoic acid

Buffers to Dissolve TCEP

• You can use many types of common buffers during preparation of protein samples to dissolve TCEP at a wide range of pH levels.

• TCEP tends to be unstable around neutral pH in phosphate buffers. If you need to use phosphate buffers with TCEP, prepare it immediately before use

For examples, you can prepare TCEP in:

- Tris-HCl buffer for protein labeling or protein purification

- Borate buffer for capillary electrophoresis-laser induced fluorescence

- Hepes buffer for chromatography

How to Prepare 0.5 M TCEP Stock Solution

1.Weigh 5.73 g of TCEP (TCEP-HCL, GoldBio Catalog # TCEP)

2.Add 35 ml of cold molecular biology grade water to the vial, and dissolve the TCEP. This resulting solution is very acidic, with an approximate pH of 2.5.

3.Bring the solution to pH 7.0 with 10 N NaOH or 10 N KOH.

4.Bring the resulting solution to 40 ml with molecular biology grade water.

5.Aliquot into 1 ml into freezer tubes and store at -20°C.

Note: This protocol allows you to prepare TCEP working concentrations and 10X stock solutions in the buffer of your choice before use. You can use TCEP as a substitute for DTT at a final concentration of 50mM.

Note: TCEP cannot be used for isoelectric focusing due to its charge in solution.

Note: Cover the tubes with aluminum foil, because TCEP is light sensitive.

Note: Stock solutions are stable for 3 months at -20°C.

致谢

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