Reaction of tris(2-carboxyethyl)phosphine (TCEP) with maleimide and α-haloacyl groups:: Anomalous elution of TCEP by gel filtration
Reaction of tris(2-carboxyethyl)phosphine (TCEP) with maleimide and α-haloacyl groups:: Anomalous elution of TCEP by gel filtration
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DOI：
10.1006/abio.2000.4609
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发表时间：
2000-06-15
影响因子：
2.9
通讯作者：
Lees, A
中科院分区：
文献类型：
--
作者：
Shafer, DE;Inman, JK;Lees, A
硫代肽常用于肽偶联物的制备，特别是免疫原的制备。通常，合成的硫醇肽被部分氧化为二硫化物，必须在与携带载体的亲电基团偶联之前被还原。二硫苏糖醇（DTT） 2通常用于还原肽二硫化物，但在还原的肽可以使用之前，必须从溶液中去除该试剂，通常是通过凝胶过滤。然而，低分子量（如1500 Da）肽可能包含在凝胶过滤基质中，因此不容易通过凝胶过滤从DTT中分离出来，因为后者经常大量过量使用。因此，可能必须采用替代方法（如离子交换）来分离还原肽。三烷基膦是二硫化物的强力和选择性还原剂(1)，但直到最近，由于它们有臭味和/或不溶于水，它们还没有在生命科学中广泛使用(2)。三-（2-羧基乙基）磷酸盐酸盐（TCEP）是一种无味且可溶于水的试剂，因此使用安全方便（(2),Pierce Chemical Co., Molecular Probes）。即使在酸性条件下，TCEP也能快速和化学计量地还原大多数肽或其他二硫化物(2)。此外，TCEP的水溶液相当稳定（2,3），TCEP的功能浓度
Thiolated peptides are frequently used in the preparation of peptide conjugates, especially in the preparation of immunogens. Usually, synthetic thiol peptides are partially oxidized to disulfides and must be reduced before being coupled to a carrier-bearing electrophilic groups. Dithiothreitol (DTT) 2 is commonly employed to reduce peptide disulfides for this purpose, but this reagent must be removed from the solution, usually by gel filtration, before the reduced peptide can be used. However, low molecular weight (eg, 1500 Da) peptides may be included in the gel filtration matrix and therefore not easily separated by gel filtration from the DTT, since the latter is often used in large excess. Therefore, alternative means (eg, ion-exchange) may have to be employed to isolate the reduced peptide. Trialkylphosphines are powerful and selective reductants for disulfides (1), but until recently, they have not been commonly used in the life sciences due to their being malodorous and/or water insoluble (2). The commercial availability of tris-(2-carboxyethyl) phosphine hydrochloride (TCEP), which is odorless and water soluble, makes this reagent safe and convenient to use ((2), Pierce Chemical Co., Molecular Probes). TCEP rapidly and stoichiometrically reduces most peptide or other disulfides, even under acidic conditions (2). Furthermore, aqueous solutions of TCEP are reasonably stable (2, 3), and the functional concentration of TCEP