A comparison between the sulfhydryl reductants tris(2-carboxyethyl)phosphine and dithiothreitol for use in protein biochemistry
A comparison between the sulfhydryl reductants tris(2-carboxyethyl)phosphine and dithiothreitol for use in protein biochemistry
复制标题
DOI：
10.1006/abio.1999.4203
复制
发表时间：
1999-08-15
影响因子：
2.9
通讯作者：
Selvin, PR
中科院分区：
文献类型：
--
作者：
Getz, EB;Xiao, M;Selvin, PR
新引入的巯基还原剂三（2-羧乙基）膦（TCEP）是一种潜在的有吸引力的替代常用的二硫苏糖醇（DTT）。我们比较了DTT和TCEP在蛋白质生物化学中的重要性质，使用马达酶肌球蛋白作为示例蛋白质。还原剂同样保留肌球蛋白的酶活性，这是敏感的巯基氧化。当用外源性探针标记时，DTT抑制马来酰亚胺与肌球蛋白的连接，并且必须在标记前去除。相比之下，马来酰亚胺附着到肌球蛋白在TCEP的存在下实现，虽然效率低于没有还原剂。令人惊讶的是，碘乙酰胺附着肌球蛋白几乎不受任何还原剂在低浓度（0.1 mM）。在利用氮氧自旋标记的电子顺磁共振（EPR）光谱中，TCEP是非常有利的：自旋标记在TCEP中的稳定性是DTT的两到四倍，从而缓解了EPR中的长期问题。在蛋白质纯化过程中，从Ni 2+亲和柱洗脱的Ni 2+浓度污染的蛋白质引起DTT的快速氧化而不影响TCEP。对于蛋白质的长期储存，在缓冲液中没有金属螯合物如EGTA的情况下，TCEP比DTT显著更稳定，而如果存在金属螯合物，DTT更稳定。因此，TCEP具有优于DTT的优点，尽管还原剂的选择是特定于应用的。(C)北京：科学出版社.
The newly introduced sulfhydryl reductant tris(2-carboxyethyl)phosphine (TCEP) is a potentially attractive alternative to commonly used dithiothreitol (DTT). We compare properties of DTT and TCEP important in protein biochemistry, using the motor enzyme myosin as an example protein. The reductants equally preserve myosin's enzymatic activity, which is sensitive to sulfhydryl oxidation. When labeling with extrinsic probes, DTT inhibits maleimide attachment to myosin and must be removed before labeling. In contrast, maleimide attachment to myosin was achieved in the presence of TCEP, although with less efficiency than no reductant. Surprisingly, iodoacetamide attachment to myosin was nearly unaffected by either reductant at low (0.1 mM) concentrations. In electron paramagnetic resonance (EPR) spectroscopy utilizing nitroxide spin labels, TCEP is highly advantageous: spin labels are two to four times more stable in TCEP than DTT, thereby alleviating a long-standing problem in EPR. During protein purification, Ni2+ concentrations contaminating proteins eluted from Ni2+ affinity columns cause rapid oxidation of DTT without affecting TCEP. For long-term storage of proteins, TCEP is significantly more stable than DTT without metal chelates such as EGTA in the buffer, whereas DTT is more stable if metal chelates are present. Thus TCEP has advantages over DTT, although the choice of reductant is application specific. (C) 1999 Academic Press.