Rhee Steven S, Burke Donald H
Department of Chemistry, Indiana University, Bloomington, IN 47405-7102, USA.
Anal Biochem. 2004 Feb 1;325(1):137-43. doi: 10.1016/j.ab.2003.10.019.
We assessed the utility of the sulfhydryl reductant Tris(2-carboxyethyl)phosphine (TCEP) for both nucleic acid and thiophosphate chemistry, including its effects on organomercurial gel electrophoresis, RNA catalysis, RNA backbone stability, and the intrinsic stability of TCEP. The sulfhydryls of dithiothreitol (DTT) compete with thiophosphates for binding to the mercury within [(N-acryloylamino)phenyl] mercuric chloride (APM) polyacrylamide gels, whereas millimolar concentrations of TCEP gave no difference in the fraction of thiophosphorylated RNA retained on the APM interface relative to samples containing no reductant. Ribozyme activity in TCEP, assessed by the self-thiophosphorylating Kin.46 ribozyme, was unaffected by the presence of DTT or TCEP or by the absence of reductant, as measured on APM gels and evaluated by Michaelis-Menten kinetics. Unexpectedly, TCEP more than doubled the half-life of full-length RNA at 50 and 70 degrees C, whether in 5 or 50mM MgCl(2), relative to DTT and the absence of reductant. Under these same conditions, the 5(')-thiophosphate showed negligible decay, and TCEP was more stable than DTT. TCEP thermostability was equivalent in the presence of 5 or 50mM MgCl(2) and 10mM adenosine or ATP.
我们评估了巯基还原剂三（2-羧乙基）膦（TCEP）在核酸和硫代磷酸化学中的效用，包括其对有机汞凝胶电泳、RNA催化、RNA主链稳定性以及TCEP固有稳定性的影响。二硫苏糖醇（DTT）的巯基与硫代磷酸竞争结合[(N-丙烯酰氨基)苯基]氯化汞（APM）聚丙烯酰胺凝胶中的汞，而毫摩尔浓度的TCEP相对于不含还原剂的样品，在APM界面上保留的硫代磷酸化RNA比例没有差异。通过自硫代磷酸化的Kin.46核酶评估，TCEP中的核酶活性不受DTT或TCEP的存在或还原剂缺失的影响，这是在APM凝胶上测量并通过米氏动力学评估的。出乎意料的是，无论在5或50mM MgCl₂中，相对于DTT和无还原剂的情况，TCEP使全长RNA在50和70摄氏度下的半衰期增加了一倍多。在相同条件下，5'-硫代磷酸的衰变可忽略不计，且TCEP比DTT更稳定。在5或50mM MgCl₂以及10mM腺苷或ATP存在的情况下，TCEP的热稳定性相当。
