Quantitative determination and reversible modification of thiols using imidazolidine biradical disulfide label
Quantitative determination and reversible modification of thiols using imidazolidine biradical disulfide label
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DOI：
10.1016/s0165-022x(97)00035-3
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发表时间：
1997-09-25
影响因子：
--
通讯作者：
Zimmer, G
中科院分区：
文献类型：
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作者：
Khramtsov, VV;Yelinova, VI;Zimmer, G
在此之前，我们报道了一种定量测定巯基的ESR方法。该方法是基于应用咪唑啉双自由基二硫化物标记，R1 S-SR 1，它参与了巯基-二硫键交换反应，然后在ESR谱中发生显着变化。在生理条件下应用R1 S-SR 1的缺点之一是需要与硫醇含量相比过量的双自由基，这导致硫醇的消耗和所研究系统的不可逆损伤。本文提出了咪唑烷双自由基二硫试剂R_2S-SR_2用于巯基化合物的ESR测定，并为其应用提供了实验依据。该标记具有以前使用的二自由基二硫化物R1 S-SR 1的优点，例如即使在不透明的样品中也对低至1 μ M的硫醇具有高灵敏度，并且可能用于蛋白质和酶的可逆修饰。R2 S-SR 2的特殊性质是其ESR谱的pH敏感性、咪唑烷自由基片段对生物还原剂的更高稳定性以及足以在生理条件下进行硫醇测定的低浓度标记。后者使得有可能使用ESR光谱法在生物系统中进行非侵入性硫醇测量，包括体内应用。(C)1997年Elsevier Science B.V.
Earlier we reported an ESR method of quantitative determination of sulfhydryl groups. The method is based on the application of the imidazoline biradical disulfide label, R1S-SR1, which participates in the reaction of thiol-disulfide exchange followed by dramatic changes in ESR spectra. One of the disadvantages of the application of R1S-SR1 at physiological conditions is the requirement of excess of the biradical compared with thiol content which results in the consumption of the thiols and irreversible damage of the system under study. In the present paper we propose imidazolidine biradical disulfide reagent, R2S-SR2, for ESR determination of thiols and provide an experimental basis for its application. This label has the advantages of the previously used biradical disulfide, R1S-SR1, such as high sensitivity down to 1 mu M of thiols even in opaque samples and could possibly be used for reversible modification of proteins and enzymes. The particular properties of the R2S-SR2 are pH-sensitivity of its ESR spectrum, higher stability of the imidazolidine radical fragment towards biological reductants and low concentration of the label sufficient for thiol determination at physiological conditions. The latter makes it possible to use ESR spectroscopy for non-invasive thiol measurements in biological systems, in vivo applications included. (C) 1997 Elsevier Science B.V.