Treuheit M J, Kirley T L
Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Ohio 45267-0575.
Anal Biochem. 1993 Jul;212(1):138-42. doi: 10.1006/abio.1993.1303.
The fluorescent cysteine derivative formed upon alkylation of proteins with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) is shown to be unstable under certain conditions. Although previously thought to be an irreversible reaction, the fluorescence and absorbance associated with the ABD-cysteine can be released from the protein by heating at basic pH in the presence of reductants. Reversal of labeling is accelerated by increasing the temperature, pH, and concentration of reductant. Upon reversal of labeling, a free sulfhydryl is regenerated, since the treated protein can subsequently be realkylated (without reductant) to approximately the same level with only ABD-F. The findings reported in this work are relevant to the use of this alkylating reagent prior to any procedure involving heating or long incubation under reducing conditions, including re-electrophoresis or enzymatic deglycosylation. Investigators using ABD-F or structurally similar reagents should be aware that the cysteine derivative formed is not stable under these conditions. The reversal of cysteine derivatization may also be useful as a method for replacing the ABD-cysteine adduct with a label more suitable for subsequent specialized procedures.
蛋白质与4-（氨磺酰基）-7-氟-2,1,3-苯并恶二唑（ABD-F）烷基化反应形成的荧光半胱氨酸衍生物在某些条件下显示出不稳定。尽管以前认为这是一个不可逆反应，但在碱性pH值和存在还原剂的情况下加热，与ABD-半胱氨酸相关的荧光和吸光度可以从蛋白质中释放出来。通过提高温度、pH值和还原剂浓度可加速标记的逆转。标记逆转后，会再生出一个游离巯基，因为经处理的蛋白质随后可以（在没有还原剂的情况下）仅用ABD-F重新烷基化至大致相同的水平。这项工作中报告的发现与在任何涉及加热或在还原条件下长时间孵育的程序（包括重新电泳或酶促去糖基化）之前使用这种烷基化试剂有关。使用ABD-F或结构相似试剂的研究人员应意识到，形成的半胱氨酸衍生物在这些条件下不稳定。半胱氨酸衍生化的逆转也可能作为一种用更适合后续专门程序的标记物取代ABD-半胱氨酸加合物的方法而有用。
