Leitner Alexander, Lindner Wolfgang
Christian Doppler Laboratory for Molecular Recognition Materials, Institute of Analytical Chemistry and Food Chemistry, University of Vienna, 1090 Vienna, Austria.
Anal Chem. 2005 Jul 15;77(14):4481-8. doi: 10.1021/ac050217h.
The reactivity of arginine residues in model proteins (ubiquitin, cytochrome c, myoglobin, ribonuclease A, lysozyme) was examined using a selective tagging reaction in combination with on-line monitoring of the reaction progress by electrospray ionization mass spectrometry (ESI-MS). The kinetics of this reaction, based on the cyclization of the guanidine group of arginine with 2,3-butanedione and phenylboronic acid at pH 8-10, allow the grouping of arginines in "exposed" or "partially buried" residues, because they differ substantially in their reaction rate constants for the conversion of the guanidine groups. The method allows one to differentiate between different protein conformations as shown for myoglobin and its apo form and native and reduced ribonuclease A: Removal of the heme group in myoglobin resulted in an increased reactivity for the two partially buried arginines. For RNAse A, quantitative reduction of the disulfide bonds lead to the exposure of an additional arginine residue and two different conformations of the reduced protein were observed by ESI-MS that could be distinguished according to their charge-state distribution. Experimentally obtained accessibilities were compared with solvent-accessibility data calculated from 3D structures and substantial agreement between both techniques was observed.
利用选择性标记反应结合电喷雾电离质谱（ESI-MS）在线监测反应进程，研究了模型蛋白（泛素、细胞色素c、肌红蛋白、核糖核酸酶A、溶菌酶）中精氨酸残基的反应活性。该反应基于精氨酸胍基在pH 8 - 10条件下与2,3 - 丁二酮和苯硼酸的环化反应动力学，可将精氨酸分为“暴露”或“部分埋藏”残基，因为它们胍基转化的反应速率常数有显著差异。该方法能够区分不同的蛋白质构象，如肌红蛋白及其脱辅基形式以及天然和还原型核糖核酸酶A：肌红蛋白中血红素基团的去除导致两个部分埋藏的精氨酸反应活性增加。对于核糖核酸酶A，二硫键的定量还原导致一个额外的精氨酸残基暴露，通过ESI-MS观察到还原型蛋白的两种不同构象，可根据其电荷态分布进行区分。将实验获得的可及性与从三维结构计算得到的溶剂可及性数据进行比较，观察到两种技术之间有相当程度的一致性。
