Dage J L, Sun H, Halsall H B
University of Cincinnati, Ohio 45221, USA.
Anal Biochem. 1998 Mar 15;257(2):176-85. doi: 10.1006/abio.1997.2552.
The chemical modification reagent diethylpyrocarbonate (DEPC) was used to modify alpha 1-acid glycoprotein (orosomucoid, OMD) under various conditions. The extents of DEPC modification of the histidine and tyrosine residues were followed by UV spectrophotometry. The resulting modified OMD was analyzed using enzyme digestion, reverse-phase HPLC, electrospray ionization-mass spectrometry (ESI/MS), and matrix-assisted laser desorption ionization time-of-flight-mass spectrometry (MALDI-TOF/MS). The inherent problem of instability of DEPC-modified histidine residues was overcome by adjusting the time scale of the postreaction processing of modified OMD. There were observed differences in reactivity of histidine 97 and histidine 100 that were consistent throughout the pH range 6-8. Furthermore, several lysine residues were modified and the amount of modification increased over the pH range 6-8. These experiments show that HPLC-ESI/MS and MALDI-TOF/MS analysis coupled with enzyme digestion provide the necessary information to describe the reaction of DEPC with OMD. In addition, the results provide the carbethoxy-histidine stability and histidine reactivity information of DEPC-modified OMD necessary for the design of experiments to characterize the drug binding properties of OMD.
使用化学修饰试剂焦碳酸二乙酯（DEPC）在不同条件下修饰α1-酸性糖蛋白（类粘蛋白，OMD）。通过紫外分光光度法追踪组氨酸和酪氨酸残基的DEPC修饰程度。使用酶消化、反相高效液相色谱、电喷雾电离质谱（ESI/MS）和基质辅助激光解吸电离飞行时间质谱（MALDI-TOF/MS）对所得修饰的OMD进行分析。通过调整修饰的OMD反应后处理的时间尺度，克服了DEPC修饰的组氨酸残基不稳定的固有问题。观察到组氨酸97和组氨酸100在pH值6 - 8的整个范围内反应性存在差异。此外，几个赖氨酸残基被修饰，修饰量在pH值6 - 8范围内增加。这些实验表明，HPLC-ESI/MS和MALDI-TOF/MS分析结合酶消化提供了描述DEPC与OMD反应所需的信息。此外，结果提供了表征OMD药物结合特性的实验设计所需的DEPC修饰的OMD的乙氧羰基组氨酸稳定性和组氨酸反应性信息。
