NITROSYLATED HEME IN MYOGLOBIN & HEMOGLOBIN BY ELECT IONIZATION MASS SPECT

使用电喷雾电离质谱（ESI-MS），我们有 证明了连接到血红素部分的NO的直接观察 在马心肌红蛋白（Mb）和？-然后呢？人的锁链 血红蛋白（Hb）。 结果发现，在很窄的范围内， 条件合谋使Fe-NO相互作用的观察 挑战，这大概就是为什么早期的尝试， 其他研究小组检测完整的Fe-NO产品的质量 光谱法是不成功的。 对于Mb和Hb，质量偏移为 观察到与血红素蛋白的NO修饰一致。 图1示出了根据本发明的方法制备的样品中Mb和Hb的脱辅基蛋白部分的ESI质谱图。 NO的存在表明， 多肽骨架。 为了验证完整的假设 亚硝基化血红素基团可通过ESI-MS观察到， 研究了模型金属卟啉。 为了进一步验证我们的假设 血红素-NO峰是由于血红素亚硝基化， 不包含来自NO修改的显著贡献 多肽骨架，我们确定了必要的ESI-MS条件 用于观察Hb中Cys残基的S-亚硝化。我们的研究结果 证明（一旦建立正确的条件）ESI-MS是一种 一个强大的工具，用于检测完整的Fe-NO相互作用， 蛋白质和卟啉。 一篇描述这项工作的论文发表在 R.K. Upmacis，D.P. Hajjar，B.T. Chait和U.A.米尔扎“直接 肌红蛋白和血红蛋白中亚硝基化血红素的电镜观察 Electrospray Ionization-Mass Spectrometry”J. Am. Chem. Soc. 119 （1997）10424-10429。

Using electrospray ionization mass spectrometry (ESI-MS), we have demonstrated the direct observation of NO attached to the heme moiety in horse heart myoglobin (Mb) and in the ?- and ?- chains of human hemoglobin (Hb). It was found that a narrow range of ESI-MS conditions conspire to make observation of Fe-NO interactions challenging, and this is presumably the reason why earlier attempts by other research groups to detect intact Fe-NO products by mass spectrometry were unsuccessful. For Mb and Hb, mass shifts are observed that are consistent with NO modification of the hemoproteins. ESI mass spectra of the apoprotein portions of Mb and Hb in the presence of NO demonstrated the absence of NO modification of the polypeptide backbones. To test the hypothesis that intact nitrosylated heme groups are observable by ESI-MS, a nitrosylated model metalloporphyrin was studied. To test further our hypothesis that the hemoprotein-NO peaks are due to heme nitrosylation a nd contain no significant contributions from NO modification of the polypeptide backbone, we determined the ESI-MS conditions necessary for observing S-nitrosation of Cys residues in Hb. Our findings demonstrate that (once correct conditions are established) ESI-MS is a powerful tool for the detection of intact Fe-NO interactions in proteins and porphyrins. A paper describing this work was published R.K. Upmacis, D.P. Hajjar, B.T. Chait, and U.A. Mirza "Direct Observation of Nitrosylated Heme in Myoglobin and Hemoglobin by Electrospray Ionization-Mass Spectrometry" J. Am. Chem. Soc. 119 (1997) 10424-10429.