Bregere Catherine, Rebrin Igor, Sohal Rajindar S
Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA, USA.
Methods Enzymol. 2008;441:339-49. doi: 10.1016/S0076-6879(08)01219-6.
Oxygen and nitrogen centered reactive species can cause specific structural modifications in amino acids and proteins, such as the addition of a nitro group onto aromatic residues. Heretofore, studies on protein nitration have mainly focused on the in vitro and in vivo nitro addition to tyrosine residues (3-nitrotyrosine or 3NT), whereas the formation of nitrotryptophan in proteins in vivo and/or its functional significance has remained quite obscure. A novel structural modification, involving the addition of nitro and hydroxy groups to tryptophan, has been detected in the mitochondrial protein succinyl-CoA:3-oxoacid CoA transferase (SCOT) in rat heart. Modified SCOT accumulated progressively with age, which was associated with an elevation of its activity. The specific biochemical properties of this novel amino acid were characterized by a combination of HPLC-electrochemical detection and mass spectrometric analysis. This chapter describes the experimental steps involved in the characterizations and a procedure for the synthesis of nitrohydroxytryptophan. Similar methodology can be applied to the identification of nitrohydroxytryptophan in other proteins.
以氧和氮为中心的活性物质可导致氨基酸和蛋白质发生特定的结构修饰，比如在芳香族残基上添加硝基。迄今为止，关于蛋白质硝化作用的研究主要集中在体外和体内酪氨酸残基的硝基添加（3-硝基酪氨酸或3NT），而蛋白质中硝基色氨酸在体内的形成及其功能意义仍相当模糊。在大鼠心脏的线粒体蛋白琥珀酰辅酶A：3-氧代酸辅酶A转移酶（SCOT）中检测到一种新的结构修饰，即色氨酸上添加了硝基和羟基。修饰后的SCOT随年龄增长而逐渐积累，这与其活性升高有关。这种新型氨基酸的特定生化特性通过高效液相色谱-电化学检测和质谱分析相结合的方法得以表征。本章描述了表征过程中涉及的实验步骤以及硝基羟基色氨酸的合成方法。类似的方法可用于鉴定其他蛋白质中的硝基羟基色氨酸。
