Hazen S L, Gaut J P, Hsu F F, Crowley J R, d'Avignon A, Heinecke J W
Department of Medicine, Washington University, St. Louis, Missouri 63110, USA.
J Biol Chem. 1997 Jul 4;272(27):16990-8. doi: 10.1074/jbc.272.27.16990.
Activated human phagocytes employ the myeloperoxidase-H2O2-Cl- system to convert L-tyrosine to p-hydroxyphenylacetaldehyde (pHA). We have explored the possibility that pHA covalently reacts with proteins to form Schiff base adducts, which may play a role in modifying targets at sites of inflammation. Because Schiff bases are labile to acid hydrolysis, prior to analysis the adducts were rendered stable by reduction with NaCNBH3. Purified pHA reacted with Nalpha-acetyllysine, an analog of protein lysine residues. The reduced reaction product was identified as Nalpha-acetyl-Nepsilon-(2-(p-hydroxyphenyl)ethyl)lysine by 1H NMR spectroscopy and mass spectrometry. The compound Nepsilon-(2-(p-hydroxyphenyl)ethyl)lysine (pHA-lysine) was likewise identified in acid hydrolysates of bovine serum albumin (BSA) that were first exposed to myeloperoxidase, H2O2, L-tyrosine, and Cl- and then reduced with NaCNBH3. Other halides (F-, Br-, I-) and the pseudohalide SCN- could not replace Cl- as a substrate in the myeloperoxidase-H2O2-L-tyrosine system. In the absence of the enzymatic system, pHA-lysine was detected in reduced reaction mixtures of BSA, L-tyrosine, and reagent HOCl. In contrast, pHA-lysine was undetectable when BSA was incubated with L-tyrosine and HOBr, peroxynitrite, hydroxyl radical, or a variety of other peroxidases, indicating that the aldehyde-protein adduct was selectively produced by HOCl. Human neutrophils activated in the presence of tyrosine also modified BSA lysine residues. pHA-lysine formation required L-tyrosine and cell activation; it was inhibited by peroxidase inhibitors and catalase, implicating myeloperoxidase and H2O2 in the reaction pathway. pHA-lysine was detected in inflamed human tissues that were reduced, hydrolyzed, and then analyzed by mass spectrometry, indicating that the reaction of pHA with proteins may be of physiological importance. These observations raise the possibility that the identification of pHA-lysine in tissues will pinpoint targets where phagocytes inflict oxidative damage in vivo.
活化的人类吞噬细胞利用髓过氧化物酶-H₂O₂-Cl⁻系统将L-酪氨酸转化为对羟基苯乙醛（pHA）。我们探讨了pHA与蛋白质共价反应形成席夫碱加合物的可能性，这可能在炎症部位修饰靶标中发挥作用。由于席夫碱对酸水解不稳定，在分析之前，通过用NaCNBH₃还原使加合物稳定。纯化的pHA与蛋白质赖氨酸残基的类似物Nα-乙酰赖氨酸反应。通过¹H NMR光谱和质谱鉴定还原后的反应产物为Nα-乙酰-Nε-(2-(对羟基苯基)乙基)赖氨酸。同样在首先暴露于髓过氧化物酶、H₂O₂、L-酪氨酸和Cl⁻然后用NaCNBH₃还原的牛血清白蛋白（BSA）的酸水解产物中鉴定出化合物Nε-(2-(对羟基苯基)乙基)赖氨酸（pHA-赖氨酸）。其他卤化物（F⁻、Br⁻、I⁻）和拟卤化物SCN⁻不能替代Cl⁻作为髓过氧化物酶-H₂O₂-L-酪氨酸系统中的底物。在没有酶系统的情况下，在BSA、L-酪氨酸和试剂HOCl的还原反应混合物中检测到pHA-赖氨酸。相反，当BSA与L-酪氨酸和HOBr、过氧亚硝酸盐、羟基自由基或多种其他过氧化物酶一起孵育时，未检测到pHA-赖氨酸，这表明醛-蛋白质加合物是由HOCl选择性产生的。在酪氨酸存在下活化的人类中性粒细胞也修饰了BSA赖氨酸残基。pHA-赖氨酸的形成需要L-酪氨酸和细胞活化；它受到过氧化物酶抑制剂和过氧化氢酶的抑制，这表明髓过氧化物酶和H₂O₂参与了反应途径。在经过还原、水解然后通过质谱分析的炎症人类组织中检测到pHA-赖氨酸，这表明pHA与蛋白质的反应可能具有生理重要性。这些观察结果增加了在组织中鉴定pHA-赖氨酸将确定吞噬细胞在体内造成氧化损伤的靶标的可能性。
