McCormick M L, Gaut J P, Lin T S, Britigan B E, Buettner G R, Heinecke J W
Research Service, Veterans Affairs Medical Center, Iowa City, Iowa 52246, USA.
J Biol Chem. 1998 Nov 27;273(48):32030-7. doi: 10.1074/jbc.273.48.32030.
Phagocytes secrete the heme protein myeloperoxidase, which is present and active in human atherosclerotic tissue. These cells also generate hydrogen peroxide (H2O2), thereby allowing myeloperoxidase to generate a range of oxidizing intermediates and stable end products. When this system acts on L-tyrosine in vitro, it forms o, o'-dityrosine, which is enriched in atherosclerotic lesions. Myeloperoxidase, therefore, may oxidize artery wall proteins in vivo, cross-linking their L-tyrosine residues. In these studies, we used electron paramagnetic resonance (EPR) spectroscopy to identify an oxidizing intermediate in this reaction pathway and in parallel reactions catalyzed by horseradish peroxidase and lactoperoxidase. Using an EPR flow system to rapidly mix and examine solutions containing horseradish peroxidase, H2O2, and L-tyrosine, we detected free tyrosyl radical (a2,6H = 6.3 G, a3,5H = 1.6 G, and abetaH = 15. 0 G). We then used spin trapping techniques with 2-methyl-2-nitrosopropane (MNP) to further identify this intermediate. The resulting three-line spectrum (aN = 15.6 G) was consistent with an MNP/tyrosyl radical spin adduct. Additional MNP spin trapping studies with ring-labeled L-[13C6]tyrosine yielded a characteristic eight-line EPR spectrum (aN = 15.6 G, a13C (2) = 8.0 G, a13C (1) = 7.1 G, a13C (1) = 1.3 G), indicating that the MNP adduct resulted from trapping a carbon-centered radical located on the aromatic ring of L-tyrosine. This same eight-line spectrum was observed when human myeloperoxidase or bovine lactoperoxidase was substituted for horseradish peroxidase. Furthermore, a partially immobilized MNP/tyrosyl radical spin adduct was detected when we exposed a synthetic polypeptide composed of glutamate and L-tyrosine residues to the myeloperoxidase-H2O2-L-tyrosine system. The broadened EPR signal resulting from this MNP/polypeptide adduct was greatly narrowed by proteolytic digestion with Pronase, confirming that the initial spin-trapped radical was protein-bound. Collectively, these results indicate that peroxidases use H2O2 to convert L-tyrosine to free tyrosyl radical. They also support the idea that free tyrosyl radical initiates cross-linking of L-tyrosine residues in proteins. We suggest that this pathway may play an important role in protein and lipid oxidation at sites of inflammation and in atherosclerotic lesions.
吞噬细胞分泌血红素蛋白髓过氧化物酶，该酶存在于人类动脉粥样硬化组织中并具有活性。这些细胞还会产生过氧化氢（H2O2），从而使髓过氧化物酶能够生成一系列氧化中间体和稳定的终产物。当该系统在体外作用于L-酪氨酸时，会形成邻苯二酚二酪氨酸，其在动脉粥样硬化病变中含量丰富。因此，髓过氧化物酶可能在体内氧化动脉壁蛋白，使它们的L-酪氨酸残基发生交联。在这些研究中，我们使用电子顺磁共振（EPR）光谱来鉴定该反应途径以及辣根过氧化物酶和乳过氧化物酶催化的平行反应中的氧化中间体。使用EPR流动系统快速混合并检测含有辣根过氧化物酶、H2O2和L-酪氨酸的溶液，我们检测到了游离的酪氨酸自由基（a2,6H = 6.3 G，a3,5H = 1.6 G，abetaH = 15.0 G）。然后我们使用2-甲基-2-亚硝基丙烷（MNP）的自旋捕获技术来进一步鉴定该中间体。所得的三线谱（aN = 15.6 G）与MNP/酪氨酸自由基自旋加合物一致。用环标记的L-[13C6]酪氨酸进行的额外MNP自旋捕获研究产生了特征性的八线EPR谱（aN = 15.6 G，a13C (2) = 8.0 G，a13C (1) = 7.1 G，a13C (1) = 1.3 G），表明MNP加合物是通过捕获位于L-酪氨酸芳香环上的碳中心自由基形成的。当用人髓过氧化物酶或牛乳过氧化物酶替代辣根过氧化物酶时，观察到了相同的八线谱。此外，当我们将由谷氨酸和L-酪氨酸残基组成的合成多肽暴露于髓过氧化物酶-H2O2-L-酪氨酸系统时，检测到了部分固定化的MNP/酪氨酸自由基自旋加合物。用链霉蛋白酶进行蛋白水解消化极大地缩小了该MNP/多肽加合物产生的变宽的EPR信号，证实最初自旋捕获的自由基是与蛋白质结合的。总体而言，这些结果表明过氧化物酶利用H2O2将L-酪氨酸转化为游离的酪氨酸自由基。它们还支持游离酪氨酸自由基引发蛋白质中L-酪氨酸残基交联的观点。我们认为该途径可能在炎症部位和动脉粥样硬化病变中的蛋白质和脂质氧化中起重要作用。
