二氧化氮在水性模型体系中的反应：肽和蛋白质中酪氨酸单元的氧化

Reactions of nitrogen dioxide in aqueous model systems: oxidation of tyrosine units in peptides and proteins.

作者信息

Prütz W A, Mönig H, Butler J, Land E J

出版信息

Arch Biochem Biophys. 1985 Nov 15;243(1):125-34. doi: 10.1016/0003-9861(85)90780-5.

Abstract

By application of pulse radiolysis it was demonstrated that nitrogen dioxide (NO2.) oxidizes Gly-Tyr in aqueous solution with a strongly pH-dependent rate constant (k6 = 3.2 X 10(5) M-1 S-1 at pH 7.5 and k6 = 2.0 X 10(7) M-1 S-1 at pH 11.3), primarily generating phenoxyl radicals. The phenoxyl can react further with NO2. (k7 approximately 3 X 10(9) M-1 S-1) to form nitrotyrosine, which is the predominant final product in neutral solution and at low tyrosyl concentrations under gamma-radiolysis conditions. Tyrosine nitration is less efficient in acidic solution, due to the natural disproportionation of NO2., and in alkaline solutions and at high tyrosyl concentrations due to enhanced tyrosyl dimerization. Selective tyrosine nitration by interaction of NO2. with proteins (at pH 7 to 9) was demonstrated in the case of histone, lysozyme, ribonuclease A, and subtilisin Carlsberg. Nitrotyrosine developed slowly also under incubation of Gly-Tyr with nitrite at pH 4 to 5, where NO2. is formed by acid decomposition of HONO. It is recalled in this context that NO2.-induced oxidations, by regenerating NO2-, can propagate NO2./NO2- redox cycling under acidic conditions. Even faster than with tyrosine is the NO2.-induced oxidation of cysteine-thiolate (k9 = 2.4 X 10(8) M-1 S-1 at pH 9.2), involving the transient formation of cystinyl radical anions. The interaction of NO2. with Gly-Trp was comparably slow (k approximately 10(6) M-1 S-1), and no reaction was detectable by pulse radiolysis with Met-Gly and (Cys-Gly)2, or with DNA. Slow reactions of NO2. were observed with arachidonic acid (k approximately 10(6) M-1 S-1 at pH 9.0) and with linoleate (k approximately 2 X 10(5) M-1 S-1 at pH 9.4), indicating that NO2. is capable of initiating lipid peroxidation even in an aqueous environment. NO2.-Induced tyrosine nitration, using 50 microM Gly-Tyr at pH 8.2, was hardly inhibited, however, in the presence of 1 mM linoleate, and was not affected at all in the presence of 5 mM dimethylamine (a nitrosamine precursor). It is concluded that protein modifications, and particularly phenol and thiol oxidation, may be an important mechanism, as well as initiation of lipid peroxidation, of action of NO2. in biological systems.

摘要

通过脉冲辐解技术证实，二氧化氮（NO₂·）在水溶液中氧化甘氨酰 - 酪氨酸，其速率常数强烈依赖于pH值（在pH 7.5时k₆ = 3.2×10⁵ M⁻¹ s⁻¹，在pH 11.3时k₆ = 2.0×10⁷ M⁻¹ s⁻¹），主要生成苯氧自由基。苯氧自由基可进一步与NO₂·反应（k₇约为3×10⁹ M⁻¹ s⁻¹）形成硝基酪氨酸，在γ辐解条件下，硝基酪氨酸是中性溶液和低酪氨酸浓度时的主要最终产物。在酸性溶液中，由于NO₂·的自然歧化反应，酪氨酸硝化效率较低；在碱性溶液中以及高酪氨酸浓度下，由于酪氨酸二聚化增强，酪氨酸硝化效率也较低。在组蛋白、溶菌酶、核糖核酸酶A和枯草杆菌蛋白酶卡尔伯格的情况下，已证实在pH 7至9时，NO₂·与蛋白质相互作用可实现选择性酪氨酸硝化。在pH 4至5时，甘氨酰 - 酪氨酸与亚硝酸盐孵育时，硝基酪氨酸也会缓慢生成，此时HONO通过酸分解形成NO₂·。在此背景下需要提及的是，在酸性条件下，NO₂·诱导的氧化反应通过再生NO₂⁻，可引发NO₂/NO₂⁻氧化还原循环。NO₂·诱导的半胱氨酸硫醇盐氧化（在pH 9.2时k₉ = 2.4×10⁸ M⁻¹ s⁻¹）甚至比酪氨酸氧化更快，该过程涉及胱氨酸自由基阴离子的瞬时形成。NO₂·与甘氨酰 - 色氨酸的相互作用相对较慢（k约为10⁶ M⁻¹ s⁻¹），通过脉冲辐解未检测到NO₂·与甲硫氨酰 - 甘氨酸和（半胱氨酰 - 甘氨酸）₂或与DNA的反应。观察到NO₂·与花生四烯酸（在pH 9.0时k约为10⁶ M⁻¹ s⁻¹）和亚油酸（在pH 9.4时k约为2×10⁵ M⁻¹ s⁻¹）的反应较慢，这表明即使在水性环境中，NO₂·也能够引发脂质过氧化。然而，在pH 8.2时，使用50 μM甘氨酰 - 酪氨酸，NO₂·诱导的酪氨酸硝化在1 mM亚油酸存在下几乎不受抑制，在5 mM二甲胺（一种亚硝胺前体）存在下则完全不受影响。可以得出结论，蛋白质修饰，特别是酚类和硫醇氧化，以及脂质过氧化的引发，可能是NO₂·在生物系统中作用的重要机制。